1. 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 Discussion B.Fluorescent Probes 1.Nuclear stains 2.Ion indicators 3.Organelle probes 4.Membrane probes 5.Tracers C.Lab: Onion epithelium as a source of live cells. D.TBA Time: review for checkout

2. 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):

3. Checkout times Review 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

4. 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.

5. 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.

6. B. Fluorescent Probes Molecular Probes (Invitrogen) –www.probes.comwww.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.listserv@ubvm.cc.buffalo.edu –Archive: http://listserv/ascu.buffalo.edu/archives/confocal.ht ml http://listserv/ascu.buffalo.edu/archives/confocal.ht ml

7. 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.

8. Controlling Photobleaching: Properties of Popular Antifade Reagents Antifade Reagent Comments 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-propylgallate The 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

9. 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.

10. Common fluorochromes used for cell staining FluorochromeExcitation (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 461 525 570 620 510 570 Various Blue Green Red-orange Red Green Red Various

11. AlexaFluor Dyes Higher quantum yield, lower photobleaching, narrower excitation spectra, etc.

12. Nucleic Acid Probes NameExcitationEmissionComments Acridine orange500526 (broad spectrum) Taken up by live cells, especially acidic vesicles Propidium iodide536617Dead/fixed cells Ethidium bromide518617Dead/fixed cells, higher background UV dyes DAPI, Hoeschst 33258 358/352461/461Semi-permeant/ permeant Syto dyes (Molecular Probes) Various Cell permeant Sytox dyes (Molecular Probes) 445/504/547 + others470/523/570 + othersHighly impermeant Others listed on p. 215 of textbook.

13. Maintaining healthy cells for live imaging Cell structure and integrity –Various imaging chambers may be necessary Temperature control –Temperature control chambers pH control –Buffers O 2, CO 2 levels –A concern in enclosed chambers Control of Osmolarity –A concern in open chambers

14. 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 Ca 2+ 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.

15. Blue Ca 2+ 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.

16. Ca 2+ Release During Shrimp Egg Activation From Lindsay et al. (1992). Extracellular Mg 2+ Induces an Intracellular Ca 2+ Wave During Oocyte Activation in the Marine Shrimp Sicyonia ingentis. Dev. Biol. 152:94-102.

17. 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

18. UV Ca 2+ 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). [Ca 2+ ] ~ 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

19. UV Ca 2+ 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. [Ca 2+ ] ~ 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

20. Free Ca 2+ 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.

21. Dual Blue Ca 2+ Indicators: Fluo-3 and Fura Red Use of two dyes solves the problem, e.g. Fluo-3 (increase at 525 with increasing [Ca 2+ ]) and Fura Red (decrease at 650 upon increasing [Ca 2+ ]). Both excited by 488 nm. [Ca 2+ ] ~ Em of Fluo-3 / Em of Fura Red, independent of [dye].

22. Frog Olfactory Bulb Neurons Labeled with Fluo-3 and Fura Red Ca 2+ 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.

23. 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

24. Organelle Probes Target, NameExcitationEmissionComments Mitochondria MitoTracker dyes490/578/551516/599/576 MT Green accumulates in mitochondria regardless of MB potential, Red and Orange in active mitochondria; aldehyde fixable Golgi BODIPY FL and TR C5-ceramide 505/589511, 620 /617TR is better for double labeling, since no green emission Lysosomes LysoTracker LysoSensor Various Endoplasmic reticulum DiOC6, DiIC6 Fluorescent Brefeldin A 484/549501/565 DiO and DiI also stain membranes generally Inhibitor of protein transport Others listed on p. 224-225 of textbook.

25. Double-labeling: Mitochondria (Mitotracker Red CMXRos) and Microfilaments (BODIPY FL phallacidin)

26. 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.

27. Membrane Probes Useful for tracing cell fates without microinjecting DiI (red, D on right) DiO (green) Many others available from Molecular Probes

28. Cell Tracers NameExEmComments CellTrackerVarious Permeant, then attaches to cellular proteins, aldehyde fixable Lucifer yellow488500-600 Yellow Common gap junction & neuronal tracer, aldehyde fixable Membrane probesVarious Can label by touching dye crystal against cell. Fluorescent dextrans Various Require microinjection

29. Lucifer yellow and gap junctions E.g. lucifer yellow Bigger than gap junction cutoff

30. 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.

31. 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. EPGFP DNA GFP mRNA GFP protein GF TF

32. GFP mice and frogs P crystallin GFP

33. GFP Fusion Proteins To determine sub-cellular location and dynamics of proteins

34. 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.DiOC 6 : 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 mitochondria 3.Lysotracker Red for lysosomes Dilute both of these to 250 nM in water, stain 5-10 min, remove, rinse 4.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.

35. 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, 3 rd ed. New York: Springer.