Announcements and Outline

• Sequential scanning reports due today.

• Midterm exam is next Monday, Feb. 26.

– After paper discussion– Mostly multiple choice test on the

lecture content.• Project proposals are also due next

Monday. Provide:– Group members (up to 3)– Organism– Probe– Question

• Checkouts during TBA time next week (see schedule)

– Review using grading rubric

A. Paper DiscussionB. Fluorescent Probes

1. Nuclear stains2. Ion indicators3. Organelle probes4. Membrane probes5. Tracers

C. Lab: Onion epithelium as a source of live cells.

D. TBA Time: review for checkout

A. Paper Discussion

• Today (Rachel): Tan et al. 2005• Feb. 26 (Ellen):• March 12 (Emily)• March 19 (Amy)• March 26 (Amanda)• April 2 (Andrea)• April 9 (Brittaney)• April 16 (Lauren)• April 23 (Joe and Molly):

Checkout timesReview checklist and practice

• Group 1 (Tuesday)– Amy 10:00-10:30– Lauren 10:30-11:00– Rachel 11:00-11:30

• Group 2 (Thursday)– Andrea 10:00-10:30– Emily 10:30-11:00– Molly 11:00-11:30

• Group 3 (Thursday)– Becky 1:00-1:30– Ellen 1:30-2:00– Katie 2:00-2:30

• Group 4 (Friday)– Amanda 2:00-2:30– Brittaney 2:30-3:00– Joe 3:00-3:30

Another project idea

• You might consider a “teaching” project instead of a “research” project.

• For example, prepare your own cryostat section of mouse kidney or intestine and do a demonstration of correct controls for double or triple labeling.– Background control: no label– Bleed-through control: single labeled– Double-labeled sample– Simultaneous versus sequential scanning

• Make a poster for prominent display in the Microscope facility, described how to do controls for double-labeling.

Controls for Double Labeling

• Background control: specimen without secondary antibody or fluorochrome– Controls for autofluorescence

• Bleed-through controls: specimens labeled with each fluorochrome separately. To determine maximum gain before bleed-through:1. Image green-labeled sample w/488 in Ch. 1, look for

cross-over in Ch. 2.2. Image red-labeled sample w/543 in Ch. 2, look for

crossover in Ch. 1.3. Using these settings, image double-labeled sample

(same stain concentrations as above) using sequential scan.

B. Fluorescent Probes

• Molecular Probes (Invitrogen) – www.probes.com– Catalog contains thousands of fluorescent probes,

with valuable technical information.

• Confocal Microscopy Listserver– To subscribe, send message “subscribe confocal”

to listserv@ubvm.cc.buffalo.edu with nothing in the subject line, without the quotation marks.

– Archive: http://listserv/ascu.buffalo.edu/archives/confocal.html

Fluorescein-5-isothiocyanate (FITC)

• Advantages– Excitation peak (495 nm) closely

matches 488 laser line– Bright, soluble, conjugates are stable

• Disadvantages– A relatively high rate of

photobleaching.– pH-sensitive fluorescence (pKa

~6.4) that is significantly reduced below pH 7.

– A relatively broad fluorescence emission spectrum, limiting their utility in some multicolor applications.

Controlling Photobleaching: Properties of Popular Antifade Reagents

Antifade ReagentComments

p-phenylene-diamine

The most effective reagent for FITC. Also effective for Rhodamine. Should be adjusted to 0.1% p-phenylenediamine in glycerol/PBS for use. Reagent blackens when subjected to light exposure so it should be stored in a dark place. Skin contact is extremely dangerous.

DABCO(1,4-diazabi-cyclo-2,2,2-

octane)

Highly effective for FITC. Although its effect is slightly lower than p-phenylenediamine, it is more resistant to light and features a higher level of safety.

n-propylgallateThe most effective reagent for Rhodamine, also effective for FITC. Should be adjusted to 1% propylgallate in glycerol/PBS for use.

2-mercapto-ethylamine

Used to observe chromosome and DNA specimens stained with propidium iodide, acridine orange, or Chromomysin A3. Should be adjusted to 0.1mM 2-mercaptotheylamine in Tris-EDTA

Tetramethylrhodamine (TRITC)

• Traditional second dye in double-labeling.– 568 nm krypton-argon– 543 nm green HeNe– But: 488 causes some bleed through into channel 1.

• So other red-emitting fluorophores are more commonly used now.

Common fluorochromes used for cell staining

Fluorochrome Excitation (nm) Emission (nm) Color

DAPI

Hoechst 33258

Fluorescein

Rhodamine

Texas Red

Cy2

Cy3(Amersham)

BODIPY dyes (Molecular Probes)AlexaFluor dyes (Molecular Probes)

358

352

495

552

596

492

550

Various

Various

461

461

525

570

620

510

570

Various

Various

Blue

Blue

Green

Red-orange

Red

Green

Red

Various

Various

AlexaFluor Dyes

• Higher quantum yield, lower photobleaching, narrower excitation spectra, etc.

Nucleic Acid Probes

Name Excitation Emission Comments

Acridine orange 500 526 (broad spectrum)

Taken up by live cells, especially acidic vesicles

Propidium iodide 536 617 Dead/fixed cells

Ethidium bromide 518 617 Dead/fixed cells, higher background

UV dyes DAPI, Hoeschst 33258

358/352 461/461 Semi-permeant/ permeant

Syto dyes (Molecular Probes)

Various Various Cell permeant

Sytox dyes (Molecular Probes)

445/504/547 + others 470/523/570 + others Highly impermeant

Others listed on p. 215 of textbook.

Maintaining healthy cells for live imaging

• Cell structure and integrity– Various imaging chambers may be necessary

• Temperature control– Temperature control chambers

• pH control– Buffers

• O2, CO2 levels– A concern in enclosed chambers

• Control of Osmolarity– A concern in open chambers

Physiological Probes (Fluorescent Ion Indicators)

• These molecular probes specifically bind an ion, which causes a change in either the intensity or wavelength of emission of the probe.

• Changes in concentration of the ion can therefore be monitored.

• There are indicators for most ions, but Ca2+ and pH are most commonly used.

• Salt or dextran forms must be microinjected; ester derivatives can be taken up by cells where they are converted to impermeant form.

Blue Ca2+ Indicators: Fluo-3 has single Ex and Em wavelengths

• A visible light excitable dye (488 nm), so Argon laser can be used.

• Emission at 525 nm.• OK for qualitative

detection but not quantitative.

Ca2+ Release During Shrimp Egg Activation

• From Lindsay et al. (1992). Extracellular Mg2+ Induces an Intracellular Ca2+ Wave During Oocyte Activation in the Marine Shrimp Sicyonia ingentis. Dev. Biol. 152:94-102.

Problems with Quantitative Single Wavelength Fluorescence Imaging

• Compartmentalization of probe in intracellular organelles.

• Uneven loading of dye in the cell.

• Instrumentation noise

• Sample geometry (thickness)

• Solution: ratio imaging of the dye

UV Ca2+ Indicators: Indo-1 is a fixed excitation ratiometric dye

• UV-excitable at fixed excitation wavelength (338 nm).

• Collect at two emission wavelengths (400 and 475 nm).

• [Ca2+ ] ~ ratio of Em400 / Em475, independent of dye concentration, etc.– Big num/small dem > [hi]– Small num/big dem > [lo]

Em at 400

Em at 475

UV Ca2+ Indicators: Fura-2 is a fixed emission ratiometric dye

• Flip excitation back and forth between 340 and 380 nm.

• Fixed emission at 510 nm is different for 340 v. 380 nm.

• [Ca2+] ~ Fluorescence at Ex340/Ex380, independent of dye concentration, etc.

• But: UV-excitable dye requires quartz optics, UV laser or conventional epiflourescence.

Ex at 380 nm

Ex at 340 nm

Free Ca2+ Concentration in a Purkinje Neuron from Embryonic Mouse Cerebellum

• Neurons were loaded with fura-2.

• Neurons were stimulated with glutamate receptor agonist.

• The composite image represents the ratio of images obtained with excitation at 340 nm and 380 nm.

Dual Blue Ca2+ Indicators:Fluo-3 and Fura Red

• Use of two dyes solves the problem, e.g. Fluo-3 (increase at 525 with increasing [Ca2+]) and Fura Red (decrease at 650 upon increasing [Ca2+]).

• Both excited by 488 nm.• [Ca2+ ] ~ Em of Fluo-3 / Em

of Fura Red, independent of [dye].

Frog Olfactory Bulb Neurons Labeled with Fluo-3 and Fura Red

• Ca2+ response to treatment with KCl and nifedipine. Image is a ratio of fluo-3 and Fura Red fluorescence.

• Images acquired with a Leica confocal laser scanning microscope.

The pH Indicator SNARF-1

• Used at near-neutral pH. Lysosensor dye can be used for acidic compartments.

• Can be excited by either 488 or 568 nm.

• At 488, emission is measured at 580 and 640 nm.

• pH ~ Em580 / Em640, independent of [dye].

Em at 640

Em at 580

Organelle Probes

Target, Name Excitation Emission Comments

Mitochondria

MitoTracker dyes 490/578/551 516/599/576

MT Green accumulates in mitochondria regardless of MB potential, Red and Orange in active mitochondria; aldehyde fixable

Golgi

BODIPY FL and TR

C5-ceramide

505/589 511, 620 /617 TR is better for double labeling, since no green emission

Lysosomes

LysoTracker

LysoSensor

Various

Various

Various

Various

Endoplasmic reticulum

DiOC6, DiIC6

Fluorescent Brefeldin A

484/549 501/565

DiO and DiI also stain membranes generally

Inhibitor of protein transport

Others listed on p. 224-225 of textbook.

Double-labeling: Mitochondria (Mitotracker Red CMXRos) and

Microfilaments (BODIPY FL phallacidin)

Golgi Apparatus and Lysosomes

Viable MDCK cells sequentially stained with BODIPY FL C5-ceramide, LysoTracker Red DND-99 and Hoechst 33258. Green-fluorescent BODIPY FL C5-ceramide localized to the Golgi apparatus, red-fluorescent LysoTracker Red stain accumulated in the lysosomes and blue-fluorescent Hoechst 33258 dye stained the nuclei.

Membrane Probes

• Useful for tracing cell fates without microinjecting

• DiI (red, D on right)• DiO (green)• Many others

available from Molecular Probes

Cell Tracers

Name Ex Em Comments

CellTracker Various Various Permeant, then attaches to cellular proteins, aldehyde fixable

Lucifer yellow 488 500-600

Yellow

Common gap junction & neuronal tracer, aldehyde fixable

Membrane probes Various Various Can label by touching dye crystal against cell.

Fluorescent dextrans

Various Various Require microinjection

Lucifer yellow and gap junctions

E.g. luciferyellow

Bigger than gapjunction cutoff

Green Fluorescent Protein (GFP)

• See pp. 232-238 of textbook.• From jellyfish Aequorea

victoria• Ex = 488 nm, Em = 509 nm.• Enhanced and colored

derivatives of GFP have been made.

• dsRED is from the coral Discosoma, with Ex = 558 nm and Em = 583 nm.

• Applications: reporter genes, fusion tags, lineage tracers, etc.

Promotor constructs

• Both can be used as reporters for promoter constructs. For example, transform an embryo with DNA coding for promoter/enhancer for growth factor fused to GFP to look for tissues that normally express the growth factor.

E P GFP DNA

GFP mRNA

GFP protein

GFTF

GFP mice and frogs

Pcrystallin GFP

GFP Fusion Proteins• To determine sub-cellular location and

dynamics of proteins

C. Lab: Onion epithelium prep(Dailey et al. 2007; Murphy, 2001)

• Cut a small square of a layer from the onion using a razor blade.• Use a forceps to peel off the thin epithelium on the inner surface of the

onion layer.• Put the epithelium on a microscope slide, cover with 1-2 drops of

staining solution.1. DiOC6: mitochondria and endoplasmic reticulum

• 0.5 mg/ml stock in EtOH, dilute 1:1000 in water on day of expt., stain 5 min, remove dye, rinse with water

2. Mitotracker Red for mitochondria3. Lysotracker Red for lysosomes

• Dilute both of these to 250 nM in water, stain 5-10 min, remove, rinse4. BODIPY-ceramide for Golgi apparatus

– Add 5 uM BODIPY-ceramide, stain 15 min, replace with water; should be in endosomes after 30 min and Golgi after 60 min; can also fix and stain as below

5. (All) DAPI for nuclei• Fix w/ 0.5% gluteraldehyde 5 min; remove fixative, rinse, 0.5 mg/ml DAPI, stain

• Cover with a coverslip.• Live motion (groups 1, 2, 3) can be collected in consecutive 1 s scans.

References

• Murphy, D.B. 2001. Fundamentals of light microscopy and electronic imaging. New York: Wiley-Liss.

• Dailey, M.E. et al. 2007. Confocal microscopy of living cells. In: Pawley, J.B., (ed.). Handbook of biological confocal microscopy, 3rd ed. New York: Springer.