Advanced fluorescence imaging

1Life Technologies™ Proprietary |

For Research Use Only. Not for use in diagnostic procedures.

Nick Dolman, Ph.D.Senior Staff Scientist

Advanced fluorescence imaging



• Fluorescent moieties

• Foundational platforms

• Qdots®

• BacMam

• Click-iT®

Topics to be Covered

• New approaches

• pHrodo™

• Autophagy

• Apotposis

• Oxidative stress

• Live/Dead



Fluorescent moieties



Fluorescence Labeling & Detection

UV to Infrared Live and/or fixed cells Photolabile

Antibody & SA conjugates Cell Tracking Ion & voltage indicators Organelle stains

Blue to Near-Infrared Live cells, fixable Photolabile

Protein expression Functional “Biosensors” Organelle targeting

Antibody & SA conjugates Cell Tracking In vivo imaging Electron Microscopy

Green to Infrared Live and/or fixed cells Very Photostable

Fluorescent Nanocrystals

Fluorescent Dyes

Fluorescent Proteins



Foundational platforms: Qdots®



What are quantum dots?

Highly fluorescent, nanometer-size, crystals of semiconductor materials

Cadmium plus selenium

or tellurium

Zinc sulfide



Many Detection Modalities and Applications for Qdot® Technology

Live Cell Tracking

(QTracker™ kits)

In vivo Imaging

Flow

CytometryWesternDot™ kits

525 565 585 625605 655545

New!

Microscopy Tissue

Electron microscopy



Marketing for Qtracker® Cell Labeling

Qtracker® Component A(Qdot® Probe Solution)

Qtracker® Component B(Carrier Solution) Mix comp. A and B

Incubate 5 min Add cell growth media

Vortex 30 sec

5 μL/mL

5 μL/mL

-or-

Add to suspended or adhered cells

Incubate at 37 °C for 30-60 min

Wash and imaging or flow cytometry to

check labeling



Qtracker® Labeling of Primary Cells: Migration Study

Qtracker® Cell Labeling did not significantly affect migration

of primary cells over a 20 h period



Foundational platforms: Click-iT®



Click Chemistry Small, bio-orthogonal and

“interchangeable” functional groups

Chemoselective ligation reaction

Two-step procedure:

1 ) Label molecule of interest metabolically, enzymatically or chemically

2) Versatile detection, compatible with any readout (fluorescence, absorbance), on any platform (blot, gel, flow cytometer, imaging, mass spectrometer)

Copper catalyzed click reaction between an alkyne and an azide



Breakthrough cell proliferation assay with Click-iT®

EdU

• Non-radioactive

• Multiplex compatible but, strand separation requirement for anti-

BrdU access, can affect:

• Ability for other antibodies to bind

• Morphology

• Ability for dyes that require dsDNA to bind efficiently, i.e.,

cell cycle dyes

• Non-radioactive

• No DNA denaturation required

• Simplified protocol

• Small molecule detection

• Multiplex compatible, including

• Other antibodies

• Dyes for cell cycle analysis



Click-iT® EdU labeling birth-

dates differentiated cells in

mouse olfactory epithelium F.

Chehrehasa et al. J Neurosci

Method (2009)

Click-iT® EdU Across Species (and Kingdoms)

Neural tube and otocyst

labeling with Click-IT®

EdU in chick embryos. M.

Warren et al. Dev.

Dynamics (2009)

Proliferating germ cells in

C. elegans with Click-iT®

EdU. M. Dorsett et al.

Genetics (2009)

Image courtesy of Sarah

Cheesman, University of

Oregon

Methods (2009) 48:8-13Image courtesy of Julian P.S.

Smith III, Winthrop Microscopy

Facility, Winthrop University

E. colimarine flatworm

zebrafish larva

Medicago sativa (alfalfa)

suspension cultures labeled

with Click-iT® EdU. Image

courtesy of Ferhan Ayadin,

Cellular Imaging Laboratory,

Biological Research Center

Image courtesy of Sarah

A. Sabatinos, University

of Southern California

S. pombe

marine flatworm



Click chemistry EdU Protocol

Click-iT™ detection

reaction

30 minutes

Wash 2X 10 minutes

Nuclear counterstain 10 minutes

Wash 1X 5 minutes

Image TOTAL

TIME 55

minutes

Incubate with EdU or BrdU, fix & permeabilize sample

With Click chemistry

• Measure proliferation in cells or tissue

• Time to complete: <1 hour

• Detect by fluorescence microscopy, flow

cytometry or high-throughput imaging (HCS)

BrdU Protocol

Wash 3X 15 minutes

HCl Denaturation 40 minutes

Neutralize 12 minutes

Wash 3X 15 minutes

Block 60 minutes

Anti-BrdU incubation 1-2 hours

Wash 3X 15 minutes

Secondary antibody

incubation

120 minutes

Wash 3X 15 minutes

Nuclear counterstain 10 minutes

Wash 1X 5 minutes

Image TOTAL TIME

6-7 hours

Click-iT® EdU vs. BrdU



Protein synthesis: Click-iT® AHA/HPG

HeLa

0.0

0001

0.0

001

0.0

01

0.0

1

0.1 1

10

100

1000

0

100

200

300 Cytoplasmic

Nuclear

Log [cyclohexamide] uM

Mean

(Rin

g/C

irc)

Avera

ge In

ten

sit

y

0.03mM 200mM

Cyclohexamide

Click-iT® HPG



0

50

100

150

200

250

Co

nt

90

uM

CQ

5u

M

MG

132

HP

G C

yto

pla

sm

Inte

nsity

*

*

Buffer

MG-132

Chloroquine

Protein degradation: Click-iT® AHA/HPG



RNA synthesis: Click-iT® EU Assay (5-thynyluridine)

α-Amanitin 250nM

α-Amanitin 0nM

-6 -5 -4 -3 -20

20

40

60

80

EC50=17pM

Actinomycin D (pM)

Cir

cS

po

t A

vera

ge In

ten

sit

y

ArrayScan VTI



Apoptosis in HepG2 cellsApoptosis: Click-iT® TUNEL Alexa Fluor® Imaging Assay

No Treatment 2 mM Staurosporine

Control 5 µM Camptothecin 40 µM Camptothecin

-5 -4 -3 -2 -1 0 1 2 3

30

40

50

60

70

80

90

100

EC50 = 9.5 mM

Log (mM ), Camptothe cin

% M

ax

imu

m o

f T

UN

EL

sta

inin

g

-4 -3 -2 -1 0 1 2 3200

300

400

500

600

EC50 = 8 nM

Log (nM), Nocodazole

Nu

cle

ar

Inte

ns

ity

(T

UN

EL

)

Arrayscan VTI



Click-iT® Detection Assays…

Nascent DNA

synthesis/Cell

Proliferation

Click-iT® EdU Cell

Proliferation Assay

Nascent RNA Synthesis

Click-iT® RNA Assay

Nascent Protein Synthesis

Click-iT® AHA

Click-iT® HPG

Detection of Damage

Click-iT® TUNEL

Click-iT® Lipid

Peroxidation

…fast, sensitive, non-toxic, and non-radioactive method of detection

Post-translational

Modifications

Click-iT® sugars

Click-iT® palmitic acid

Click-iT® farnesyl alcohol

…many more



Foundational platforms: Cell Structure



What Cell Shape & Structure Can Tell Us

Predictive of function

Changes reflect cell health

and potential progression to

a disease state

Highly dynamic information

Well-suited to imaging



Organelle-Selective Dyes

A wide variety of fluorescent dyes that stain cellular

structures in living cells based on their chemical nature

Examples include…

MitoTracker® dyes for mitochondria

Nucleic acid stains for nuclei and nucleoli

LysoTracker ® & LysoSensor ® dyes for acidic compartments

Lipophilic dyes for membranes

ER-Tracker™ dyes for endoplasmic reticulum.

Ceramide conjugates for the Golgi apparatus

Vast toolbox, ideal for shorter-term

live cell imaging experiments (some fixable)



Tracker® dyesGolgi Lysosomes

Mitochondria ER



Foundational platforms: BacMam



BacMam Gene Delivery Technology

Insect BACulovirus with MAMmalian promoter

Easy to use, just add virus to cells

Titratable and transient expression at high levels

Non-toxic to cells and BSL1

Non-replicating in mammalian cells

Flexibility of batch transduction and cryopreservation

Transduces hard-to-transfect cells

− Primary cells

− Stem cells

− Neurons

Fluorescent proteins for live cell biology, accessible to all with

BacMam gene delivery CellLigh® Reagents & Premo™ Sensors



Simple workflow….

….enabling longer-term,

higher content live cell imaging



BacMam-mediated Transduction of “Difficult” Cells

Human umbilical vein

endothelial cellHuman airway epithelial

smooth muscle

Human Aortic Smooth

MuscleHuman mesenchymal

stem cell

Human KeratinocytesHuman Melanocytes Human Mammary Epithelial Cells

Human Adipose-derived Stem Cells

Mouse ES

Cardiomyocytes

Mouse hippocampal neurons Mouse hippocampal neuronsOmura,Costigan and Woolf

Harvard UniversityRutten and Gregory,

Oregon Medical Laser Center

Adult rat DRG neurons



CellLight® Targeting Strategy for Illuminating Cell Structure

Leader sequence of E1a

pyruvate dehydrogenase

Human actin monomer

Human Golgi-resident enzyme

N-acetylgalactosaminyltransferase-2

Human tubulin monomer

MAP4

LAMP1

Nuclear export sequence

(N- or C- terminus)

Peroxisomal C-terminal

targeting sequence

Myristoylation/palmitoylation

sequence from Lck tyrosine kinase

Signal sequence of Calreticulin

and KDEL

Late Endosome

Rab7a

Histones

Histone 2BSV40 nuclear localization

sequence (N- or C- terminus)

Synapse

Synaptophysin

Early Endosome

Rab5a

Human C-terminus of talin

Talin



Live cell imaging of nuclei and the cytoskeleton

during division with CellLight® Reagents

CellLight® Reagents

Enable Longer-Term Live Cell Imaging of Cell Structure

Image acquisition every 5 min for 16 hours Image acquisition every 15 min for 14 hours

MAP4-GFP/H2B-RFP Tubulin-GFP/Actin-RFP



CellLight® Mito-GFP and 50nM TMRM Transient localized depolarization and global

irreversible deploarization (induced by CCCP treatment)

CellLight® Reagents and Fluoresenct Dyes in Combination

Decreased signal response from mitochondrial membrane potential dyes

Mitochondrial movement, rupture, or depolarization?

Remove ambiguity of results: “Functionally-independent”

organelle markers with “function-dependent “dyes

CellLight® Mito-GFP and TetraMethylRhodamine Methyl Ester (TMRM)



Premo™ FUCCI Cell Cycle Sensor

GFP-Geminin

+

RFP Cdt1

=

FUCCI



Premo™ FUCCI Cell Cycle Sensor

GFP-Geminin

+

RFP Cdt1

=

FUCCI



New approaches: LIVE/DEAD



Cell Health: Cells Exist Anywhere on a Continuum Between Healthy and Dead

Changes in cell health:

− Underlie disease progression

− Report the effectiveness of a drug or other therapeutic intervention

− Inform us of the importance of a gene in a siRNA/KO study

− Represent important considerations regardless of area of study or model

Live Dead

ProliferationMembrane

Potential

Enzyme

Activity

Membrane

Integrity

Apoptosis

Autophagy

Necrotic

Fluorescence microscopy provides spatial and temporal resolution for

high content assays of cell health-related parameters and dynamics



LIVE/DEAD® Cell Imaging Kit *488/570*

• Fast, simple and sensitive determination of live and dead cells

• Measures recognized parameters of cytotoxicity and cell viability

• Ideal for FITC and Texas Red® filters

Readout based on intracellular esterase activity and plasma membrane integrity



New approaches: pHrodo™



Internalization with Fluorogenic pHrodo™ Green and Red

Dyes



pHrodo Red and pHrodo Green Fluorogenic pH Sensor

DyespHrodo RED

lmax Excite 550nm

lmax Emit 585nm

pHrodo Green

lmax Excite 505nm

lmax Emit 525nm

pKa = 6.8

Reactive dyes and conjugates

Multiplex and Pulse-Chase Experimental Configurations



Live cell imaging of pHrodo™

s.aureus

zymosan

e.coli



Endocytosis with pHrodo™10K-dextran

Initial signal seen in endosomes @ 10 mins

Endosome-GFP

Lysosome-GFP

…Trafficked into lysosomes @ 30 mins

Sorted away from Golgi and Peroxisomes

Peroxisome-

GFP

Golgi-GFP

1)

2)

3)



CellLight™

OVERLAY Endosome-GFP

AF-647-TfN pHrodo™ Dextran

pHrodo™ Multiplexing with Transferrin

pHrodo™ Dextran conjugates are found

mainly in lysosomes

Transit through endosomes quickly

Emission is brighter in lysosomes than

endosomes

Lysosomal pH is more acidic

Labeled Transferrin trafficking



Antibody Labeling with Reactive pHrodo™ Dyes

2 dyes:protein gives signal amplification

No dye–dye interactions and resultant

self-quenching

No modification of antigen binding sites

Loss of function caused by antigen

binding site modification

Dye–dye interactions = self-quenching

= no fluorescence increase

Precipitation due to masking of protein

surface

Just Right(DOL = 2)

Too Much(DOL = 6)

► Molar ratio of dyes:protein is called the DOL (degree of labeling)

► Optimization of DOL is important!



Pretreat control Pretreat Unlabeled EGF 10 ug/mL

EGF internalization: pHrodo™ Red avidin & EGF-Biotin



New approaches: Oxidative stress



Click-iT® Lipid Peroxidation Imaging Kit - Alexa Fluor® 488

Control

200 uM Cumene hydroperoxide

BPAE cells

With lipid peroxidation in cells, incorporated LAA is oxidized

and its products form click-able adducts on proteins and DNA



Plate cells Stain with Image-iT® lipid peroxidation sensor Treatment

Control 200 µM CH

U2-OS cells

0

0.5

1

1.5

2

2.5

3

3.5

Fold

ch

ange

(R

ed/G

reen

)

Control Control + Toc

Menadione Menadione + Toco

TBHP TBHP + Toco

CH CH + Toco

Localization in cellular membranes

Red to Green ex/em shift upon

oxidation (580/590 485/510 nm)

Stable stock solution, simple

workflow, positive control provided

Image-iT® Lipid Peroxidation Imaging Kit



CellROX™ Deep Red Reagent for Detection of Oxidative Stress

CellROX™ Deep Red reagent added to live bovine pulmonary artery

endothelial cells in complete media

Untreated 100 µM Menadione 100 µM Menadione +

MnTBAP

Reduced fluoresceins, traditionally used for cellular ROS

measurements, must be added to serum-free media and suffer

from high background, photo-oxidation, and photo-instability

0

20

40

60

80

100

120

0 2 4 6 8 10 12 14

Time (sec)

No

rma

lize

d F

luo

res

ce

nc

e In

ten

sit

y

CellROX™ Deep Red Reagent H2-DCFDA

CellROX™ Deep Red signal is

retained (~2 hr) after formaldehyde-

based fixation in U-2 OS cells

Liv

eF

ixed

MenadioneUntreated



Control 200 µM TBHP

0

50

100

150

200

250

300

350

Mea

n s

ign

al in

ten

sity

Control Control + Ebselen TBHP TBHP + Ebselen

***b

***a

CellROX™ Orange ReagentExcitation and emission of 545/565 nm

Photostable

Compatible with GFP-expressing cells

Multiplexibility with other cell health markers

CellROX™ Green ReagentExcitation and emission of 485/550 nm

Formaldehyde fixable and detergent resistant

Compatible with RFP-expressing cells

Multiplexibility with other cell health markers

Control 100 µM Menadione

0

100

200

300

400

Mea

n s

ign

al in

ten

sity

Control Control + NAC

Menadione Menadione + NAC

BPAE cells BPAE cells



CellRox™ green & TMRM

Menadione treated U-2 0S cells



H2-DCFDA DHECellROX™ Deep

RedCellROX™

OrangeCellROX™

Green

Add in Complete

mediaNo Yes Yes Yes Yes

Fixable No No Yes No Yes

Detergent

resistantNo No No No Yes

Multiplexibility Yes No Yes Yes Yes

GFP compatible No No Yes Yes No

RFP compatible Yes No Yes No Yes

Photostability * N/A *** **** **

CellROX™ Reagents for Oxidative Stress Detection



New approaches: Autophagy



Autophagy

Adapted from Klionsky et al 2007 Nat Rev Mol Cell Biol

PAS expansion

‘LC3B’MEMBRANE SOURCES Rab7/vAMP

Low pH

ATG3

Membrane recycling

mTOR



Visualization of Autophagomes

LC3B Antibody Kit for Autophagy

(includes autophagosome inducer)

Control

50 mM Chloroquine Premo™ Autophagy Sensor LC3B-FP (Includes G120A

mutant LC3B-FP control & autophagosome inducer)



Autophagy in Hippocampal Neurons

LC3B-GFP Mutant LC3B G120A

Premo™ Autophagy Sensor LC3B-GFP kit Kit includes G120A mutant LC3B-GFP control & autophagosome inducer



New approaches: Apoptosis



Apoptosis

Adapted from Taylor et al 2008 Nat Rev Mol Cell Biol

Perforin

Granzyme B

BID

tBID

Bax‘BH3’ domain proteins

*INTRINSIC PATHWAY*

BCl-2 subfamily

-ve

+ve

Apoptossome

(APAF-1/C9)

C8

C7

C10

C2

C3

C8

FADD

Death receptor

FasL/TNFa

C3

C6

*EXTRINSIC PATHWAY*

*Granzyme B PATHWAY*

Cyt C



CellEvent™ Caspase 3/7 Green Reagent for Apoptosis

Fluorogenic Caspase 3/7 Substrate

A nucleic acid dye conjugated to a DEVD peptide

Non-fluorescent until active caspase 3/7cleaves the DEVD peptide and the free nucleic acid dye binds to DNA.

ADVANTAGES:

− Live cell amenable, no-wash protocol

− May be added to complete growth media

− Retained after fixation and permeabilization

− May be multiplexed with other live or fixed cell probes

DEVD

Active

Caspase-3/7 Enzyme

Non-fluorescent

No DNA binding

Bound

DNA dyeDNA dye

Read

Add

CellEvent™

reagent

Incubate

30 min



CellEvent™ Caspase 3/7 Green Reagent in Live and Fixed cells

Traditional Fluorescence Microscopy and High Content Imaging

StaurosporineNo Treatment

Live HeLa cells• 4 hr staurosporine treatment

• CellEvent™ Caspase 3/7 Green

Fixed and permeabilized U-2 OS cells 18 hr etoposide treatment

• CellEvent™ Caspase 3/7 Green

• AlexaFluor® 546 Phalloidin ,

• MitoTracker® Deep Red

•Hoechst



0

20

40

60

80

100

CellEvent™ Caspase 3/7 Green Specificity and Robustness:

No-wash Apoptosis Detection

No Inhibitor 20 µM Inhibitor

CellEvent™ signal is absent in cells

treated with Z-DEVD-FMK caspase inhibitor

% P

osit

ive A

po

pto

tic C

ells

Fragile apoptotic cells are easily lost

by plate washes, introducing assay

variance and error



Multiplex Time Lapse Imaging of Apoptosis and

Mitochondrial Health: CellEvent™ Caspase 3/7 Green and TMRM

Red: TMRM mitochondrial

membrane potential indicator

Fades with apoptosis

Green: CellEvent™ Caspase 3/7

Fluorogenic with apoptosis

HeLa cells, 0-7 hrs treatment with 0.5 mM staurosporine

Documents

Advanced fluorescence imaging