Index [] Index A AAQ (acryl-azobenzene-quaternary ammonium), 38 AAV. SeeAdeno-associated virus AAV-G-CaMP3, 68 AAV plasmid preparation (recipe), 604–605 Ablation, for in situ histology

1061

Index

AAAQ (acryl-azobenzene-quaternary

ammonium), 38AAV. See Adeno-associated virusAAV-G-CaMP3, 68AAV plasmid preparation (recipe), 604–605Ablation, for in situ histology of brain tissue

with femtosecond laser pulses,437–445

Acepromazine, 515, 526Acetoxymethyl (AM)-conjugated indicators

dextran-conjugated indicators compared,131–132, 132f, 137

limitations of, 131loading cerebellar granule cell axons in

brain slices (protocol), 123–129experimental method, 124–125imaging calcium transients in parallel

fiber presynaptic terminals, 125imaging setup, 123loading granule cell presynaptic terminals

in rodent transverse cerebellarbrain slices, 124–125, 125f

materials, 123–124preparation of AM-conjugated calcium

indicator loading solution, 124recipes, 129troubleshooting, 126

presynaptic calcium measurements usingbulk loading, 121–129

loading conditions, determiningappropriate, 128, 128f

overview, 121–122protocol, 123–126selection of indicator, 127–128, 127f

Acetoxymethyl (AM) ester calcium indicatorsimaging action potentials with, 369–374loading of Bergmann glia with (protocol),

716–717Acousto-optical deflector (AOD)-based

patterned UV neurotransmitteruncaging, 255–270

application, 258–260, 259fglutamate, patterned uncaging of,

258–260, 259f

implementation of AOD-based system,258

comparison with other beam-steeringapproaches, 258

comparison with two-photonuncaging system, 258

implementing beam steering by softwarecontrol of AOD, 267

analog outputs, 267daily recalibration, 267parking the beam, 267stimulus design, 267

overview, 255–257acousto-optic deflectors, 256–257characteristics of caged compounds,

256one- and two-photon imaging, 256spatial resolution, 257

protocols, 261–270alignment and implementation of

uncaging, 264–267aligning uncaged and imaging

beams, 266course alignment, 265discussion, 266–267experimental method, 264–265implementing beam steering by

software control of AOD, 267materials, 264preparation of caged fluorescein

dried sample, 264troubleshooting, 265–266

integrating AOD-based patterneduncaging with UV light into atwo-photon microscope,261–263

assembling UV beam path andincorporating the AODs, 263

basic control of AODs, 262imaging setup, 262overview of UV beam path,

261–262, 261fpreparing and handling caged

compound solutions, 268–270application of caged compounds,

269

bath application, 268caged glutamate, preparation of,

268capillary application, 269discussion, 269–270, 269fexperimental method, 268–269focal application through capillary

tubing, 269–270, 269fmaterials, 268

Acousto-optical deflector (AOD)-based two-photon microscopy for high-speed calcium imaging ofneuronal population activity,543–555

overview, 543–544random-access pattern scanning

(protocol), 545–555application example, 554discussion, 554–555experimental method, 548–552

alignment and optimizationprocedures, 549–550

animal preparation, 550microscope setup, 548–549random access pattern scanning,

550–552, 551fimaging setup, 545–547, 547fmaterials, 547–548troubleshooting, 552–553

Acousto-optical deflectors (AODs), 256–257application to astrocyte imaging, 6953D laser scanning and, 539limitations of, 544patterned UV neurotransmitter uncaging

and, 255–270Acousto-optical tunable filter (AOTF), for

confocal spot detection, 142Acryl-azobenzene-quaternary ammonium

(AAQ), 38ACSF. See Artificial cerebrospinal fluidAction potentials

Aplysia abdominal ganglion, 472, 473f,476–477

back-propagated action potential (bAP),293

calcium dynamics and, 63, 66, 70, 71

Page references followed by f denote figures; page references followed by t denote tables.

1061-1082_Index_IMG2_Imaging 2/14/11 9:43 AM Page 1061

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1062 / Index

Action potentials (Continued)imaging neuronal population activity,

839–849action potential detection, 845–847application example, 847, 848fdata analysis, 843–847, 844fexperimental procedures, 840–843future prospects, 848–849

imaging with calcium indicators, 369–374advantages, 374application example, 373, 373finferring spikes, 373, 374flimitations, 374protocol

experimental method, 370–372imaging setup, 370loading embryonic and neonatal

acute cortical slices, 371loading juvenile and adult acute

cortical slices, 371materials, 370pressure injection, 371–372

interferometric detection of, 195–199advantages and limitations, 199application example, 198–199, 198fprinciples of interferometric detection,

195–198secretion-coupled light scattering and,

161–168AdEasy-1, 718, 719Adeno-associated virus (AAV)

in Alzheimer’s disease imaging, 991f, 992,993, 994f

for opsin expression in mammalian brain,867, 869, 871–872

recombinant (rAAV), 588–590, 588f–589fAdenosine triphosphate (ATP), as

gliotransmitter, 639Adenovirus, recombinant for gene transfer

into astrocytes, 707, 708f,710–712, 718

Adenylyl cyclases, photoactivated, 867ADVASEP-7, 179, 188Aldehyde dehydrogenase (Aldh1L1) promoter,

687Alexa dyes

Alexa-488 dextran, 138Alexa-594 dextran, 138Alexa Fluor 488

two-photon calcium imaging ofdendritic spines, 275, 276f

Alexa Fluor 594for detection of astrocyte vacuole-like

vesicles, 644–645, 645ffor imaging neuronal population

activity, 842for two-photon calcium imaging of

dendritic spines, 274–276, 275fAlexa Fluor 568 hydrazide, 132f

Algebraic reconstruction technique, 1028Alkaline trapping, 668, 669fAllen Brain Atlas, 377Allen Institute for Brain Science, 116

All-optical in situ histology of brain tissue,437–445

advantages and limitations, 443–444application example, 443, 444flaser alignment, 442–443overview, 437–438, 440fprotocol, 438–445

blocking and staining, 442discussion, 442–444experimental method, 441–442imaging setup, 439, 440fiterative imaging and ablation, 442,

443tmapping region of interest, 441–442materials, 439, 441perfusing with fluorescent gel, 442recipes, 445subblocking and mounting, 442

All-trans retinal, 864αCaMKII promoter, 585, 586fα-chloralose, 931Alzheimer’s disease

imaging neural networks in mouse model(protocol), 1000–1009

discussion, 1007–1008, 1008fexperimental method, 1001–1005

multicolor imaging, 1004f, 1005plaque-staining procedure, 1002,

1003fstaining neurons and glia with

calcium indicator dyes,1002–1003

surgical procedure, 1001–1002imaging setup, 1000materials, 1000–1001recipes, 1008–1009troubleshooting, 1005–1006

imaging of structure and function in,989–995

mouse models of, 990, 1000–1009near-infrared fluorescence (NIRF)

tomography, 989, 990overview, 989, 999postmortem diagnosis, 989two-photon imaging, 990–995, 1000–1009

amyloid plaques, 991–992calcium imaging, 993–994, 994ffunctional in vivo imaging, 993–995,

994f, 995fneurofibrillary tangles, 992–993oxidative stress, 994, 995fstructural in vivo imaging, 991–993

AMPA receptor (AMPAR), 7, 8fimaging with two-photon uncaging

microscopy, 251–252, 251fmapping distribution using two-photon

photostimulation, 429Amplex Red, 994, 995FAmyloid β, 989–995, 999. See also Alzheimer’s

diseaseAmyloid plaques, labeling of, 1002, 1003f, 1007Amyloid precursor protein (APP), 989, 992,

999

Anesthesiacat, 514–515, 526Drosophila, 561mouse, 308, 322, 324, 492, 504, 534, 550,

569, 595, 602–603, 615, 688,711, 714, 724, 726, 728, 871,952, 965, 985, 1001, 1015, 1016,1019, 1026

rat, 534, 871, 931rodent, 514, 526zebrafish, 569

Anesthetic for cats (recipe), 526Anesthetic for rodents (recipe), 526AngioSense, 1025Antigen presenting cells (APCs), preparation

of, 984Antioxidants, to decrease photodynamic

damage, 337AODs. See Acousto-optical deflectors (AODs)AOTF (acousto-optical tunable filter), for

confocal spot detection, 142APCs (antigen presenting cells), preparation

of, 984Aplysia abdominal ganglion action potentials,

472, 473f, 476–477APP (amyloid precursor protein), 989, 992,

999Arc lamp, 1037–1038Artificial cerebrospinal fluid (ACSF) (recipe),

129, 244, 315, 415, 424,486–487, 527, 652, 681–682,696, 705, 733, 760, 976, 988

HEPES-buffered, 719high-divalent, 425holding, 486–487modified, 487modified, free of carbonate and

phosphate, 936Ascorbate, 337ASLV (avian sarcoma and leucosis virus), 85,

96Astrocytes, 521

current status of in vivo imaging of, 695imaging astrocyte calcium and vacuole-

like vesicles, 639–653detection of calcium signals by two-

photon microscopy, 642–644,643f

detection from astrocytes infusedwith membrane-impermeablecalcium indicators, 643–644,643f

detection from multiple astrocytesin slices, 642, 643f

detection from photolysis of cagedcompound in single astrocyte,644, 644f

detection of vacuole-like vesicles bytwo-photon microscopy,644–647, 645f

using Fluo-4 AM, 646using Fluo-4 potassium and Alexa

Fluor 594, 644–645, 645f





Index / 1063

using FM1-43, 646–647, 647fidentification of astrocytes and

neurons in hippocampal slices,640–642, 641f

by biocytin staining, 642by electrophysiological properties,

642by GRAP staining, 642morphology confirmation, 640,

641f, 642imaging setup, 640protocols, 648–653

biocytin staining procedure, 648recipes, 652–653two-photon imaging of acute brain

slices preincubated with Fluo-4AM, 649

two-photon imaging of astrocytesfilled with photolabile cagedcalcium compound, 651

monitoring exocytosis in, 655–670overview, 655–656protocols

identification and staining ofdense-core granules (DCGs),659–660

identification and staining oflysosomes, 661

identification and staining ofsynaptic-like microvesicles(SLMVs), 657–658

imaging exocytosis and recycling atthe whole-cell level, 667–669

imaging exocytosis at the singlevesicle level with EWi, 663–666,665f

transfection of, 658, 659–660two-photon imaging astrocytic excitation

in cerebellar cortex of awakemobile mice, 745–760

in vivo calcium imaging of cerebellarcerebellar craniotomy for in vivo

imaging (protocol), 713–715injection of recombinant adenovirus

for gene transfer of FCIP G-CaMP2 into astrocytes ofcerebellar cortex (protocol),710–712

experimental method, 711–712materials, 710–711troubleshooting, 712

overview, 707–709, 708fpreferential loading of Bergmann glia

with synthetic acetoxymethylcalcium dyes (protocol),716–717

in vivo imaging of structural andfunctional properties, 685–696

future developments in imaging, 695overview, 685–687protocols

imaging of calcium dynamics incortical astrocytes: loading with

calcium indicator dyes, 690imaging of calcium dynamics in

cortical astrocytes: loading withphotolabile caged calciumcompounds, 691

in vivo imaging of astrocytesstained with SR101, 688–689

in vivo imaging of redox stateusing NADH intrinsicfluorescence with cellularresolution, 692–694, 693f

in vivo labeling of cortical astrocytes withsulforhodamine 101, 673–682,674f

application example, 680, 689foverview, 673–674, 674fprotocol, 675–682

ATP (adenosine triphosphate),gliotransmitter, 639

Atropine, 514, 515, 526Autofluorescence, of flavoproteins, 919Automated imaging and analysis of behavior

in Caenorhabditis elegans,763–775

Avertin, 1015, 1016, 1019Avian sarcoma and leucosis virus (ASLV), 85,

96Awake animals, imaging in

fiber-optic calcium monitoring ofdendritic activity, 903, 904f

imaging neuronal population activity,839–849

application example, 847, 848fdata analysis, 843–847, 844f

action potential detection, 845–847correction for movement artifacts,

845estimation of noise levels and

neuropil contamination, 845normalization of fluorescence

values, 843–844experimental procedures, 840–843

cell-attached electrophysiology,842–843, 844f

dye preparation for injection,842–843

habituation and training for awakeexperiments, 840

head plate design, 840–841, 841fhead plate implantation, 841–842surgical considerations, 842targeting sensory areas, 842

future prospects, 848–849miniaturization of two-photon

microscopy for imaging infreely moving animals, 851–860

optical imaging based on intrinsic signals,912

optogenetics in freely moving mammals,877–885

overview, 851–862two-photon imaging/microscopy of

astrocytic and neuronal

excitation in cerebellar cortexof awake mobile mice, 745–760

two-photon imaging of neural activity inawake mobile mice, 827–836

data acquisition and processing,831–834, 833f

examples of imaging in different brainregions, 834, 835f

experimental apparatus, 828–829, 828ffuture outlook, 836surgical procedures and dye loading,

829–831, 830fvoltage-sensitive dye imaging of cortical

spatiotemporal dynamics inawake behaving mice, 817–824

voltage-sensitive dye imaging ofneocortical activity of corticaldynamics in behaving monkeys,810–811

Axonscorrelated light and electron microscopy

of green fluorescent protein–labeled, 227–236

interferometric detection of actionpotentials, 195–199

loading cerebellar granule cell axons inbrain slices with acetoxymethyl(AM)-conjugated calciumindicators, 123–129

sodium imaging in, 201–206Azobenzene, 34f, 35–38

BBAC (bacterial artificial chromosome)

advantages for labeling genetically definedcell types, 116

Cre mice, 116, 118–119transgenic mice, 113–119

application to neuroimaging studies,116–118, 117f, 118f

generation of, 114–115, 115fGENSAT Project database, 118–119ordering mouse lines, 119

Back-propagated action potential (bAP), 293Bacteriorhodopsin

light-induced dipole switching, 43foverview, 42f, 866photocycle, 43f, 44–45, 48structural and functional homologies with

channelrhodopsin, 46f, 47Bafilomycin A1, 668, 669fBallistic delivery of dyes, 447–456

advantages of, 455application examples, 454, 454fdisadvantages of, 455–456overview, 447–448protocol, 449–456

discussion, 454–456experimental method, 450–452imaging setup, 449labeling cells with water-soluble dyes,

452materials, 449–450





1064 / Index

Ballistic delivery of dyes (Continued)preparation and delivery of lipophilic

dyes, 450–452bullet preparation, 450–451coating tungsten beads, 450dye diffusion, tissue mounting, and

imaging, 451–452dye preparation, 450particle delivery, 451, 451f

troubleshooting, 452–453BAP (back-propagated action potential), 293BBS (BES-buffered saline 2x) (recipe), 605Beam splitter, dichroic, 1033f, 1034Behavior

automated imaging and analysis ofbehavior in Caenorhabditiselegans, 763–775

fiber-optic dendritic calcium monitoringduring, 897–905

imaging motor behavior in zebrafish,783–789

optogenetic modulation of dopamineneurons, 877–886, 879f

functional integration during opticalcontrol, 884–885, 885f

integrating behavioral readouts withreward circuit modulation,880–884

conditioned place preference,881–883, 882f

operant conditioning, 883–884two-photon imaging of astrocytic and

neuronal excitation incerebellar cortex of awakemobile mice, 745–760

habituation of mice (protocol), 750–751optical window preparation and tissue

(protocol), 752–754surgical implantation of a head plate

(protocol), 747–749, 748ftwo-photon imaging, 755–760

discussion, 759–760example data, 758fexperimental method, 756–757imaging setup, 755materials, 756recipe, 760troubleshooting, 757


voltage-sensitive dye imaging of corticalspatiotemporal dynamics inawake behaving mice, 817–824

voltage-sensitive dye imaging ofneocortical activity, long-termin behaving monkeys, 810–811

Benzonase, 600, 602Bepanthen, 724Bergmann glia

imaging calcium waves in, 699–705, 704fpreferential loading with synthetic

acetoxymethyl calcium dyes(protocol), 716–717

in vivo calcium imaging with syntheticand genetic indicators,707–719, 708f

Biocytin-filled cells, histologicalreconstruction of, 381

Biocytin staining of astrocytes, 641f, 642, 648Bioluminescence imaging, for monitoring

doxycycline (Dox)-controlledgene expression, 585, 586f, 592,595

Bipolar cells, loading and TIRF imaging ofdissociated, 187–188

experimental method, 187–188materials, 187troubleshooting, 188

Birefringent plate, in interferometry, 196Bleaching

G-CaMP and, 558, 558fin stimulated emission depletion (STED),

242voltage-sensitive dyes, 480

Blocking solution (recipe), 445Blood–brain barrier, 990, 1011–1012, 1021Blood flow

optical imaging based on intrinsic signals,913–917

optically induced occlusion of single bloodvessels in neocortex, 939–947

application example, 947histology, 945–946, 945flimitations of, 947overview, 939–940protocol, 941–946

two-photon imaging of blood flow incortex, 927–936, 930f, 933f

two-photon imaging of neuronalstructural plasticity in miceduring and after ischemia,949–957

overview, 949protocol, 950–957

discussion, 957experimental method, 951–955,

951f, 953fimaging setup, 950materials, 950–951troubleshooting, 955–956

Blowfly, in vivo dendritic calcium imaging invisual system, 777–780

[Ca2+] measurements, 779–780, 780fcell labeling, 778imaging, 779imaging setup, 778–779, 778fwhole-mount preparation, 777–778

Blue dyes, in vivo voltage-sensitive dyeimaging of mammalian cortexusing, 481–483

BOLD (blood-oxygenation-level-dependent)functional magnetic resonanceimaging (fMRI), 915, 916

Bovine chromaffin cells, transfection andTIRF imaging of cultured,189–190


BPB (bromophenol blue), 180, 180fBrainbow

advantages, 109color-assisted circuit tracing, 102, 103colors, 101–102generation and imaging of Brainbow mice

(protocol), 105–110discussion, 109experimental method, 106–108imaging setup, 105materials, 106recipe, 109–110Reconstruct software, 105, 107–108troubleshooting, 108

GRE mice, 104limitations, 109overview of approach, 99–100, 100fstrategies, 100–101, 102ttransgene generation, 103–104

elements for optimized expression, 104fluorescent proteins, 103FRT site, 104lox sites, 103promoter, 103subcellular localization signals, 103–104

transgenic micegeneration and imaging of, 105–110lines, 104

Brain slicescircuit mapping by ultraviolet uncaging of

glutamate, 417–425imaging astrocyte calcium and vacuole-

like vesicles, 639–653imaging microglia in, 735–742infrared-guided laser stimulation of

neurons in, 388–390loading AM-conjugated indicators in

cerebellar granule cell axons,123–129

maintaining live slices, 331–338mapping connections in, 432–434, 433f,

434foptical measurement of dopamine nerve

terminals in, 884–885voltage-sensitive dye imaging of

population signals in, 478–480Brain tumor imaging, 1011–1021

fluorescence molecular tomography,1023–1029

imaging brain metastasis using a brain-metastasizing breastadenocarcinoma (protocol),1018–1021

discussion, 1021experimental method, 1019–1020

analysis of tumor metastasis byfluorescence imaging,1019–1020, 1020f

preparation of cells for injection,1019





Index / 1065

tumor cell injection by intracardiacinjection, 1019

imaging setup, 1018materials, 1018–1019

live imaging of glioma by two-photonmicroscopy (protocol),1013–1017


cranial window preparation, 1015postimaging analysis, 1016preparation of cells for injection,

1014tumor cell injection into brain,

1015two-photon imaging, 1016, 1016f

imaging setup, 1013materials, 1013–1014troubleshooting, 1017

Breast cancer, metastatic, 1012, 1018–1021,1020f

Breathing artifacts, 10066-bromo-7-hydroxycoumarin-4-ylmethoxy-

carbonyl-glutamate (Bhc-glu),246, 246f

Bromophenol blue (BPB), 180, 180fBungarotoxin (BTX), 221–222Buprenorphine, 324, 712, 872, 902, 1015,

1019

CCAA (cerebral amyloid angiopathy), 992, 994,

995fCaenorhabditis elegans, automated imaging

and analysis of behavior in,763–775

Caffeine, dorsal root ganglion (DRG) neuronresponse to, 343, 343f

Ca2+-free Ringer’s solution (recipe), 509Caged calcium chelators, 393–401

chemical properties of, 393–397, 394f,394t, 395f, 396f

detection from photolysis of cagedcompound in single astrocyte,644, 644f, 651

illumination systems, 399–401light sources, 399–400measuring effectiveness of photolysis,

401setup and accessories, 400

overview, 393practical challenges to use in neurons,

397–399cell loading, 397–398estimating the efficiency of photolysis,

398quantification of ∆[Ca2+]i, 398–399,

399fCaged compounds, characteristics of, 256Caged glutamate stock solution (recipe), 425Calcium (Ca2+)

confocal spot detection of Ca2+ domains,141–149

application to study of presynapticCa2+ microdomains, 147–149,148f

hardware overview, 142–143, 143foptical conditions for, 143–147

Ca2+ indicator choice forappropriate brightness andsensitivity, 144–146

characterization of confocalvolume, 144

point spread function (PSF), 144rapidly responding indicator,

146–147shortcomings of, 147signal-to-noise ratio (SNR), 147–149,

148feffects on light scattering associated with

secretion from peptidergicnerve terminals, 163–164, 163f

fiber-optic monitoring of dendriticactivity in vivo, 897–905

single compartment model of calciumdynamics, 355–366

uncaging calcium in neurons, 393–401uncaging in nerve terminals, 151–159

examining transmitter release at calyxof Held synapse, 158–159, 158f

overview, 151–152three-point calibration procedure

(protocol), 153–159calculation of Keff, 157determination of post-flash

calibration constants, 157experimental method, 154–157imaging setup, 153in-cell calibration, 155–157, 156fmaterials, 153, 154tratiometric measurements of

[Ca2+]i, 154–155recipes, 159in vitro calibration, 155, 156f

in vivo recordings and channelrhodopsin-2 activation through an opticalfiber, 889–895

application examples, 895overview, 889protocol, 890–894, 893f

anesthesia, 892experimental method, 892–893, 893fimaging setup, 890, 891fimplantation and fixation of

optical fiber, 892–893materials, 890–891skull preparation, 892troubleshooting, 894

wide-field charge coupled device (CCD)camera based imaging ofcalcium waves and sparks incentral neurons

loading cells and detecting [Ca2+]ichanges (protocol), 282–285

application examples, 284–285,284f, 285f

discussion, 284–285experimental method, 283materials, 282–283

Calcium chelators. See Caged calciumchelators

Calcium dynamicsimaging in cortical astrocytes

loading with calcium indicator dyes,690

loading with photolabile caged calciumcompounds, 691

simultaneous recording of tectal Ca2+

dynamics and motor behaviorin zebrafish, 785

single compartment model of, 355–366application examples, 363dynamics of single [Ca2+]i transients,

360extensions to model, 364–366

buffered calcium diffusion, 364deviations from linear behavior,

364–365, 365fmeasurement of calcium-driven

reactions, 365–366saturation of buffers and pumps,

364slow buffers, 365

materials, 356model application, 361–363

estimates of amplitude and totalcalcium charge, 362–363

estimates of endogenous Ca2+-binding ratio and clearancerate, 361–362, 362f

estimates of unperturbed calciumdynamics, 363

model assumptions and parameters,356–360, 358t–359t

buffering, 358clearance, 360influx, 357–358

overview, 355–356schematic of model, 357fsummation of [Ca2+]i transients during

repetitive calcium influx, 360–361Calcium Green-1 (CG-1)

two-photon calcium imaging of dendriticspines, 278

for two-photon imaging of neural activityin awake mobile mice, 831

Calcium Green-1 (CG-1) dextran, 132fballistic delivery of, 452, 454, 454fcalcium imaging in olfactory neurons, 566,

566fimaging neural activity in zebrafish larvae,

793, 796, 796fCalcium imaging

acousto-optical deflector (AOD)-basedtwo-photon microscopy forhigh-speed imaging of neuronalpopulation activity, 543–555

in Alzheimer’s disease, 993–994, 994f,1002–1005





1066 / Index

Calcium imaging (Continued)confocal imaging of neuronal activity in

larval zebrafish, 791–797in Drosophila olfactory system with a

genetic indicator, 557–563functional neuron-specific expression of

genetically encoded fluorescentcalcium indicator proteins inliving mice, 583–606

imaging astrocyte calcium and vacuole-like vesicles, 639–653

imaging calcium waves and sparks incentral neurons, 281–285

in intact olfactory system of zebrafish andmouse, 565–571

NADH imaging in combination with, 694of neuronal activity and motor behavior

in zebrafish, 783–789in neuronal endoplasmic reticulum,

339–344application example, 343, 343foverview, 339, 340fprotocol, 341–344

advantages and limitations,343–344

discussion, 343–344imaging setup, 341intracellular calibration of

MagFura-2 signals, 342materials, 341real-time video imaging, 342recipes, 344staining neurons with MagFura-2

AM and Fluo-3, 342of neuronal population activity in awake

and anesthetized rodents,839–849

of neurons in visual cortex using troponinC–based indicator, 611–620,612f

in populations of olfactory neurons byplanar illumination microscopy,573–580

of two-photon of dendritic spines,273–278

overview, 273, 278protocol, 274–278

estimating changes in calciumconcentration from ∆F/F, 277

experimental method, 274–277imaging calcium transients in

dendritic spines, 276–277loading neurons with calcium

indicators and Alexa dyes,274–276, 275f, 276f

materials, 274recipe, 278

in vivo dendritic imaging in fly visualsystem, 777–780

in vivo local dye electroporation, 501–509in vivo of cerebellar astrocytes, 707–719in vivo two-photon imaging in visual

system, 511–527

in vivo two-photon using multicell bolusloading of fluorescentindicators, 491–499

Calcium indicatorsconfocal spot detection of presynaptic

Ca2+ domains, 141–149detection of calcium signals from

astrocytes by two-photonmicroscopy, 643–644, 643f

imaging action potentials with, 369–374imaging neuronal activity with genetically

encoded calcium indicators(GECIs), 63–73

imaging of calcium dynamics in corticalastrocytes, 690

with photolabile caged calciumcompounds, 691

imaging presynaptic calcium transientsusing dextran-conjugatedindicators, 131–138

influence of rate constant on speed ofcalcium-dependentfluorescence response, 146

loading into olfactory sensory neurons,566–567

bolus loading of neurons downstreamfrom glomeruli, 567

dextran-coupled (protocol), 568–571genetically encoded indicators, 567selective loading of neurons projecting

to glomeruli, 566multicell bolus loading, 491–499presynaptic calcium measurements using

bulk loading of acetoxymethylindicators, 121–129

signal-to-noise ratio, 64, 145–146single compartment model of calcium

dynamics, 356two-photon calcium imaging of dendritic

spines, 273–278in vivo calcium imaging of cerebellar

astrocytes, 707–719, 708fCalcium phosphate transfection, 597, 599–600Calcium waves

imaging in central neurons, 281–285imaging in cerebellar Bergmann glia,

699–705, 707, 708f, 709application example, 704, 704foverview, 699–700protocol, 701–705

discussion, 704experimental method, 701–702imaging setup, 701materials, 701recipes, 705troubleshooting, 702–703

Calibrating solutions (recipe), 344Calliphora vicina, in vivo dendritic calcium

imaging in visual system,777–780

[Ca2+] measurements, 779–780, 780fcell labeling, 778imaging, 779

imaging setup, 778–779, 778fwhole-mount preparation, 777–778

Calmodulin (CaM), 583, 584Cameleon

development of Yellow Cameleons,583–584

neuronal imaging in Caenorhabditiselegans, 764

CameraCCD camera

electron-multiplying (EMCCD), 578imaging calcium waves in Bergmann

glia, 699–705, 704fobjective-coupled planar illumination

(OCPI) microscopy, 578use in dendritic voltage imaging, 288,

292f, 293wide-field charge coupled device

(CCD) camera-based imaging,of calcium waves and sparks incentral neurons, 281–285

choice for voltage-sensitive dye imaging,475

infrared (IR), 378Camgaroo-1, 584Camgaroo-2, 584, 585, 586f–587fCamKIIα promoter, 867Capase indicators, 995Capillary tubing, application of caged

compounds through, 269–270Carbon dioxide, for Drosophila anesthesia, 5614-carboxymethoxy-5,7-dinitroindolinyl-

glutamate (CDNI-glu), 246,246f

Carprofen, 324, 615Cat

anesthesia induction, 514–515surgery, 515

craniotomy, 516durotomy, 517

in vivo two-photon calcium imaging invisual system, 511–527

Cautions, 1051–1058CCD camera

electron-multiplying (EMCCD), 578imaging calcium waves in Bergmann glia,

699–705, 704fobjective-coupled planar illumination

(OCPI) microscopy, 578use in dendritic voltage imaging, 288,

292f, 293wide-field charge coupled device (CCD)

camera-based imaging, ofcalcium waves and sparks incentral neurons, 281–285

CCLp, 584CD63, 661cDNA, recovery of replication-competent

rabies virus from (protocol),87–92

cell culture, 89fixation of cells and direct

immunofluorescence, 90





Index / 1067

materials, 88–89transfection and virus recovery, 89–90virus stock preparation, 92virus titration, 91, 91f

CDNI-glu (4-carboxymethoxy-5,7-dinitro-indolinyl-glutamate), 246, 246f

Cell-attached electrophysiology, combiningimaging with, 842–843, 844f

Cell populationslabeling genetically defined using BAC

transgenic mice, 113–119in vivo calcium imaging using multicell

bolus loading of fluorescentindicators, 491–499

Cerebellar craniotomy, 713–715Cerebral amyloid angiopathy (CAA), 992, 994,

995fCerebral blood flow, two-photon imaging of,

927–936, 930f, 933fCerebrospinal fluid, artificial. See Artificial

cerebrospinal fluidCFP. See Cyan fluorescent proteinCG-1. See Calcium Green-1 (CG-1)Channelrhodopsin (ChR), 864

biophysical properties, 48–49ChR1, 46–47, 48–49ChR2, 46f, 47, 48–49

activation spectrum of, 864activation through an optical fiber,

889–895, 893fdeactivation rate, 866light required for activation, 868

light-gated ion channels, 46–47light-induced dipole switching, 43foverview, 42f, 46–47step-function opsin genes (SFOs), 47structural and functional homologies with

bacteriorhodopsin, 46f, 47Channelrhodopsin-2-assisted circuit mapping

(CRACM), 424Chelators. See Caged calcium chelatorsChemical two-photon uncaging, 25–31

disadvantage of, 30overview, 25–26, 26fprotocol, 27–30

application example, 29, 29fdiscussion, 29–30experimental method, 28imaging setup, 27materials, 27–28troubleshooting, 28

Chlamydomonas reinhardtii, 46–47, 864Chloride imaging using Clomeleon, 75–80

advantages, 79application example, 79, 80flimitations, 79–80mode of action, 75protocol, 77–78

calibration procedure, 78imaging setup, 77materials, 77slice preparation, 77–78

structure of, 75, 76f

Chloride imaging using MQAE, 623–632advantages and limitations, 630application example, 627f, 628f, 630overview, 623–624protocol, 624–631

discussion, 630experimental method, 626–628imaging setup, 625intracellular calibration of MQAE,

627–628materials, 625–626recipes, 630–631staining cells or tissue slices via bath

application, 626, 627fstaining of neurons and glia using

multicell bolus loading, 626,628f

troubleshooting, 629two-photon imaging of stained cells,

626Choleratoxin B-Alexa (CTB-Alexa), 384ChR. See Channelrhodopsin (ChR)Chrysamine-G, 1007Ciona intestinalis, 53, 57Circuit mapping by ultraviolet uncaging of

glutamate, 417–425overview, 417–418protocol, 419–425

analyzing traces, 422discussion, 424experimental method, 420–422, 421fimaging setup, 419mapping a neuron’s excitation profile,

422mapping a neuron’s synaptic inputs,

420–421, 421fmaterials, 420preparation of brain slices, 420recipes, 424–425troubleshooting, 422–423

Circuit tracingcolor-assisted in Brainbow studies, 102,

103in vivo local dye electroporation for,

501–509Circularly permuted fluorescent proteins, 584Classical conditioning, for assessing reward-

related behavior, 881Cleaning optical equipment, 1039–1040Clomeleon, 75–80

chloride imaging, 623–624advantages, 79application example, 79, 80flimitations, 79–80mode of action, 75protocol, 77–78

structure of, 75, 76fCNS-1 cell line, rat, 1011–1017, 1016fCollagenase type IA, 593Common carotid artery occlusion, 951–954,

951fComplementation, rabies virus vectors and,

84–85, 85f

Conditioned place preference, 881–883, 882fConfocal calcium imaging of neuronal activity

in larval zebrafish, 791–797Confocal spot detection of Ca2+ domains,

141–149application to study of presynaptic Ca2+

microdomains, 147–149, 148fhardware overview, 142–143, 143foptical conditions for, 143–147

Ca2+ indicator choice for appropriatebrightness and sensitivity,144–146

characterization of confocal volume,144

point spread function (PSF), 144rapidly responding indicator, 146–147

shortcomings of, 147signal-to-noise ratio (SNR), 147–149, 148f

Congo Red, 1007Contact lens, 516Continuous-wave (CW) lasers

use in dendritic voltage imaging, 289use in stimulated emission depletion

(STED), 243Coomassie Brilliant Blue staining solution

(recipe), 605CoroNa Green, 202, 303Cortex buffer (recipe), 328, 620Cortical blood volume (CBV), activity-

dependent increase in, 914–915Cortical spatiotemporal dynamics, voltage-

sensitive dye (VSD) imaging inawake behaving mice, 817–824

Cortical stress response, 322cpVenus, 584CRACM (channelrhodopsin-2-assisted circuit

mapping), 424Cranial window

imaging through chronic, 319–328fluorophore choice, 320overview, 319–320protocol, 321–328

application example, 325fexperimental method, 322–326image acquisition, 324, 325fimage analysis, 324, 326imaging setup, 321materials, 321–322recipe, 328surgery, 322–324, 323ftroubleshooting, 326

quantification of morphologicalchanges, 327

transcranial imaging compared, 314implantation for calcium imaging using

TN-XXL, 615preparation for glioma imaging, 1015,

1015fpreparation for imaging excitation in

cerebellar cortex, 753–754preparation in rat for two-photon imaging

of blood flow in cortex(protocol), 929–936, 930f, 933f





1068 / Index

Cranial window (Continued)application example, 933–934, 933fexperimental method, 931–932materials, 929–931setup, 930f

for two-photon imaging of microglia, 968Craniotomy

for Alzheimer’s disease imaging, 1002bleeding during, 1005cat, 516cerebellar, 713–715rodent, 516, 516f

Cre/lox recombinationin Brainbow strategies, 99–104CreER/tamoxifen system, 101, 106–107, 108doxycycline (Dox)-controlled gene

expression, 585in Thy1 mice, 211–213

Cre mice, 104, 116, 118–119Cre recombinase

in optogenetics studies, 878, 879fuse in optogenetics, 867–868

Crick, Francis, 886CTB-Alexa (choleratoxin B-Alexa), 384Culture medium (recipe), 192Cutting solution (recipe), 425CW lasers. See Continuous-wave (CW) lasersCyan fluorescent protein (CFP)

in Clomeleon, 75, 76f, 77, 79–80, 80fFRET and, 583, 584in VSFPs (voltage-sensing fluorescent

proteins), 55, 55t, 56f, 57Cysteine-maleimide reactivity, 38Cytomegalovirus (CMV) immediate-early

(IE) promoter, 707, 708f, 718

DDAB (diaminobenzidine), 176–178, 176fDark fringe method, 196Dark noise, in voltage-sensitive dye imaging,

472, 474DCGs. See Dense-core granulesD3cpv, 584, 590, 591fD3cpVenus, 64, 65f, 67, 67tDCU (dispersion compensation unit),

546–547, 547f, 549–550Dendrites

correlated light and electron microscopyof green fluorescentprotein–labeled, 227–236

fiber-optic calcium monitoring ofdendritic activity in vivo,897–905

infrared-guided neurotransmitteruncaging on dendrites, 387–391

single compartment model of calciumdynamics in, 355–366

sodium imaging in, 201–206in vivo calcium imaging in fly visual

system, 777–780Dendritic spines

GFP expression levels required to detect,320

induction of structural plasticity at single,252, 253f

multiphoton stimulation of, 411–415stimulated emission depletion (STED)

imaging of, 237–244two-photon calcium imaging of, 273–278two-photon imaging and uncaging of,

247–250two-photon sodium imaging in, 297–304

Dendritic voltage imaging, 287–294imaging setup, 288–289overview, 287–288staining individual vertebrate neurons

(protocol), 290–294advantages and limitations, 294example, 292f, 293experimental method, 290–291, 291fmaterials, 290recipes, 294troubleshooting, 291

Dense-core granules (DCGs)identification, 659staining (protocol), 659–660

2-deoxyglucose (2-DG), 908Deoxyhemoglobin, 914, 915Destaining solution (recipe), 605Developmental studies, using optical imaging,

912Dexamethasone, 322, 515, 526, 615Dextran-conjugated indicators

acetoxymethyl (AM)-conjugated indicatorscompared, 131–132, 132f, 137

advantages of, 137imaging presynaptic calcium transients

using dextran-conjugatedindicators, 131–138

overview, 131–133presynaptic imaging of projection

fibers by in vivo injection ofdextran-conjugated calciumindicators (protocol), 134–136

experimental method, 135imaging setup, 134loading solution preparation, 135materials, 134–135preparation of brain slices, 135presynaptic imaging, 135–136troubleshooting, 136in vivo injection of dextran

indicators, 135properties of, 137–138red, 138

Diaminobenzidine (DAB), 176–178, 176fDi-8-ANEPEQ dye, 484Diazo-2, 393, 394f, 397Dichroic beam splitter, 1033f, 1034DIC microscopy, for identification of

astrocytes and neurons inhippocampal slices, 640–642,641f

DiD, 450Diffractive optical element (DOE), 430–431Digital micromirror device (DMD), 258

DiI, 450DiO, 450Dispersion compensation unit (DCU),

546–547, 547f, 549–550Dissection medium (recipe), 742DMD (digital micromirror device), 258DMEM3+ (recipe), 98DM-nitrophen (DMNP), 151–153, 154t, 156f,

157, 159, 393, 394f–396f, 394t,395–401

DOE (diffractive optical element), 430–431Dopamine, optogenetic modulation of

neurons, 877–886, 879ffunctional integration during optical

control, 884–885, 885foptical measurement of dopamine

nerve terminals in brain slices,884–885

optical measurement of dopaminerelease in vivo, 885


conditioned place preference, 881–883,882f

operant conditioning, 883–884Doxycycline (Dox)-controlled gene

expression, 585–586, 586f–587f,592, 595–596

DQ gelatin, 995, 995fDrd1, 878Drosophila

anesthesia, 561antennae–brain preparation, 558f, 561calcium imaging in olfactory system,

557–563Drosophila adult hemolymph-like saline

(AHLS) (recipe), 561D-serine, as gliotransmitter, 639, 657DsRed, for astrocyte imaging, 707, 708fDurotomy, 517Dye-containing pipette solution (recipe), 352Dye loading with whole-cell recordings,

347–353dye loading with patch pipettes (protocol),

348–353discussion, 352experimental method, 349–350, 350fimaging setup, 348materials, 348–349recipes, 352–353troubleshooting, 350–351

overview, 347Dye-making recipe, 527Dyes. See also Fluorescent dyes; FM dyes;

Voltage-sensitive dye; specificapplications; specific dyes

ballistic delivery of, 447–456organic, labeling and imaging receptors

using, 2, 4–7, 4tDye solution (recipe), 905Dynamic high bandwidth atomic force

microscopy (HBAFM), 164





Index / 1069

EEar biopsy, 593eGFP. See Enhanced green fluorescent proteinElectromagnetic pulse (EMP), 1038Electromagnetic spectrum, 1031, 1032fElectron microscopy

correlated light and electron microscopyof green fluorescentprotein–labeled axons anddendrites, 227–236

imaging green fluorescentprotein–labeled neurons usinglight and electron microscopy(protocol), 228–236

discussion, 235experimental protocol, 230–234finding imaged neuron in sections,

231–232, 232ffixation and immunochemistry,

230fixation of imaged slices, 230–231imaging dendrites in EM, 234, 235fimaging setup, 228materials, 228–229recipes, 236resin embedding for EM, 231serial sectioning and imaging in

EM, 233–234, 233ftroubleshooting, 234–235

overview, 227FM dyes as endocytic markers for,

176–178, 176fElectron-multiplying CCD (EMCCD) camera,

578Electro-optic modulator, in interferometry,

196, 197fElectroporation

targeted in vivo, 463–464, 464fin utero, 213, 612, 613in vivo local dye electroporation for

calcium imaging and neuronal-circuit tracing, 501–509

overview, 501–502protocol, 503–509

application examples, 504, 506f,507f, 508f

discussion, 509experimental method, 504, 505fimaging setup, 503materials, 503recipe, 509troubleshooting, 506–508

Embryo medium (recipe), 789EMCCD (electron-multiplying CCD) camera,

578Emission filter, 1033–1034, 1033fEmission maxima, for fluorochromes,

1035t–1036tEMP (electromagnetic pulse), 1038Endocytosis and imaging of synaptic vesicle

recycling using FM dyes,171–181

Endoplasmic reticulum, calcium imaging inneuronal, 339–344

Enhanced green fluorescent protein (eGFP)in Alzheimer’s disease imaging, 1005in BAC (bacterial artificial chromosome)

transgenic mice, 113–114,116–119, 117f, 118f

dense-core granule staining, 659–660expression in astrocytes, 687in imaging synaptic-like microvesicles

(SLMVs), 658immune cell expression of, 987, 987fneuropeptide Y–enhanced green

fluorescent protein (NPY-eGFP), 183, 185, 189, 191, 192f

in rabies virus vectors, 84f, 85, 87–96, 95fEnvA, of avian sarcoma and leucosis virus

(ASLV), 85, 96Envelope switching, rabies virus vectors and,

85Ephus, 419Epifluorescence illumination (EPIi), 574, 574f,

662, 667–669, 669fEpifluorescent microscopy

for fiber optic calcium monitoring ofdendritic activity in vivo, 897,899–900, 899f

for voltage-sensitive dye (VSD) imaging ofcortical spatiotemporaldynamics in awake behavingmice, 818–823, 818f

Epigenetic silencing, 595EPSCs (excitatory postsynaptic currents), 259EPSPs (excitatory postsynaptic potentials),

411, 432, 433f, 434fEvanescent field, 183, 184fEvanescent field fluorescence microscopy. See

Total internal reflectionfluorescence microscopy

Evans solution (recipe), 789EWi (evanescent wave illumination), 662–670,

665f, 669fExcitation filter, 1033, 1033fExcitation in mouse cerebellar cortex,

imaging, 745–760Excitation maxima, for fluorochromes,

1035t–1036tExcitation-secretion coupling, peptidergic

nerve terminals and, 161–168Excitatory postsynaptic currents (EPSCs), 259Excitatory postsynaptic potentials (EPSPs),

411, 432, 433f, 434fExocytosis

imaging with total internal reflectionfluorescence microscopy(TIRFM), 183–193, 655–670

kiss and run fusion, 665, 665fmonitoring in astrocytes, 655–670regulated, 655–656

Experimental autoimmune encephalomyelitis,981, 987f

Expression of genetically encoded calciumindicators, 583–606

Expression vector, Thy1, 208–209, 208fExternal solution (recipe), 352–353

FFast Green (1% stock solution) recipe, 129,

138Fast-scan cyclic voltammetry (FSCV), to

measure dopamine release, 884,885f

FCIP. See Fluorescent calcium indicatorprotein

Fentanyl, 615Fiber optics

calcium monitoring of dendritic activityin vivo, 897–905


bolus loading, 902–903discussion, 905experimental method, 902–903,

904fhead mount, 903imaging setup, 899–900, 899f, 900fmaterials, 901–902recipes, 905troubleshooting, 903–904

periscope, 900, 900f, 905in vivo calcium recordings and

channelrhodopsin-2 activationthrough an optical fiber,889–895


Fiberscope. See Two-photon fiberscopesFilamentTracer, 378Filter culture medium (FCM) (recipe), 742Filters, fluorescence microscopy, 1033–1034,

1033fFirefly luciferase activity, for monitoring

doxycycline (Dox)-controlledgene expression, 585, 586f, 592,595

Fixative (recipe), 652FlaSh (fluorescent Shaker), 54, 55t, 56f, 486Flavoproteins, autofluorescence of, 919Flp recombinase, use in Brainbow studies,

101, 104Fluo-3, use in calcium imaging in neuronal

endoplasmic reticulum,339–343, 340f

Fluo-4for detection of astrocyte vacuole-like

vesicles, 644–645, 645ftwo-photon calcium imaging of dendritic

spines, 275, 275f, 276ftwo-photon imaging of astrocytes filled

with, 650Fluo-4 AM

for detection of astrocyte vacuole-likevesicles, 646

imaging astrocyte calcium activity, 642,643f, 646, 690





1070 / Index

Fluo-4 AM (Continued)for in vivo calcium imaging of cerebellar

astrocytes, 707, 716–717two-photon imaging of acute brain slices

preincubated with, 649Fluo-4 dextran, 138Fluo-5F AM, for in vivo calcium imaging of

cerebellar astrocytes, 707, 708f,716–717

Fluorescein, caged, 264–266Fluorescence, normalization of values,

843–844Fluorescence microscopy. See also specific

applicationsbrain tumor metastasis, 1018–1020, 1020ffilters, 1033–1034, 1033ffluorescence molecular tomography of

brain tumors, 1023–1029illumination methods in, 574fsafe operation of fluoresence microscope,

1037sodium imaging in axons and dendrites,

201–206stimulated emission depletion (STED),

237–244total internal reflection fluorescence

microscopy (TIRFM), imagingexocytosis with, 183–193

Fluorescence molecular tomography of braintumors in mice

experimental method, 1026–1028, 1026finitial image processing, 1027tomographic reconstruction, 1028

imaging setup, 1024–1025, 1025fautomation and data acquisition, 1025illumination, 1024, 1025timaging, 1025mounting, 1025

materials, 1025–1026troubleshooting, 1028

Fluorescent calcium indicator protein (FCIP)calcium indicator protein expression using

mouse transgenesis (protocol),592–596

experimental method, 593–595preparation of mouse ear

fibroblasts and quantitativegene expression assay, 593, 595

screening for transgenic fortransgenic mouse founderswith high FCIP expression, 593

imaging setup, 592materials, 592–593table of transgenic mice, 594t

calcium indicator protein expression usingrAAVs (protocol), 597–603


calcium phosphate–mediated genetransfection of HEK cells,599–600

purification of rAAV by heparincolumn, 601–602

purification of rAAV by iodixanolgradient, 600–601

stereotactic injection, 602in vivo imaging in olfactory bulb,

603in vivo two-photon imaging, 603

imaging setup, 597materials, 597–599

development of, 583–584functional neuron-specific expression of

proteins in living mice, 583–606future prospects, 604ratiometric, 584recipes, 604–605single GFP-based, 584strategies for expression, 585–591

using mouse transgenesis, 585–587,586f–587f

viral delivery using rAAV, 588–590,588f–589f

in vivo calcium imaging of cerebellarastrocytes, 707–712, 708f, 718

Fluorescent dyes. See also specific applications;specific dyes

ballistic delivery of, 447–456free radical generation by, 176loading via pipettes in whole-cell patch-

clamp method, 347–353for membrane voltage measurement,

53–54sodium-binding benzofuran isophthalase

(SBFI) for sodium imaging,202–206, 205f

Fluorescent gel solution (recipe), 445Fluorescent proteins

Brainbow mice, imaging multicolor,99–109

circularly permuted, 584genetically encoded calcium indicators

(GECIs), 63–71for imaging synaptic-like microvesicles

(SLMVs), 658for labeling and imaging receptors, 2, 4–7,

4tfor membrane voltage measurements,

53–59Fluorescent Shaker (FlaSh), 54, 55t, 56f, 486Fluorochromes

emission maxima, 1035t–1036texcitation maxima, 1035t–1036t

FM1-43, for detection of astrocyte vacuole-like vesicles, 646–647, 647f

FM dyeschemical structure, 172f, 173destaining, 175, 179for imaging synaptic-like microvesicles

(SLMVs), 658imaging synaptic vesicle recycling with,

171–181application examples, 179–181, 180fexocytosis imaging, 179–181, 180foverview, 171–173, 172fprotocols

FM dye photoconversion, 176–178,176f

recipes, 181staining and destaining synaptic

vesicles with FM dyes, 174–175slice preparation, 179

loading of astrocytes with, 661properties and mechanism of action, 172f,

173total internal reflection fluorescence

microscopy (TIRFM) forimaging exocytosis, 183–193,191f

FMRI. See Functional magnetic resonanceimaging

Förster resonance energy transfer. See FRETFree radicals, generation by fluorescent dyes,

176Frequency, 1031FRET

Clomeleon use as chloride indicator, 75,79

described, 583fluorescent calcium indicator protein

(FCIP) development and,583–584

genetically encoded calcium indicators(GECIs), 64, 65f, 67

in membrane voltage measurement, 54neuronal imaging in Caenorhabditis

elegans, 764TN-XXL and, 611, 613, 618

FRT sites, in Brainbow constructs, 101, 104FSCV (fast-scan cyclic voltammetry), to

measure dopamine release, 884,885f

Functional magnetic resonance imaging(fMRI)

BOLD (blood-oxygenation-level-dependent), 915, 916

contrast-agent-based, 917low spatial resolution of, 908perfusion-based, 917

Functional neuron-specific expression ofgenetically encoded fluorescentcalcium indicator proteins inliving mice, 583–606

Fungizone, 593Fura-2

calcium wave imaging in cerebellarBergmann glia, 701–702

detection of changes in amplitude ofpresynaptic calcium influx,127–128, 127f

Fura-2 AM for action potential imaging,370–373, 373f, 374f

Fura-2 dextran, 137Fura-2FF use in Ca2+ uncaging in nerve

terminals, 151, 154–156, 154t,156f, 158

GGABA (γ-aminobutyric acid), 623





Index / 1071

caged use in optical fiber-based uncagingsystem, 405–408

receptors, 389Gal4/UAS gene expression system

calcium imaging in Drosophila olfactorysystem, 557, 558f, 563

Galvanometers, voltage-controlled mirror, 419Galvanometric scanning, 258G-CaMP

benefits of, 584calcium imaging in Drosophila olfactory

system, 557–563G-CaMP2, 68, 69f, 707–712, 708f, 718G-CaMP3, 64, 65f, 67, 67t, 68, 69fneuronal imaging in Caenorhabditis

elegans, 764in vivo calcium imaging of cerebellar

astrocytes, 707–712, 708f, 718GECIs. See Genetically encoded calcium

indicatorsGelfoam, 323Gene gun for ballistic delivery of dyes,

447–456Genetically defined neuronal populations,

structure–function analysis of,377–385

Genetically encoded calcium indicators(GECIs), 63–73

application examples, 64calcium imaging in Drosophila olfactory

system with a genetic indicator,557–563

functional neuron-specific expression ofproteins in living mice, 583–606

future design, 71to measure neural activity, 71optimization, 68–70

G-CaMP3, 68, 69fGECI expression, 68practical improvement, 68subcellular targeting, 70

overview, 63–64properties influencing performance, 64–67

expression level, 66–67fluorescence properties, 67, 67tunderlying calcium dynamics, 64, 66

structure of, 64, 65ftesting standardization, 70TN-XXL, calcium imaging of neurons in

visual cortex using, 611–620,612f

GENSAT (Gene Expression Nervous SystemAtlas) Project, 113–119, 377

GFAP (glial fibrillary acidic protein), 640,641f, 642, 687

GFP. See Green fluorescent proteinGH146-Gal4, 557, 558fGlia. See also Astrocytes

ballistic delivery of dyes, 447–456Bergmann

imaging calcium waves in, 699–705,704f

preferential loading with synthetic

acetoxymethyl calcium dyes(protocol), 716–717

in vivo calcium imaging with syntheticand genetic indicators,707–719, 708f

imaging microglia in brain slices and slicecultures, 735–742

two-photon imaging/microscopy in spinalcord in vivo, 721–733

two-photon imaging of microglia inmouse cortex in vivo, 961–976

Glial fibrillary acidic protein (GFAP), 640,641f, 642, 687

Glial fibrillary acidic protein (GFAP)promoter, 687, 708f, 867

Glioblastoma, 1012, 1017Glioma, 1011–1017, 1016fGliotransmitters, 639–640, 655, 657Glossary, 1041–1050Glutamate

cagedacousto-optical deflector (AOD)-based


circuit mapping by ultravioletuncaging, 417–425

methoxy-7-nitroindolinyl-glutamate(MNI-glu), 246, 246f, 249–253,251f, 253f, 268, 277, 431–432

optical fiber-based uncaging system,405–408

properties of caged glutamate for two-photon photolysis, 246, 246f

ruthenium-bipyridine-trimethylphosphine-glutamate(RuBi-glutamate), 431–432

two-photon calcium imaging ofdendritic spines, 276f, 277

two-photon mapping of neuralcircuits, 429–434, 430f, 432t

using patterned uncaging to simulatenetwork activity in vitro,258–260

as gliotransmitter, 639, 657Glutamate receptors

light-activated ionotropic glutamatereceptor (LiGluR), 36

spatially resolved flash photolysis viachemical two-photon uncaging,29, 29f

Glutamate transporter 1 (GLT1) promoter, 687Glycine, 623Glycosylphosphatidylinositol (GPI), 208GlyR (glycine receptor) lateral diffusion,

measuring using quantum dots,15, 16f

GMEM4+ (recipe), 98Gold nanoparticles

detection by photothermal heterodyneimaging (PHI), 2

labeling and imaging receptors using, 2,4–7, 4t

tracking using single-nanoparticle

photothermal tracking(SNaPT), 2

G-protein-coupled receptors (GPCRs)in chimeric opsins, 867of type II opsins, 44

Green fluorescent protein (GFP). See alsoEnhanced green fluorescentprotein

in Alzheimer’s disease imaging, 991f, 992correlated light and electron microscopy

of green fluorescent protein–labeled axons and dendrites,227–236

in FlaSh (fluorescent Shaker), 54, 55t, 56flevel required for dendritic spine

detection, 320pHluorins, 19–20SPARC (sodium channel protein-based

activity reporting construct),55, 55t, 56f

HHabituation and training for awake

experiments, 840Halobacterium salinarum, 45Halorhodopsin

light-induced dipole switching, 43fmode of action, 45overview, 42f, 866

Hazardous materials, 1053–1058HBAFM (high bandwidth atomic force

microscopy), dynamic, 164Head plate

for imaging neuronal population activityhead plate design, 840–841, 841fhead plate implantation, 841–842

implantation protocol, 747–749, 748ffor two-photon imaging of neural activity

in awake mobile mice, 828–829,829f

Heat filter, 1034Held synapse, using Ca2+ uncaging to examine

transmitter release at calyx of,158–159, 158f

Hemoglobin, oxygen saturation of, 913–914,915–916

Heparin column, purification of rAAV by,601–602

HEPES-buffered culture medium (HCM)(recipe), 742

HEPES-KRH buffer (recipe), 670Hidden Markov model (HMM), 832, 845High bandwidth atomic force microscopy

(HBAFM), dynamic, 164High-divalent ACSF (recipe), 425High-K+ Ringer’s solution (recipe), 192Histology, in situ of brain tissue with

femtosecond laser pulses,437–445

HIV-1 Env, 85HMM (hidden Markov model), 832, 845Human clinical studies, using optical imaging,

912–913





1072 / Index

IIce cube model, 910, 911fIGOR Pro, 267ImageJ, 736, 1000, 1016, 1020Immune cells, two-photon imaging of,

981–988overview, 981protocol, 982–988

cocultures of acute hippocampal sliceswith immune cells, 984–985

discussion, 987–988, 987fexperimental method, 984–985imaging acquistion and analysis, 985imaging setup, 982, 983fmaterials, 982–984preparation of brain stem of

anesthetized mice for intravitalimaging, 985

troubleshooting, 986Imspector software package, 241Incident light, noise in, 472Infrared (IR) camera, 378Infrared-guided laser stimulation of neurons

in brain slices, 388–390advantages and limitations of photolytic

neurotransmitter activation,390

applications of targeted stimulation, 389,390f

experimental method, 389–390imaging setup, 388materials, 388–389

Infrared-guided neurotransmitter uncagingon dendrites, 387–391

IntegriSense, 1025Interferometric detection of action potentials,

195–199advantages and limitations, 199application example, 198–199, 198fprinciples of interferometric detection,

195–198Internal pipette solution (recipe), 206Internal recording solution (recipe), 278, 467Internal solution (recipe), 192Intracellular saline containing SBFI (recipe),

303Intrapipette solution (recipe), 344, 824Intrinsic imaging

basics of, 634chronic, 634–637, 635fof functional map development in

mammalian visual cortex,633–637, 635f, 636f

overview, 633–634Intrinsic signals

optical imaging based on, 907–919combining with other techniques, 917,

918flimitations of, 919outlook for, 917, 919overview, 908–913

sources of, 913–915

In utero electroporation, 213, 612, 613Inverse pericam, 586f–587f, 587In vitro calibration solutions (recipe), 631Iodixanol gradient, purification of rAAV by,

600–601Ion channels

engineering light-regulated, 33–40modifications to existing light-gated

channels, 37–38photoswitchable tethered ligands

(PTLs), 35practical considerations, 38strategies, 33, 34ftargeted proteins, 36

light-gated, 46–47Ion pumps, light-gated, 44–46Ischemia



Isoflurane, 492, 514, 515, 714, 728, 753, 871,892, 902, 931, 952, 965, 985,1001, 1026

Isolectin B4 (IB4), 737–739, 741Isolectin B4 (IB4) derived from Giffonia

simplicifolia seeds (recipe), 742

JJackson Laboratory, 211

KKatushka, 1023Ketamine, 308, 315, 322, 324, 514, 515, 526,

569, 595, 602–603, 688, 711,714, 871, 892, 952

Kiss-and-run fusion, 171, 179–181, 180fKrebs-Ringer solution (recipe), 391Kwik-sil, 830–831KX mixture (recipe), 315

LLabVIEW, 267, 492, 531Lamp1, 661Laser

light sources for photolysis, 399–400mode-locked, 248, 307, 411–412pulsed nitrogen, 400safe operation of, 1037–1038ultraviolet, 258, 261, 262, 399–400

Laser pulses, in situ histology of brain tissuewith, 437–445

advantages and limitations, 443–444application example, 443, 444flaser alignment, 442–443overview, 437–438, 440fprotocol, 438–445

blocking and staining, 442discussion, 442–444experimental method, 441–442imaging setup, 439, 440f

iterative imaging and ablation, 442,443t

mapping region of interest, 441–442materials, 439, 441perfusing with fluorescent gel, 442recipes, 445subblocking and mounting, 442

Laser scanning photostimulation (LSPS)circuit mapping by ultraviolet uncaging of

glutamate, 417–425hardware, 419software, 419

Laser-targeted focal photothrombotic strokemodel, 954–955, 955f

Lead citrate (recipe), 236Lentivirus, for opsin expression in

mammalian brain, 867, 869,871–872

Lidocaine, 492, 603, 902, 931, 952, 965Light-activated ionotropic glutamate receptor

(LiGluR), 36Light-gated ion channels, 46–47Light-gated ion pumps, 44–46Light-regulated ion channels, engineering,

33–38modifications to existing light-gated

channels, 37–38engineering ion channel targets, 37photochromic ligands, 37–38photoswitch tuning, 37

photoswitchable tethered ligands (PTLs),35

ligand, 35photoswitch, 35reactive moiety, 35

practical considerations, 38cysteine-maleimide reactivity, 38light, 38

strategies, 33, 34ftargeted proteins, 36

ionotropic glutamate receptor, 36nicotinic acetylcholine receptor, 36voltage-gated potassium channel, 36

Light scatteringcalcium effects on, 163–164, 163fsecretion-coupled from peptidergic nerve

terminals, 161–168effects of Ca2+ on intrinsic optical

signal components, 163–164,163f

measuring intrinsic optical signalsfrom mammalian nerveterminals (protocol), 166–168

overview, 161–163, 162funderlying mechanisms, 164–165, 164f

Light-sheet microscopy, 573–575Line averaging, 336–337Lipofectamine 2000, 595Lipofuscin, 1008Lipophilic dyes, ballistic delivery of, 447–456Live cells and tissue slices, maintaining,

331–338ill health indicators, 337





Index / 1073

maintaining in the imaging setup, 332–337collecting images, 336–337gas and pH condition maintenance

after mounting, 334, 334f, 335fmedium considerations, 332mounting specimens for microscopic

observation, 332–335, 333ftemperature considerations, 336

overview, 331–332Lobster, interferometry of walking leg nerve,

198, 198f, 199Local dye electroporation for calcium imaging

and neuronal-circuit tracing,501–509


application examples, 504, 506f, 507f,508f

discussion, 509experimental method, 504, 505fimaging setup, 503materials, 503recipe, 509troubleshooting, 506–508

Low-Ca+ Ringer’s solution (recipe), 193LSPS. See Laser scanning photostimulationLucas–Kanade algorithm, 845Luciferin, 595LY-411575, 992Lysosome identification and staining in

astrocytes, 661Lysosome marker proteins, 661

MMagFura-2 AM, use in calcium imaging in

neuronal endoplasmicreticulum, 339–344, 340f

MagFura-5 AM, 132f, 137MagIndo-1 AM, 432Magnesium Green, 127–128, 127fMagnetic resonance imaging (MRI). See also

Functional magnetic resonanceimaging

of Alzheimer’s disease, 989, 990functional brain mapping with, 913

Maleimides, cysteine-reactivity of, 38Mannitol, 711Mapping

functional based on intrinsic signals,913–917

single-condition, 916–917two-photon of neural circuits, 429–434

MATLAB, 267, 784, 935Matrix metalloproteinases (MMPs), 995, 995fMauthner cells, 796, 796fMB-231BR mouse cell line, 1012, 1018–1020,

1020fMCBL. See Multicell bolus loading (MCBL) of

fluorescent indicatorsmCherry

in fluorescence molecular tomography,1023, 1028

for imaging synaptic-like microvesicles

(SLMVs), 658synaptic-like microvesicles (SLMVs)

imaging, 663–666, 665fVGLUT1 tagged with, 663, 664–665, 665f

Mechanical drift, in stimulated emissiondepletion (STED), 242

Medetomidine, 615MEM (recipe), 670Membrane voltage

fluorescent proteins for measurement of,53–59

applications, 58, 58fdiscussion of, 59experimental setup, 58FlaSh (fluorescent Shaker), 54, 55t, 56fmechanism of fluorescence change,

56f, 57–58Mermaid, 55t, 57, 58, 58fSPARC (sodium channel protein-based


VSFPs (voltage-sensing fluorescentproteins), 54–55, 55t, 56f, 57

optical approaches to studying, 53–54fluorescent dyes as sensors, 53–54protein-based sensors, 54

MEM medium for quantum dot imaging(recipe), 17

MEMS (microelectromechanical system), 856MEPSCs (miniature excitatory postsynaptic

currents), 251–252, 251fMEQ (6-methoxy-N-ethylquinolinium

chloride), 624Mercury arc lamp, 1037, 1038

as light sources for photolysis, 399–400Mermaid, 55t, 57, 58, 58fMetastasis, 1012, 1018–1021, 1020fMethohexital, 724–725Methoxy-7-nitroindolinyl-glutamate (MNI-

glu), 246, 246f, 249–253, 251f,253f, 268, 277, 431–432

Methoxy-XO4, 990, 991f, 992, 1007Mice

Alzheimer’s disease model, 991anesthesia, 308, 322, 324, 492, 504, 534,

550, 569, 595, 602–603, 615,688, 711, 714, 724, 726, 728,871, 952, 965, 985, 1001, 1015,1016, 1019, 1026

BAC transgenic, 113–119Brainbow, generation and imaging

multicolor, 99–111calcium imaging in intact olfactory system

of, 565–571craniotomy, 516, 516ffluorescence molecular tomography of

brain tumors, 1023–1029fluorescent calcium indicator protein

(FCIP) expression intransgenic, 585–587, 586f–587f,594t

functional neuron-specific expression ofgenetically encoded fluorescent

calcium indicator proteins inliving mice, 583–606

GENSAT (Gene Expression NervousSystem Atlas) Project, 113–119

head plate implantation, 747–749, 748f,963–965, 964f

imaging neocortical neurons through achronic cranial window,319–328

intubation, 725MB-231BR cell ine, 1012, 1018–1020,

1020fmeasuring intrinsic optical signals from

mammalian nerve terminals(protocol), 166–168

Mutant Mouse Regional Resource Center(MMRRC), 119

ordering mouse lines, 119Thy1 lines, 207–225transgenic

BAC, 113–119with constitutive promoters driving

FCIP expression, 594tfluorescent calcium indicator protein

(FCIP) expression in, 585–587,586f–587f, 594t

for targeting reward circuitry, 879ttet-activator (tTA), 594ttet-responder, 594t

two-photon imaging of astrocytic andneuronal excitation incerebellar cortex of awakemobile mice, 745–760

two-photon imaging of microglia inmouse cortex in vivo, 961–976


two-photon imaging of neurons and gliain spinal cord in vivo, 721–733

in vivo two-photon calcium imaging invisual system, 511–527

voltage-sensitive dye imaging of corticalspatiotemporal dynamics inawake behaving mice, 817–824

Microbial opsins. See also Opsinsbiophysical properties, 48–49homologies, 44light-gated ion channels, 46–47light-gated ion pumps, 44–46for optical control of neural activity, 41–50overview, 41–42, 42f

Microelectromechanical system (MEMS), 856Microglia

imaging in brain slices and slice cultures,735–742

imaging setup, 735–736migration of microglia, 740fprotocol, 737–742

confocal and two-photon imaging,739

discussion, 740–741, 740f, 741fexperimental method, 737–739labeling with fluorescent lectin, 738





1074 / Index

Microglia (Continued)materials, 737mounting tissue slices for imaging,

738–739recipes, 742tissue slice preparation, 737–738troubleshooting, 739

two-photon imaging in mouse cortex invivo, 961–976

discussion, 975–976optical window preparation (protocol),

966–968materials, 966–967sealed craniotomy method, 968thinned skull method, 967–968troubleshooting, 968

overview, 961–962surgical implantation of head plate in

preparation for imaging(protocol), 963–965

experimental method, 964–965materials, 963–964setup, 964f

two-photon imaging (protocol),969–974

experimental method, 970–972,971f, 973f

imaging setup, 969–970materials, 970troubleshooting, 972–974

Microinjection buffer (recipe), 223Microinjection solution (recipe), 696Microscope objective, 1039–1040

for confocal spot detection, 142selection for total internal reflection

fluorescence microscopy(TIRFM), 185

Microscopy. See also specific applicationsacousto-optical deflector (AOD)-based

two photon microscope,545–547, 547f

objective-coupled planar illumination(OCPI), 573–580, 576f, 578f

photothermal imaging microscope, 3planar illumination microscopy, 573–580single-molecule epifluorescence

microscope, 3stimulated emission depletion (STED),

237–244total internal reflection fluorescence

microscopy (TIRFM), 183–193two-photon uncaging, 245–253

Midazolam, 615Miniature excitatory postsynaptic currents

(mEPSCs), 251–252, 251fMiniaturization of two-photon microscopy

for imaging in freely movinganimals, 851–860

application examples, 857–859, 858ffluorescent dye labeling in vivo, 857imaging setup, 852–857, 853t, 854f

components, 852, 853fdesign considerations, 852–857, 854f

fluorescence detection, 856mechanical attachment to animal, 857miniaturized fiber-scanning devices,

855–856small microscope objectives, 856two-photon excitation through optical

fibers, 853, 855MitoMice, 211fmKOκ (monomeric kusabira orange), 57MLCKp (myosin light chain kinase), 583MMPs (matrix metalloproteinases), 995, 995fMMPSense, 1025MMRRC (Mutant Mouse Regional Resource

Center), 119MNI-glu (methoxy-7-nitroindolinyl-

glutamate), 246, 246f, 249–253,251f, 253f, 268, 277, 431–432

Mode-locked laser, 248, 307, 411–412Modified balanced salt solution (MBSS)

(recipe), 742MOG peptide (myelin oligodendrocyte

glycoprotein peptide), 984MOM (movable objective microscope), 828Monkey, voltage-sensitive dye imaging of

neocortical activity of corticaldynamics in behaving, 810–811

Monomeric kusabira orange (mKOκ), 57Monomeric umi-kinoko green (mUKG), 57Monosynaptic tracing, with recombinant

fluorescent rabies virus vectors,83–98, 85f

Motor behavior, imaging in zebrafish,783–789

Mouse model of Alzheimer’s disease, 999–1009Mouse Ringer’s solution (recipe), 168Movable objective microscope (MOM), 828Movement artifacts, 845, 1006Mowiol 4-88 mounting medium (recipe), 385MPScope, 439MQAE (N-(6-methoxyquinolyl) acetoethyl

ester), chloride imaging using,623–632

advantages and limitations, 630application example, 627f, 628f, 630overview, 623–624protocol, 624–631

discussion, 630experimental method, 626–628imaging setup, 625intracellular calibration of MQAE,

627–628materials, 625–626recipes, 630–631staining cells or tissue slices via bath

application, 626, 627fstaining of neurons and glia using

multicell bolus loading, 626, 628ftroubleshooting, 629two-photon imaging of stained cells,

626MQAE cuvette calibration solutions (recipe),

631mRaspberry, 1023

MRI. See Magnetic resonance imagingMS-222, 569–570mUKG (monomeric umi-kinoko green), 57Multicell bolus loading (MCBL) of fluorescent

indicators, 491–499advantages and limitations, 497–498in Alzheimer’s disease imaging,

1002–1003, 1007–1008application examples, 495–497, 495f, 496foverview, 491protocol for in vivo two-photon calcium

imaging, 492–499experimental method, 493–494imaging setup, 492materials, 492–493troubleshooting, 494

in vivo calcium imaging of cerebellarastrocytes, 707, 708f, 716–717

Multiphoton imaging, in olfactory system ofzebrafish and mouse, 566f, 567,568

Multiphoton stimulation of neurons andspines, 411–415

advantages of, 415application example, 414, 414f, 415fexperimental protocol, 412–413imaging setup, 412limitations of, 414–415materials, 412

Multiple sclerosis, murine models of, 981Multi-worm Tracker, 770–773, 773fMutant Mouse Regional Resource Center

(MMRRC), 119Myelin oligodendrocyte glycoprotein peptide

(MOG peptide), 984Myosin light chain kinase (MLCKp), 583

NNADH intrinsic fluorescence, in vivo imaging

of redox state using, 692–694,693f

Nanolabels, tracking receptors usingadvantages, 7limitations, 7overview, 1–2protocol, 3–7

analysis, 6application example, 7, 8f.experimental method, 4–6image acquisition, 6imaging setup, 3labeling cells, 4–5, 4tmaterials, 4particle tracking, 6recipe, 7signal characterization, 5

National Center for Biotechnology (NCBI),119

Natronomonas pharaonis, 45, 46f, 866NCAM (neuronal cell adhesion molecule), 84Near-infrared fluorescence (NIRF)

tomography, of Alzheimer’sdisease, 989, 990





Index / 1075

Nematodes. SeeWorm trackingNembutal, 504Neocortical activity, voltage-sensitive dye

imaging of, 799–812Neocortical neurons, imaging through a

chronic cranial window,319–328

Nerve terminalsCa2+ uncaging in, 151–159light scattering changes associated with

secretion from peptidergic,161–168

single compartment model of calciumdynamics in, 355–366

Neural circuits, two-photon mapping,429–434

overview, 429–431practical examples, 431–434

choice of caged glutamate, 431–432,432t

mapping connections in brain slices,432–434, 433f, 434f

Neural networkacousto-optical deflector (AOD)-based

two-photon microscopy forhigh-speed calcium imaging ofneuronal population activity,543–555

three-dimensional imaging of activity,529–540

application example, 538, 538fmeasuring neuronal population

activity using 3D laser scanning(protocol), 531–537

animal preparation and 3Dimaging procedure, 534

assigning fluorescence signals tocells within the scan volume,535f

creating 3D scan trajectories,532–533, 532f

discussion, 539experimental method, 532–536imaging setup, 531laser intensity adjustment, 533–534materials, 531–532, 532frecipe, 540signal assignment and 3D

visualization of networkdynamics, 534, 536

troubleshooting, 536–537overview, 529–530principles of 3D laser scanning, 532f

voltage-sensitive dye imaging ofneocortical activity, 799–812

NeuroCCD-SM Imaging System, 288Neurointermediate lobe, isolation of, 166–167Neurolucida, 378Neuromantic, 378Neuromuscular explant, imaging of Thy1 lines

using an acute, 221–222experimental method, 222imaging setup, 221

materials, 221Neuronal activity

confocal calcium imaging in larvalzebrafish, 791–797

imaging activity and motor behavior inzebrafish, 783–789

Neuronal cell adhesion molecule (NCAM), 84Neuronal membrane receptors, labeling single

receptors with quantum dots,13–17

experimental method, 14imaging setup, 13interpretation of imaging data, 15, 16fmaterials, 13measuring GlyR (glycine receptor) lateral

diffusion, 15, 16frecipes, 17troubleshooting, 14

Neuronal population activity, imaging inawake and anesthetizedrodents, 839–849

application example, 847, 848fdata analysis, 843–847, 844f

action potential detection, 845–847correction for movement artifacts, 845estimation of noise levels and neuropil

contamination, 845normalization of fluorescence values,

843–844experimental procedures, 840–843

cell-attached electrophysiology,842–843, 844f

dye preparation for injection, 842–843habituation and training for awake

experiments, 840head plate design, 840–841, 841fhead plate implantation, 841–842surgical considerations, 842targeting sensory areas, 842

future prospects, 848–849Neuronal structural plasticity, two-photon

imaging of, 949–957NeuronIQ, 378Neurons

ballistic delivery of dyes for structure-function studies, 447–456

calcium imaging in neuronal endoplasmicreticulum, 339–344

imaging action potentials with calciumindicators, 369–374

imaging calcium waves and sparks incentral, 281–285

imaging green fluorescent protein–labeledusing light and electronmicroscopy (protocol), 228–236

discussion, 235experimental protocol, 230–234finding imaged neuron in sections,

231–232, 232ffixation and immunochemistry, 230fixation of imaged slices, 230–231imaging dendrites in EM, 234, 235fimaging setup, 228

materials, 228–229recipes, 236resin embedding for EM, 231serial sectioning and imaging in EM,

233–234, 233ftroubleshooting, 234–235


imaging neuronal activity with geneticallyencoded calcium indicators(GECIs), 63–73

microbial opsins for optical control ofneural activity, 41–50

multiphoton stimulation of, 411–415optogenetic modulation of dopamine,

877–886, 879frecombinant fluorescent rabies virus

vectors for tracing, 83–98structure-function analysis of genetically

defined neuronal populations,377–385

two-photon imaging neuronal excitationin cerebellar cortex of awakemobile mice, 745–760

type-specific staining with voltage-sensitive dye, 486

uncaging calcium in neurons, 393–401NeuronStudio, 378Neuropeptide Y (NPY), 659Neuropeptide Y–enhanced green fluorescent

protein (NPY-eGFP), 183, 185,189, 191, 192f

NeuroPlex, 288, 479Neurosurgery, use of optical imaging in, 912Neurotransmitter uncaging. See UncagingNeutral-density filter, 1034NGM agar medium (recipe), 775

low-peptone, 775Nicotinic acetylcholine receptor (nAChR),

photoisomerizable, 36Ni-DAQ Tools, 267NIRF (near-infrared fluorescence)

tomography, of Alzheimer’sdisease, 989, 990

Nitrophenyl-EGTA (NP-EGTA), 393, 394f–396f,395–399, 399f, 644, 644f, 651

NK3630, 479–480NK2367 dye, 162, 162fNoise

signal-to-noise ratio (SNR)of calcium indicators, 64, 145–146confocal spot detection, 147–149, 148fdefined, 64GCaMPs, 558, 584improving

by increasing incident illumination,336

by increasing pixel dwell time,336–337

by line averaging, 336–337random-access pattern scanning,

554–555





1076 / Index

Noise (Continued)of voltage-sensitive dyes, 801, 802, 810

voltage-sensitive dye, imaging withdark noise, 472, 474noise in incident light, 472shot noise, 474, 474fvibrational noise, 472

Normal rat Ringer (NRR) solution, 540, 555NP-EGTA (nitrophenyl-EGTA), 393, 394f–396f,

395–399, 399f, 644, 644f, 651NpHR, 866, 868NRR (recipe), 540, 555Nystatin, 593

OObjective-coupled planar illumination

(OCPI) microscopy, 573–580,576f, 577t, 578f

Objective lens, 856, 1039–1040Odor coding pattern transformation with

olfactory bulb, 501–502, 504,507f, 508f

OGB-1. See Oregon Green BAPTA-1Olfactory receptor neurons, synapto-pHluorin

expression in, 21–22, 22fOlfactory system

calcium imaging in Drosophila with agenetic indicator, 557–563

overview, 557–558, 558f, 559fprotocol, 560–563

antennae–brain preparation, 561discussion, 563imaging setup, 560materials, 560–561nerve stimulation preparation,

561–562olfactory stimulation and imaging,

561recipes, 563

calcium imaging in intact olfactory systemof zebrafish and mouse,565–571

imaging methods, 567multiphoton imaging, 567wide-field fluorescence imaging,

567loading calcium indicators, 566–567

bolus loading of neurons down-stream from glomeruli, 567

dextran-coupled (protocol),568–571

genetically encoded indicators, 567selective loading of neurons

projecting to glomeruli, 566overview, 565–566, 566f

calcium imaging in populations ofolfactory neurons by planarillumination microscopy,573–580

overview, 573–575, 574f, 576fprotocol, 577–580, 577t, 578f

fluorescent calcium indicator protein(FCIP) expression, 586f, 597, 603

Operant conditioning, for assessing reward-related behavior, 883–884

Opsins. See also Optogeneticsbiophysical properties, 48–49chimeric, 867, 878gene expression and targeting systems,

867–868light-gated ion channels, 46–47light-gated ion pumps, 44–46in neuroscience, 864–867overview, 41–42, 42fphotoreaction mechanisms, 43fprinciples of operation, 41–42, 43–44step-function, 866table of, 864t–865ttype I (microbial type), 44type II (animal type), 44

Optical equipment, cleaning, 1039–1040Optical fiber

fiber-optic calcium monitoring ofdendritic activity in vivo,897–905


uncaging system, optical fiber-based,405–408

advantages of, 408assembly and optimization, 406, 407fexperiments utilizing the system,

406–408, 407flimitations of, 408materials, 406

in vivo calcium recordings and channel-rhodopsin-2 activation throughan optical fiber, 889–895


Optical imaging based on intrinsic signals,907–919

combining with other techniques, 917,918f

limitations of, 919outlook for, 917, 919overview, 908–913

in awake behaving animals, 912chronic imaging, 910developmental studies, 912example studies, 908, 910, 911fexperimental setup, 908, 909fhuman clinical studies, 912–913

sources of intrinsic signals, 913–915Optical imaging spectroscopy, 913Optically induced occlusion, of single blood

vessels in neocortex, 939–947Optical neural interface, fiber-optic-based,

868–873, 870fOptical parametric oscillator, 982Optical window. See also Cranial window;

Craniotomychoice of type, 975–976preparation protocol, 966–968

materials, 966–967

sealed craniotomy method, 968thinned skull method, 967–968troubleshooting, 968

Optodes, 912Optogenetics

Cre mice for targeting reward circuitry,878, 879t

described, 41, 42establishing a fiber-optic-based optical

neural interface (protocol),869–873, 870f

experimental method, 871–873material, 869–871, 870ftroubleshooting, 873

in freely moving mammals, 877–885future outlook, 874, 886modulation of dopamine neurons,

877–886, 879ffunctional integration during optical

control, 884–885, 885foptical measurement of dopamine

nerve terminals in brain slices,884–885




experimental design, 880–881operant conditioning, 883–884

overview, 863–868tools, table of, 864t–865t

OptoXRs, 867, 878Oregon Green BAPTA-1, 521–523, 531, 534,

535f, 566, 579in Alzheimer’s disease imaging, 1000in vivo recordings and channelrhodopsin-

2 activation through an opticalfiber, 890, 891f

Oregon Green BAPTA-1 AM, 531, 534, 535fin Alzheimer’s disease imaging, 993–994,

994f, 1002–1005, 1003f, 1004fimaging calcium excitation in cerebellar

cortex, 758f, 759imaging neuronal activity and motor

behavior in zebrafish, 784, 787multicell bolus loading of, 495, 495f, 497in vivo recordings and channelrhodopsin-

2 activation through an opticalfiber, 893f

Oregon Green BAPTA-6F, 278Oregon Green BAPTA-5N, 147Organic dyes, labeling and imaging receptors

using, 2, 4–7, 4tORION, 378OSN dye solution (recipe), 571Oxidative stress, in Alzheimer’s disease, 994,

995fOxonol dyes

NK3630, 479–480RH155, 476





Index / 1077

Oxygen saturation of hemoglobin, 913–914,915–916

PParaformaldehyde (recipe), 385Paraformaldehyde solution 8% (recipe), 652Parallel fibers, 699, 702–704Parallel Worm Tracker, 773Patch-clamp pipette filling solution (recipe),

391Patch-clamp recording

calcium imaging in neuronal endoplasmicreticulum, 339–344

dye loading technique, 347–353infrared-guided neurotransmitter

uncaging on dendrites, 387–391two-photon targeted patching and

electroporation in vivo,459–467

targeting single cells with patchpipettes in vivo (protocol),461–467

advantages and limitations,466–467

application examples, 466experimental method, 462–464,

463f, 464fmaterials, 461practical considerations, 464–465recipes, 467

Patch pipettesdye loading with, 348–353targeting single cells with patch pipettes in

vivo (protocol), 461–467Pavolvian conditioning. See Conditioned place

preferencePBS. See Phosphate-buffered salinePentobarbital, 603, 724Perfusion system, for live cell and tissue slice

imaging, 334, 334f, 335fPET. See Positron-emission tomographyPFA (paraformaldehyde) (recipe), 385PHI (photothermal heterodyne imaging), 2, 6pHluorins, 19–24

advantages and limitations, 22–23application examples, 21–22, 22fecliptic, 19–20gene expression, 21optical imaging, 21overview, 19–20phogrin tagged with, 660ratiometric, 20synapto-pHluorins, 19–24targeting module, 20VGLUT1 tagged with, 658, 663–666, 665f,

667–669, 669fPhogrin, 660Phosphate-buffered saline (PBS) (recipe), 7,

110, 181, 193, 605PBS-MK 1x (recipe), 60510x, 223, 605

Photoactivation of Rose Bengal, 949, 954–955,955f, 957

Photobleaching, G-CaMP and, 558, 558fPhotoconversion, FM dye, 176–178, 176fPhotodamage

G-CaMP and, 558, 558freducing with antioxidants, 337SNR compromise, 336–337

Photoisomerizationlight-regulated ion channels, engineering,

33–38of retinal, 43–44, 43f

Photolysisacousto-optical deflector (AOD)-based


detection calcium signals from photolysisof caged compound in singleastrocyte, 644, 644f

infrared-guided laser stimulation ofneurons in brain slices, 388–390

light sources for, 399–400measuring effectiveness of, 401spatially resolved flash photolysis via

chemical two-photon uncaging,25–31

overview, 25–26, 26fprotocol, 27–30

two-photon, 246, 246f, 256uncaging calcium in neurons, 393–401

Photolyzable Ca2+ chelator, 151–152, 156f, 157Photon energy, 1031Photorhodopsin, 45Photoswitchable molecules and engineering

light-regulated ion channels,33–40

modifications to existing light-gatedchannels, 37–38

photoswitchable tethered ligands (PTLs),35

practical considerations, 38strategies, 33, 34ftargeted proteins, 36

ionotropic glutamate receptor, 36nicotinic acetylcholine receptor, 36voltage-gated potassium channel, 36

Photoswitchable tethered ligands (PTLs), forlight-regulated ion channelengineering, 35

ligand, 35modifications to existing light-gated

channels, 37–38engineering ion channel targets, 37photochromic ligands, 37–38photoswitch tuning, 37

photoswitch, 35practical considerations, 38

cysteine-maleimide reactivity, 38light, 38

reactive moiety, 35Photothermal heterodyne imaging (PHI), 2, 6Photothermal imaging microscope, 3Photothrombosis, 939, 941, 942f, 943–944,

945f, 947, 949, 954–955, 955fPhototoxicity

in FM dye imaging, 174in stimulated emission depletion (STED),

242voltage-sensitive dye s, 480

Piezoelectric bending element, 855Pipette solution for biocytin staining (recipe),

653Pipette solution for Fluo-4 staining (recipe),

653Pipette staining solution (recipe), 631Pittsburg compound B (PiB), 990, 992, 1003f,

1007Planar illumination microscopy, calcium

imaging in populations ofolfactory neurons by, 573–580

overview, 573–575, 574f, 576fprotocol, 577–580, 577t, 578f

Plaques. See Alzheimer’s diseasePlasma-mediated ablation, 940, 941, 942f,

944–945, 947Pluronic F-127 in DMSO, 492p75 neurotrophin receptor (p75NTR), 84Pockels cell, 412, 534Point spread function (PSF), 144Positron-emission tomography (PET)

of Alzheimer’s disease, 989, 990low spatial resolution of, 908

Potassium channelfluorescent Shaker (FlaSh), 54synthetic photoisomerizable azobenzene

regulated, 36ppHcrt promoter, 867Presenilins, 999Pressure injection, of AM calcium indicators,

371–372Promoters

aldehyde dehydrogenase (Aldh1L1), 687αCaMKII, 585, 586ffor Brainbow construct expression, 103CamKIIα, 867cytomegalovirus (CMV) immediate-early

(IE), 707, 708f, 718for expression in astrocytes, 687glial fibrillary acidic protein (GFAP), 687,

708f, 867glutamate transporter 1 (GLT1), 687ppHcrt, 867tetracycline, 585, 588, 588f–589f, 590, 593UAS, 557

Pronuclear injection, generation of Thy1constructs for, 214–216

cloning of fluorescent reporter proteinsequence, 215–216

experimental method, 215–216materials, 214–215preparation of DNA for pronuclear

injection, 216troubleshooting, 216

ProSense, 1025Proteorhodopsins, 45Proton pumps, light-driven, 45Pseudotyping, of rabies virus, 84f, 85, 95–96PSF (point spread function), 144





1078 / Index

Psychtoolbox, 784PTLs. See Photoswitchable tethered ligandsPulsed nitrogen laser, 400

QQD streptavidin conjugate solution (recipe),

17QDT. See Quantum dot trackingQuantum dots

imaging single receptors with, 11–17labeling neuronal membrane receptors

(protocol), 13–17experimental method, 14imaging setup, 13interpretation of imaging data, 15,

16fmaterials, 13measuring GlyR (glycine receptor)

lateral diffusion, 15, 16frecipes, 17troubleshooting, 14

overview, 11–12limitations to quantum dot staining, 12properties of, 11–12

blinking, 12color, 11fluorescence strength, 11mercury lamp excitation, 11

Quantum dot tracking (QDT)advantages and limitations, 7application example, 7, 8fdescription, 2image acquisition, particle tracking, and

analysis, 6labeled cell preparation, 5signal characterization, 5

RrAAV. See Recombinant adeno-associated

virusRabies virus

genome organization, 84–85, 84fneurotropism of, 83protocols, 87–98

application examples, 97, 97fdiscussion, 97pseudotyping, 95–96recipes, 97–98recovery of G-gene deleted virus,

93–94recovery of replication-competent

rabies virus from cDNA, 87–92pseudotyping, 84f, 85, 95–96recombinant fluorescent vectors for

tracing neurons and synapticconnections, 83–98

biosafety issues, 86complementation of gene-deleted

vectors, 85, 85fenvelope switching, 85genetic engineering of, 86protocols, 87–96pseudotyping, 84f, 85

recovery of G-gene deleted virus(protocol), 93–94

experimental method, 93–94materials, 93

recovery of replication-competent rabiesvirus from cDNA (protocol),87–92

cell culture, 89fixation of cells and direct

immunofluorescence, 90materials, 88–89transfection and virus recovery, 89–90virus stock preparation, 92virus titration, 91, 91f

titration of, 91, 91ftransynaptic transmission, 83–84vaccination, 86

Random-access multiphoton (RAMP)microscopy, 695

Random-access pattern scanning (RAPS)technique, 543, 545–555

Ratanesthesia, 534, 871, 931CNS-1 cell line, 1011–1017, 1016fdurotomy, 517

recA gene, 115Recipes

AAV plasmid preparation, 604–605anesthetic for rodents, 526artificial cerebrospinal fluid (ACSF), 129,

244, 315, 415, 424, 486–487,527, 652, 681–682, 696, 705,733, 760, 976, 988

HEPES-buffered, 719high-divalent, 425holding, 486–487modified, 487modified, free of carbonate and

phosphate, 936BES-buffered saline 2x (BBS), 605blocking solution, 445caged glutamate stock solution, 425calibrating solutions, 344Coomassie Brilliant Blue staining solution,

605cortex buffer, 328, 620culture medium, 192cutting solution, 425destaining solution, 605dissection medium, 742DMEM3+, 98DM-Nitrophen, 159Drosophila adult hemolymph-like saline

(AHLS), 561dye-containing pipette solution, 352dye-making recipe, 527dye solution, 905embryo medium, 789Evans solution, 789external solution, 352–353Fast Green (1% stock solution), 129, 138filter culture medium (FCM), 742fixative, 652

fluorescent gel solution, 445GMEM4+, 98HEPES-buffered culture medium (HCM),

742HEPES-KRH buffer, 670high-divalent ACSF, 425high-K+ Ringer’s solution, 192internal pipette solution, 206internal recording solution, 278, 467internal solution, 192intracellular saline containing SBFI, 303intrapipette solution, 344, 824isolectin B4 (IB4) derived from Giffonia

simplicifolia seeds, 742Krebs-Ringer solution, 391KX mixture, 315lead citrate, 236low-Ca+ Ringer’s solution, 193MEM, 670MEM medium for quantum dot imaging,

17microinjection buffer, 223microinjection solution, 696modified balanced salt solution (MBSS), 742Mowiol 4-88 mounting medium, 385MQAE cuvette calibration solutions, 631Na+-free extracellular saline containing

ionophores, 304NGM agar medium, 775NGM agar medium, low-peptone, 775NRR, 540, 555OSN dye solution, 571paraformaldehyde (PFA), 385paraformaldehyde solution 8%, 652patch-clamp pipette filling solution, 391PBS-MK 1x, 605PBS 1x, 605PBS 10x, 605phosphate-buffered saline (PBS), 7, 110,

181, 193, 223, 605pipette solution for biocytin staining, 653pipette solution for Fluo-4 staining, 653pipette staining solution, 631QD streptavidin conjugate solution, 17recombinant adenovirus containing FCIP

sequence, 719Ringer’s solution, 824

Ca2+-free, 509high-K+, 192Krebs-Ringer solution, 391low-Ca2+, 193mouse, 168normal rat Ringer (NRR), 540, 555standard, 1931x, 22310x, 223

RPMI 1640 cell culture medium, 988SDS-PAGE gel, 605–606SDS-PAGE Laemmli buffer (5x), 606SDS-PAGE 10x SDS running buffer, 606slice solution, 653standard external saline, 499, 631, 1009standard extracellular saline, 344





Index / 1079

standard extracellular solution forvertebrate neurons, 294

standard intracellular solution forvertebrate neurons, 294

standard pipette solution, 499, 1009Terrific Broth (TB-A), 604Terrific Broth (TB-B), 604titration to estimate purity of DM-

Nitrophen, 159in vitro calibration solutions, 631

Recombinant adeno-associated virus (rAAV)fluorescent calcium indicator protein

(FCIP) expression and,588–590, 588f–589f

in optogenetics studies, 878, 879fReconstruct software, 105, 107–108Red fluorescent protein (RFP), for dense-core

granule staining, 659–660RESOLFT (reversible saturable optical

fluorescence transitions), 238Retinal, 43–44, 43fRetinal Schiff base, 43, 43f, 44Retrograde staining with voltage-sensitive

dyes, 484–485Reverse tetracycline transactivator (rtTA), 585,

587, 589f, 590, 595Reversible saturable optical fluorescence

transitions (RESOLFT), 238Reward circuits

genetic strategies for controlling, 878,879f, 879t

optogenetic modulation of dopamineneurons, 877–886, 879f

functional integration during opticalcontrol, 884–885, 885f

optical measurement of dopaminenerve terminals in brain slices,884–885




operant conditioning, 883–884reagents and transgenic mouse lines for

targeting, 879tRFP (red fluorescent protein), for dense-core

granule staining, 659–660RH1691, 481–482RH155 dye, 476Rhod-2 AM, 690Rhod-2 dextran, 138Rhodopsin, 43. See also Microbial opsinsRinger’s solution recipes, 824

high-K+, 192low-Ca+, 193mouse, 168normal rat Ringer (NRR) solution, 540, 555standard, 1931x, 22310x, 223

Rose Bengal, 944, 949, 950, 953f, 954–955,955f, 956, 957

RPMI 1640 cell culture medium (recipe), 988rtTA. See Reverse tetracycline transactivatorRuthenium-bipyridine-trimethylphosphine-

glutamate (RuBi-glutamate),431–432

SSample chamber, for live cell and tissue slice

imaging, 332–334, 333fSample drift, in stimulated emission depletion

(STED), 242SBFI (sodium-binding benzofuran

isophthalate), 202–206, 205f,297–302, 297–303, 301f

Scanning confocal microscopes, limits ofcurrent, 142

SDS-PAGE gel (recipe), 605–606SDS-PAGE Laemmli buffer (5x) (recipe), 606SDS-PAGE 10x SDS running buffer (recipe),

606Secretogranin II, 659Semiconductor quantum dots, labeling and

imaging receptors using, 2, 4–7,4t

Semliki Forest virus, 613Sensory rhodopsin I, 42f, 45–46Sensory rhodopsin II, 42f, 45–46SFOs (step-function opsin genes), 47Shadowpatching, 459–467Shot noise, in voltage-sensitive dye imaging,

474, 474fSignal-to-noise ratio (SNR)

of calcium indicators, 64, 145–146confocal spot detection, 147–149, 148fdefined, 64GCaMPs, 558, 584improving

by increasing incident illumination, 336by increasing pixel dwell time, 336–337by line averaging, 336–337

random-access pattern scanning, 554–555of voltage-sensitive dyes, 801, 802, 810

Single compartment model of calciumdynamics, 355–366

application examples, 363dynamics of single [Ca2+]i transients, 360extensions to model, 364–366

buffered calcium diffusion, 364deviations from linear behavior,

364–365, 365fsaturation of buffers and pumps,

364slow buffers, 365

measurement of calcium-drivenreactions, 365–366

materials, 356model application, 361–363

estimates of amplitude and totalcalcium charge, 362–363

estimates of endogenous Ca2+-bindingratio and clearance rate,

361–362, 362festimates of unperturbed calcium

dynamics, 363model assumptions and parameters,

356–360, 358t–359tbuffering, 358clearance, 360influx, 357–358

overview, 355–356schematic of model, 357fsummation of [Ca2+]i transients during

repetitive calcium influx,360–361

Single-condition mapping, 916–917Single-molecule epifluorescence microscope, 3Single-molecule tracking (SMT), for labeling

and imaging receptors usingnanolabels

advantages and limitations, 7application example, 7, 8fdescription, 2image acquisition, particle tracking, and


Single-nanoparticle photothermal tracking(SNaPT), for labeling andimaging receptors usingnanolabels

advantages and limitations, 7application example, 7, 8fdescription, 2image acquisition, particle tracking, and


Single photon confocal spot detection, 142Single-photon emission tomography

(SPECT), of Alzheimer’sdisease, 990

Skull holder, 308f, 309, 312–313Skull thinning, 967–968, 975–976Slice cultures, imaging microglia in, 735–742Slice solution (recipe), 653SLM (spatial light modulator), 258, 695SLMVs. See Synaptic-like microvesiclesSMT. See Single-molecule trackingSNaPT. See Single-nanoparticle photothermal

trackingSNR. See Signal-to-noise ratioSodium-binding benzofuran isophthalate

(SBFI), 202–206, 205f, 297–303,301f

Sodium channel protein-based activityreporting construct (SPARC),55, 55t, 56f

Sodium-free extracellular saline containingionophores (recipe), 304

Sodium imagingone-photon imaging in axons and

dendrites, 201–206loading cells and detecting [Na+]i

changes (protocol), 202–206





1080 / Index

Sodium imaging (Continued)application example, 204, 205fcalibration of SBFI fluorescence

changes, 204discussion, 204–206experimental method, 203limitations of method, 204–206materials, 202–203recipes, 206

overview, 201–202two-photon imaging in dendritic spines,

297–304calibration and measurement of [Na+]i

within neurons using SBFI(protocol), 300–304

discussion, 303experimental method, 300–302, 301fimaging setup, 300materials, 300recipes, 303–304troubleshooting, 302

overview, 297–299Sodium methohexital, 724–725SPARC (sodium channel protein-based


SPARK (synthetic photoisomerizableazobenzene regulatedpotassium) channel, 36

Spatial light modulator (SLM), 258, 695Spatially optimized line scans, generation of,

934–935Spatially resolved flash photolysis via chemical

two-photon uncaging, 25–31overview, 25–26, 26fprotocol, 27–30

Spatial resolution, of voltage-sensitive dye,801–802

SPECT (single-photon emission tomography),of Alzheimer’s disease, 990

Spinal cord, two-photon imaging/microscopyof neurons and glia in, 721–733

overview, 721–722preparation of mouse spinal column for

repetitive imaging using 2pLSM(protocol), 727–728

preparation of mouse spinal column forsingle imaging using 2pLSM(protocol), 722–726

anesthetizing and intubating themouse, 724–725

experimental method, 724–726materials, 723–724preparing the spinal cord for imaging,

725–726, 725ftroubleshooting, 726

in vivo two-photon imaging of mousespinal cord (protocol), 729–732

experimental method, 730–731imaging setup, 729materials, 729microscopy and image acquisition,

730–731, 731f

mounting the vertebral column, 730preparation for imaging, 730

SPQ (6-methoxy-N-(3-sulphopropyl)quinolinium),624

SR101. See Sulforhodamine 101Standard external saline (recipe), 499, 631,

1009Standard extracellular saline (recipe), 344Standard extracellular solution for vertebrate

neurons (recipe), 294Standard intracellular solution for vertebrate

neurons (recipe), 294Standard pipette solution (recipe), 499, 1009STED. See Stimulated emission depletionStep-function opsins (SFOs), 47, 866Stimulated emission depletion (STED),

237–244application to astrocyte imaging, 695dyes, STED-compatible, 243future prospects for, 243image acquisition and analysis, 240–241imaging of dendritic spines, 237–244

application example, 243, 243fin living hippocampal tissue

(protocol), 239–244discussion, 243experimental protocol, 241imaging setup, 239–241materials, 241recipes, 244troubleshooting, 242

laser sources, 243microscopy setup, 239f, 240overview of, 237–238principles of microscopy, 239

Strokeimaging long-term changes caused by

induced, 957laser-targeted focal photothrombotic

model, 954–955, 955fStructure–function analysis of genetically

defined neuronal populations,377–385

overview, 377protocol, 378–385

application examples, 381–384, 382fcircuit analysis, long-range

projections, and inputmapping, 384

histological reconstruction ofbiocytin-filled cells, 381

reconstruction of 3D morphology,381, 383f, 384

single-cell electrophysiology andmorphological analysis ofgenetically defined neurons,381, 382f

discussion, 384–385experimental method, 379–380imaging setup, 378materials, 378–379recipes, 385

Structure–function studies, ballistic deliveryof dyes for, 447–456

Styryl dyes. See FM dyesSubcellular targeting, using genetically

encoded calcium indicators(GECIs), 70

Sulforhodamine, 179Sulforhodamine 101 (SR101), 497, 498

chemical structure, 674ffor imaging calcium excitation in

cerebellar cortex, 758f, 759specificity of, 677fstaining of astrocytes in a brain slice, 640,

641f, 642for three-dimensional imaging of

neuronal network activity, 534,535f

for two-photon calcium imaging in visualsystem, 521–523

for two-photon imaging of neural activityin awake mobile mice, 830–831

in vivo imaging of astrocytes (protocol),688–689

in vivo labeling of cortical astrocytes with,673–682, 674f

application example, 680, 680foverview, 673–674, 674fprotocol, 675–682

Synaptic connections, recombinantfluorescent rabies virus vectorsfor tracing, 83–98

Synaptic inhibition, imaging with a geneticallyencoded chloride indicator,75–80

Synaptic-like microvesicles (SLMVs)identification of, 657imaging exocytosis and recycling in

astrocytes, 662staining (protocol), 657–658

Synaptic vesicle recycling, imaging with FMdyes, 171–181

Synapto-pHluorins, 19–24advantages and limitations, 22–23application examples, 21–22, 22fgene expression, 21optical imaging, 21overview, 19–20targeting module, 20

Synthetic photoisomerizable azobenzeneregulated potassium (SPARK)channel, 36

TTamoxifen

induced recombination in mice, 106–107toxicity, 106–107

Tau protein, 989, 992–993, 999tdRFP, immune cell expression of, 987, 987fTellurium dioxide, 257, 261Temporal resolution, of voltage-sensitive dye,

801–802Terrific Broth (TB-A) (recipe), 604Terrific Broth (TB-B) (recipe), 604





Index / 1081

Tetracycline (Tet)-inducible system, forfluorescent calcium indicatorproteins (FCIPs), 583, 585–587,586f–587f

Tetracycline promoters, 585, 588, 588f–589f,590, 593

Tetracycline (Tet) transactivator (tTA), 585,587, 588f–589f, 594t, 595

Tetrodotoxin (TTX), 433, 497, 497fTexas Red, 991fTexas Red dextran, 132f, 138, 952T helper 17 (Th17) cells, 981, 984Thinned skull imaging, 305–315. See also

Transcranial two-photonimaging of living mouse brain

Thioflavin-S, 991–993, 991f, 1000, 1002, 1003,1003f, 1004f, 1005, 1007

Three-dimensional imaging of neuronalnetwork activity, 529–540

application example, 538, 538fmeasuring neuronal population activity

using 3D laser scanning(protocol), 531–537

animal preparation and 3D imagingprocedure, 534

assigning fluorescence signals to cellswithin the scan volume, 535f

creating 3D scan trajectories, 532–533,532f

discussion, 539experimental method, 532–536imaging setup, 531laser intensity adjustment, 533–534materials, 531–532, 532frecipe, 540signal assignment and 3D visualization

of network dynamics, 534, 536troubleshooting, 536–537

overview, 529–530principles of 3D laser scanning, 532f

Thy1 mice, 207–225expression vector, 208–209, 208flines, 211–213

generating, 212–213overview of, 211–212table of, 212t

overview, 207–211protocols

generation of Thy1 constructs forpronuclear injection, 214–216

cloning of fluorescent reporterprotein sequence, 215–216

experimental method, 215–216materials, 214–215preparation of DNA for pronuclear

injection, 216troubleshooting, 216

generation of tissue sections forscreening Thy1 lines, 217–220

experimental method, 218–220imaging setup, 217materials, 217–218troubleshooting, 220

imaging of Thy1 lines using an acuteneuromuscular explant,221–222

experimental method, 222imaging setup, 221materials, 221

recipes, 223stochastic subset labeling, 209–211, 210f,

211fTIRFM. See Total internal reflection

fluorescence microscopyTN-XL, 584TN-XXL, 64, 65f, 67, 67t, 584

calcium imaging of neurons in visualcortex, 611–620, 612f

in vivo expression of, 612–613Total internal reflection fluorescence

microscopy (TIRFM)advantages of, 191imaging exocytosis with, 183–193, 184f

application examples, 191, 191f, 192fimaging setup, 185–186loading and TIRF imaging of

dissociated bipolar cells(protocol), 187–188


overview, 183–185, 184frecipes, 192–193transfection and TIRF imaging of

cultured bovine chromaffincells (protocol), 189–190


limitations of, 191–192microscope objective selection, 185monitoring exocytosis in astrocytes,

655–670optical design considerations, 184optical design for dual-shutter prismless,

663principle of, 183–184safe operation of microscope, 1037

Transcranial two-photon imaging of livingmouse brain, 305–315

application example, 310f, 311f, 314challenges of preparing an animal for,

314–315cranial window imaging compared, 314overview, 305–306protocol, 307–315

discussion, 314–315experimental method, 308–312imaging setup, 307mapping imaging area for future

relocation, 310, 310fmaterials, 307–308recipes, 315recovery, 312re-imaging, 312thinned skull preparation, 308–310, 308f

TPLSM imaging of neuronal or glialstructures, 310f, 311, 311f

troubleshooting, 312–313Transfection

of astrocytes, 658, 659–660calcium phosphate, 597, 599–600genetically encoded calcium indicators,

595, 597, 599–600Transsynaptic tracing, with recombinant

fluorescent rabies virus vectors,83–98, 85f

Tricaine methanesulfonate, 569–570Troponin

TN-XL, 584TN-XXL, 584, 611–619

TTA. See Tetracycline (Tet) transactivatorTTX (tetrodotoxin), 433, 497, 497fTwo-photon fiberscopes, 851–860

application examples, 857–859, 858ffluorescent dye labeling in vivo, 857imaging setup, 852–857, 853t, 854f

components, 852, 853fdesign considerations, 852–857, 854ffluorescence detection, 856mechanical attachment to animal, 857miniaturized fiber-scanning devices,

855–856small microscope objectives, 856two-photon excitation through optical

fibers, 853, 855Two-photon imaging/microscopy

acousto-optical deflector (AOD)-basedmicroscopy for high-speedcalcium imaging of neuronalpopulation activity, 543–555

of astrocytic and neuronal excitation incerebellar cortex of awakemobile mice, 745–760

of blood flow in cortex, 927–936, 930f, 933fcalcium imaging in Drosophila olfactory

system with a genetic indicator,557–563

calcium imaging in vivo dendritic imagingin fly visual system, 777–780

calcium imaging of dendritic spines,273–278

calcium imaging of neurons in visualcortex using troponin C–basedindicator, 611–620, 612f

chloride imaging using MQAE, 623–632chloride imaging with Clomeleon, 77, 79electron microscopy reimaging of green

fluorescent protein–labeledaxons and dendrites, 227–236

fluorescent calcium indicator protein(FCIP) expression, 597, 603

of genetically encoded calcium indicators(GECIs), 70

for glioma imaging, 1013–1017, 1016fimaging action potentials with calcium

indicators, 373, 373fimaging astrocyte calcium and vacuole-

like vesicles, 639–653





Two-photon imaging/microscopy (Continued)imaging neocortical neurons through a

chronic cranial window,319–328

of immune cells in neural tissue, 981–988mapping of neural circuits, 429–434measuring neuronal population activity

using 3D laser scanning(protocol), 531–537

of microglia in mouse cortex in vivo,961–976

miniaturization for imaging in freelymoving animals, 851–860

multiphoton stimulation of neurons andspines, 412

of neural activity in awake mobile mice,827–836

of neural networks in mouse model ofAlzheimer’s disease, 999–1009

of neuronal population activity in awakeand anesthetized rodents,839–849

of neuronal structural plasticity in miceduring and after ischemia,949–957

of neurons and glia in spinal cord in vivo,721–733

sodium imaging in dendritic spines,297–304

staining of astrocytes in the intact rodentcortex with SR101 (protocol),675–682

of structural and functional properties ofastrocytes, 685–696, 686f

of structure and function in Alzheimer’sdisease, 989–995

targeted patching and electroporation invivo, 459–467

transcranial of living mouse brain,305–315

in vivo calcium imaging in visual system,511–527

in vivo calcium imaging using multicellbolus loading of fluorescentindicators, 491–499

Two-photon laser-scanning microscopy(TPLSM, 2pLSM). See alsoTwo-photonimaging/microscopy

all-optical in situ histology of brain tissue,439–444, 440f, 444f


mouse spinal cord preparation forrepetitive imaging, 727–728

NADH intrinsic fluorescence imaging,692–694, 693f

of neurons and glia in spinal cord,721–733

overview, 721–722preparation of mouse spinal column

for repetitive imaging using

2pLSM (protocol), 727–728preparation of mouse spinal column

for single imaging using 2pLSM(protocol), 722–726

in vivo two-photon imaging of mousespinal cord (protocol), 729–732

Two-photon photolysis, 246, 246f, 256Two-photon uncaging microscopy, 245–253

application examples, 251–253, 251f, 253fimaging of AMPA receptor densities in

hippocampal neurons, 251–252,251f

induction of structural plasticity atsingle dendritic spines, 252,253f

chemical, 25–31disadvantage of, 30overview, 25–26, 26fprotocol, 27–30

application example, 29, 29fdiscussion, 29–30experimental method, 28imaging setup, 27materials, 27–28troubleshooting, 28

development of, 245–246imaging and uncaging of dendritic spines

(protocol), 247–250experimental method, 250imaging setup, 247–249

electrophysiology setup, 249microscopy setup, 247–248, 247fmode-locked laser beam, 248software, 248–249

materials, 249mapping of neural circuits, 429–434overview, 245–246properties of caged glutamate for two-

photon photolysis, 246, 246fTyrosine hydroxylase, 878, 880

UUAS promoter, 557Ultraviolet (UV) light

acousto-optical deflector (AOD)-basedpatterned UV neurotransmitteruncaging, 255–270

circuit mapping by ultraviolet uncaging ofglutamate, 417–425

infrared-guided neurotransmitteruncaging on dendrites, 387–391

optical fiber–based uncaging system,405–408

Uncagingacousto-optical deflector (AOD)-based


calcium in neurons, 393–401Ca2+ uncaging in nerve terminals, 151–159characteristics of caged compounds, 256chemical two-photon uncaging, 25–31infrared-guided neurotransmitter

uncaging on dendrites, 387–391

optical fiber–based uncaging system,405–408

two-photon mapping of neural circuits,429–434

two-photon uncaging microscopy,245–253

Urethane, 504, 534, 550, 603, 931, 952

VVaccinia virus, 86VChR1

activation spectrum of, 864deactivation rate, 866

Velate protoplasmic astrocytes, in vivocalcium imaging with syntheticand genetic indicators,707–719, 708f

Venus, 584, 1011, 1012, 1013, 1016, 1016f,1018–1020, 1020f

Vercuronium, 526Vesicle recycling. See Synaptic vesicle recyclingVesicular glutamate transporter-1 (VGLUT1),

657–658VGLUT-mCherry, 663, 664–665, 665fVGLUT-pHluorin, 663–666, 665f,

667–669, 669fVessel occlusion



Vibrational noise, in voltage-sensitive dyeimaging, 472

Viral delivery using rAAV, 588–590, 588f–589fVisual cortex

calcium imaging of neurons in usingtroponin C–based indicator,611–620, 612f

future perspectives, 618–619overview, 611–612repeated two-photon imaging of TN-

XXL-labeled neurons(protocol), 614–620

application examples, 612f, 618,619f

cranial window implantation, 615data analysis, 618experimental method, 615–617imaging setup, 614imaging visually driven calcium

signals, 616mapping of visual cortex, 615–

616materials, 614–615recipes, 620repeated imaging, 617two-photon imaging, 616

intrinsic optical imaging of functionalmap development inmammalian, 633–637, 635f,636f

1082 / Index





Index / 1083

Visual systemorientation preference in 3D neural

networks of visual cortex, 538,538f

in vivo dendritic calcium imaging in fly,777–780

in vivo two-photon calcium imaging,511–527


anesthesia induction in cats,514–515

anesthesia induction in rodents,514

contact lens insertion, 516craniotomy in cats, 516craniotomy in rodents, 516, 516fdiscussion, 526durotomy, 517dye loading, 517–519, 518fexperimental method, 514–522imaging setup, 513initial surgery in cats, 515initial surgery in rodents, 515materials, 513–514neuropil signal, 521–522, 521frecipes, 526–527signal from astrocytes, 521somatic signal from neurons, 520troubleshooting, 522–525visual stimulation, 519–520

Voltage-gated potassium channel, syntheticphotoisomerizable, 36

Voltage imaging, dendritic, 287–294imaging setup, 288–289overview, 287–288staining individual vertebrate neurons

(protocol), 290–294advantages and limitations, 294example, 292f, 293experimental method, 290–291, 291fmaterials, 290recipes, 294troubleshooting, 291

Voltage-sensing phosphatase, 57, 58Voltage-sensitive dye (VSD)

Aplysia abdominal ganglion actionpotentials, 472, 473f, 476–477

dendritic voltage imaging, 287–288,290–291, 291f, 292f, 293

imaging of cortical spatiotemporaldynamics in awake behavingmice, 817–824

application example, 822–823, 822foverview, 817–818, 818fprotocol, 819–824

discussion, 823experimental method, 820–821materials, 819recipes, 824

imaging of neocortical activity, 799–812distributed processing, 803–804, 804f

dynamics of shape processing atsubcolumnar resolution,805–806, 806f

long-range horizontal spread oforientation selectivity controlby intracortical cooperativity,804–805, 805f

long-term imaging of corticaldynamics in behaving monkeys,810–811

microstimulation in frontal and motorcortex, 811–812

outlook, 812overview, 799–800selective visualization of neuronal

assemblies, 806, 808–809, 808fspatial and temporal resolution,

801–803from in vitro single-cell recordings to

in vivo population imaging,800–801

without signal averaging, 809–810imaging with, 471–487

future directions of, 486imaging of population signals in brain

slices (protocol), 478–480bleaching, 480data acquisition and analysis, 479dye staining, 480experimental method, 479–480imaging setup, 478–479materials, 479phototoxicity, 480sensitivity, 480slice preparation, 479–480total recording time, 480

protocols, 476–487recipes, 486–487recording action potentials from

imaging neurons ininvertebrate ganglia (protocol),476–477

retrograde staining (protocol),484–485

in vivo imaging of mammalian cortexusing “blue” dyes (protocol),481–483

neuron type-specific staining, 486overview, 471–475

camera choice, 475dark noise, 472, 474noise in incident light, 472shot noise, 474, 474fvibrational noise, 472

Voltammetry measurement of dopaminerelease, 884, 885f

Volvox carteri, 47, 864VSDI (voltage-sensitive dye imaging). See

Voltage-sensitive dyeVSFPs (voltage-sensing fluorescent proteins),

54–55, 55t, 56f, 57

WWater-soluble dyes, ballistic delivery of, 452Wavelength, 1031Whisker movement, voltage-sensitive dye

(VSD) imaging in, 812–823,822f

Wide-field calcium imaging in olfactorysystem of zebrafish and mouse,566f, 567, 568

Wide-field charge coupled device (CCD)camera–based imaging, ofcalcium waves and sparks incentral neurons, 281–285

loading cells and detecting [Ca2+]i changes(protocol), 282–285

application examples, 284–285, 284f,285f

discussion, 284–285experimental method, 283materials, 282–283

overview, 281Wollaston prism, in interferometry, 196Woodchuck hepatitis virus post-

transcriptional control element(WPRE), 588f

Worm Tracker (software), 773, 774fWorm tracking, 763–775

experimental methods, 765, 765fprinciples of single-worm tracking,

764–765protocols

illumination for worm tracking andbehavior imaging, 768–769

preparation of samples for wormtracking, 766–767

recipes, 775tracking movement behavior of

multiple worms on food,770–772

WPRE (woodchuck hepatitis virus post-transcriptional controlelement), 588f

XXenon arc lamps, as light sources for

photolysis, 399–400Xylazine, 308, 315, 322, 324, 514, 526, 569,

595, 602, 688, 711, 714, 871,892, 952

Xylocaine, 322

YYC3.6, in Alzheimer’s disease imaging, 993,

994fYC3.60, 64, 65f, 67, 584Yellow Cameleons, development of, 583–584Yellow fluorescent protein (YFP). See also

Venuscircularly permuted variants, 584in Clomeleon, 75, 76f, 77, 79–80, 80fin stimulated emission depletion (STED),

238, 241–243





1084 / Index

Thy1-YFP, 209, 210f, 211f, 212tin VSFPs (voltage-sensing fluorescent

proteins), 55, 55t, 56f, 57Yellow Cameleons, 583–584

ZZebrafish

advantages of use, 783–784anesthesia, 569calcium imaging in intact olfactory system

of, 565–571imaging methods, 567

loading calcium indicators, 566–567overview, 565–566, 566f

confocal calcium imaging of neuronalactivity in larval, 791–797

imaging setup, 792protocol, 793–797

application example, 796, 796fdiscussion, 797experimental method, 793–794materials, 793troubleshooting, 794–795

imaging neuronal activity and motorbehavior, 783–789

imaging setup, 784–785calcium imaging of tectal neuronal

population activities, 784, 785fsimultaneous recording of tectal

Ca2+ dynamics and motorbehavior, 785

visual stimulation, 784–785protocol, 786–789

discussion, 789materials, 786–787methods, 787–788recipes, 789troubleshooting, 788





Documents

Index [] Index A AAQ (acryl-azobenzene-quaternary ammonium), 38 AAV. SeeAdeno-associated virus AAV-G-CaMP3, 68 AAV plasmid preparation (recipe), 604–605 Ablation, for in situ histology