1. GFP Fluorescence/Confocal Microscope Kimberly Ching ABE Workshop Summer 2007

2. Fluorescent Microscope Multiple Labeling Multiple Labeling ID specific organelles ID specific organelles Necrotic vs. apoptotic cells Necrotic vs. apoptotic cells Verify cell membrane permeability Verify cell membrane permeability Locate antigen-specific molecules Locate antigen-specific molecules

3. Alexander Jablonski Diagram Light from the excitation filter excites the fluorochoromes to a higher energy state Light from the excitation filter excites the fluorochoromes to a higher energy state From the high state it declines slowly releasing energy From the high state it declines slowly releasing energy Transition between absorption & emission Transition between absorption & emission

4. Excitation and Emission Stokes Shift/Law Stokes Shift/Law Florescence emission wave length is longer Florescence emission wave length is longer Excitation wave length is shorter Excitation wave length is shorter

5. Light Path Light from excitation filter thru objective lens; light absorbed Light from excitation filter thru objective lens; light absorbed Light emitted goes back thru objective lens, barrier filter, then detector Light emitted goes back thru objective lens, barrier filter, then detector

6. Fluorochromes DAPI: blue dye, 4',6-diamidino-2- phenylindole, binds to DNA excited with UV, absorption max358nm, emission max 461nm DAPI: blue dye, 4',6-diamidino-2- phenylindole, binds to DNA excited with UV, absorption max358nm, emission max 461nm GFP: Green Fluorescence Protein, Aequorea victoria, absorption 488nm, emission 509nm GFP: Green Fluorescence Protein, Aequorea victoria, absorption 488nm, emission 509nmAequorea victoriaAequorea victoria Fluorescence overlap between DAPI and GFP Fluorescence overlap between DAPI and GFP

7. Immunolabeling for Fluorescence 1.Block with PBST+5% milk 1 hr 1.Block with PBST+5% milk 1 hr 2.Incubate with primary antibody in PBS or blocking solution 1-2hr, @ r.t 2.Incubate with primary antibody in PBS or blocking solution 1-2hr, @ r.t 3.Wash with PBST+5% milk 3x3 min 3.Wash with PBST+5% milk 3x3 min 4.Incubate with 2ndary antibody in PBS 1hr r.t 4.Incubate with 2ndary antibody in PBS 1hr r.t 5.Wash with PBST+5% milk 5 min 5.Wash with PBST+5% milk 5 min 6.Wash with PBS no milk 2x5 min 6.Wash with PBS no milk 2x5 min 7.Wash with dH 2 0 2x10 min 7.Wash with dH 2 0 2x10 min 8.Coverslip with Vectashield & view with fluorescence/confocal microscope 8.Coverslip with Vectashield & view with fluorescence/confocal microscope

8. Confocal Microscope Better resolution Better resolution Cells can be live or fixed Cells can be live or fixed Serial optical sections can be collected Serial optical sections can be collected

9. Laser Beam Laser goes thru aperture, then objective lens; pixel by pixel scanning Laser goes thru aperture, then objective lens; pixel by pixel scanning Light is reflected back thru objective lens, beam splitter allows laser thru, and reflects fluorescence Light is reflected back thru objective lens, beam splitter allows laser thru, and reflects fluorescence To the detector, pic can be viewed on the computer To the detector, pic can be viewed on the computer

10. Fluorochromes FITC: fluorescein isothiocyanate absorption maximum at 495 nm, 488nm excitation wavelength FITC: fluorescein isothiocyanate absorption maximum at 495 nm, 488nm excitation wavelengthfluorescein isothiocyanatefluorescein isothiocyanate TEXAS RED: 595nm excitation wavelength, 615 max absorption, red dye, marks protein TEXAS RED: 595nm excitation wavelength, 615 max absorption, red dye, marks protein

11. Florescence: Dark GFP 2SC Cotyledon 20x DAPI Root tip 40x DAPI Cotyledon 20x DAPI Root tip 40x DAPI

12. Florescence: Dark GFP 2SC Cotyledon 20x GFP Root tip 40x GFP

13. Florescence: Dark Wild Type Cotyledon 20x DAPI Root tip 40x DAPI Cotyledon 20x DAPI Root tip 40x DAPI

14. Florescence: Wild Type Cotyledon 20x GFP: chloroplasts Cotyledon 20x GFP: chloroplasts

15. Confocal: GCNC 19 GFP, Mito Tracker, Auto fluorescence GFP, Mito Tracker, Auto fluorescence

16. Confocal: GCNC 19 Animation Video Animation Video

17. Confocal: GFP Animation Video Animation Video GFP, Mito Tracker, Auto florescence

18. GFP 2SC Light Light Cotyledon 40x Light Cotyledon 40x Light Cotyledon 40x Light Cotyledon 40x

19. GFP 2SC Dark Cotyledon 40x Cotyledon 20x Cotyledon 40x Cotyledon 20x

20. References http://micro.magnet.fsu.edu/primer/techniques/ fluorescence/fluorescenceintro.html http://micro.magnet.fsu.edu/primer/techniques/ fluorescence/fluorescenceintro.html http://micro.magnet.fsu.edu/primer/techniques/ fluorescence/fluorescenceintro.html http://micro.magnet.fsu.edu/primer/techniques/ fluorescence/fluorescenceintro.html http://micro.magnet.fsu.edu/primer/lightandcol or/fluorescencehome.html http://micro.magnet.fsu.edu/primer/lightandcol or/fluorescencehome.html http://micro.magnet.fsu.edu/primer/lightandcol or/fluorescencehome.html http://micro.magnet.fsu.edu/primer/lightandcol or/fluorescencehome.html http://www.bdbiosciences.com/pharmingen/pr otocols/Fluorochrome_Absorption.shtml http://www.bdbiosciences.com/pharmingen/pr otocols/Fluorochrome_Absorption.shtml http://www.bdbiosciences.com/pharmingen/pr otocols/Fluorochrome_Absorption.shtml http://www.bdbiosciences.com/pharmingen/pr otocols/Fluorochrome_Absorption.shtml http://web.uvic.ca/ail/techniques/epi- fluorescence.html http://web.uvic.ca/ail/techniques/epi- fluorescence.html http://web.uvic.ca/ail/techniques/epi- fluorescence.html http://web.uvic.ca/ail/techniques/epi- fluorescence.html