Transcript GFP Fluorescence Microscope

GFP Fluorescence/Confocal
Microscope
Kimberly Ching
ABE Workshop
Summer 2007
Fluorescent Microscope
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Multiple Labeling
ID specific organelles
Necrotic vs. apoptotic cells
Verify cell membrane permeability
Locate antigen-specific molecules
Alexander Jablonski Diagram
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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
Excitation and Emission
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Stokes Shift/Law
Florescence emission
wave length is longer
Excitation wave length
is shorter
Light Path
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Light from excitation
filter thru objective lens;
light absorbed
Light emitted goes back
thru objective lens,
barrier filter, then
detector
Fluorochromes
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DAPI: blue dye, 4',6-diamidino-2phenylindole, 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
Immunolabeling for Fluorescence
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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
Confocal Microscope
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Better resolution
Cells can be live or fixed
Serial optical sections can be collected
Laser Beam
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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
Fluorochromes
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FITC: fluorescein isothiocyanate absorption
maximum at 495 nm, 488nm excitation
wavelength
TEXAS RED: 595nm excitation wavelength,
615 max absorption, red dye, marks protein
Florescence: Dark GFP 2SC
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Cotyledon 20x DAPI
Root tip 40x DAPI
Florescence: Dark GFP 2SC
Cotyledon 20x GFP
Root tip 40x GFP
Florescence: Dark Wild Type
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Cotyledon 20x DAPI
Root tip 40x DAPI
Florescence: Wild Type
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Cotyledon 20x GFP: chloroplasts
Confocal: GCNC 19
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GFP, Mito Tracker, Auto fluorescence
Confocal: GCNC 19
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Animation Video
Confocal: GFP
GFP, Mito Tracker, Auto florescence
Animation Video
GFP 2SC Light
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Light Cotyledon 40x
Light Cotyledon 40x
GFP 2SC Dark
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Cotyledon 40x
Cotyledon 20x
References
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http://micro.magnet.fsu.edu/primer/techniques/
fluorescence/fluorescenceintro.html
http://micro.magnet.fsu.edu/primer/lightandcol
or/fluorescencehome.html
http://www.bdbiosciences.com/pharmingen/pr
otocols/Fluorochrome_Absorption.shtml
http://web.uvic.ca/ail/techniques/epifluorescence.html