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GFP Fluorescence/ Confocal Microscope

GFP Fluorescence/ Confocal Microscope. Kimberly Ching ABE Workshop Summer 2007. Fluorescent Microscope. Multiple Labeling ID specific organelles Necrotic vs. apoptotic cells Verify cell membrane permeability Locate antigen-specific molecules.

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GFP Fluorescence/ Confocal Microscope

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

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

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

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

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

  6. Fluorochromes • 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 • Fluorescence overlap between DAPI and GFP

  7. Immunolabeling for Fluorescence • 1.Block with PBST+5% milk 1 hr • 2.Incubate with primary antibody in PBS or blocking solution 1-2hr, @ r.t • 3.Wash with PBST+5% milk 3x3 min • 4.Incubate with 2ndary antibody in PBS 1hr r.t • 5.Wash with PBST+5% milk 5 min • 6.Wash with PBS no milk 2x5 min • 7.Wash with dH20 2x10 min • 8.Coverslip with Vectashield & view with fluorescence/confocal microscope

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

  9. 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 To the detector, pic can be viewed on the computer Laser Beam

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

  11. Florescence: Dark GFP 2SC • 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

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

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

  16. Confocal: GCNC 19 • Animation Video

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

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

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

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

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