GFP Fluorescence/Confocal GFP Fluorescence/Confocal MicroscopeMicroscope
Kimberly ChingKimberly Ching
ABE WorkshopABE Workshop
Summer 2007Summer 2007
Fluorescent MicroscopeFluorescent Microscope
Multiple LabelingMultiple Labeling ID specific organellesID specific organelles Necrotic vs. apoptotic cellsNecrotic vs. apoptotic cells Verify cell membrane permeabilityVerify cell membrane permeability Locate antigen-specific molecules Locate antigen-specific molecules
Alexander Jablonski Diagram Alexander Jablonski Diagram
Light from the Light from the excitation filter excites excitation filter excites the fluorochoromes to a the fluorochoromes to a higher energy statehigher energy state
From the high state it From the high state it declines slowly declines slowly releasing energyreleasing energy
Transition between Transition between absorption & emissionabsorption & emission
Excitation and EmissionExcitation and Emission
Stokes Shift/LawStokes Shift/Law Florescence emission Florescence emission
wave length is longerwave length is longer Excitation wave length Excitation wave length
is shorteris shorter
Light PathLight Path
Light from excitation Light from excitation filter thru objective lens; filter thru objective lens; light absorbedlight absorbed
Light emitted goes back Light emitted goes back thru objective lens, thru objective lens, barrier filter, then barrier filter, then detectordetector
FluorochromesFluorochromes
DAPI: blue dye, 4',6-diamidino-2-DAPI: blue dye, 4',6-diamidino-2-phenylindole, binds to DNA excited with UV, phenylindole, binds to DNA excited with UV, absorption max358nm, emission max 461nm absorption max358nm, emission max 461nm
GFP: Green Fluorescence Protein, GFP: Green Fluorescence Protein, Aequorea victoria, absorption 488nm, , absorption 488nm, emission 509nmemission 509nm
Fluorescence overlap between DAPI and GFPFluorescence overlap between DAPI and GFP
Immunolabeling for Immunolabeling for FluorescenceFluorescence
1.Block with PBST+5% milk 1 hr1.Block with PBST+5% milk 1 hr 2.Incubate with primary antibody in PBS or blocking 2.Incubate with primary antibody in PBS or blocking
solution 1-2hr, @ r.tsolution 1-2hr, @ r.t 3.Wash with PBST+5% milk 3x3 min3.Wash with PBST+5% milk 3x3 min 4.Incubate with 2ndary antibody in PBS 1hr r.t4.Incubate with 2ndary antibody in PBS 1hr r.t 5.Wash with PBST+5% milk 5 min5.Wash with PBST+5% milk 5 min 6.Wash with PBS no milk 2x5 min6.Wash with PBS no milk 2x5 min 7.Wash with dH7.Wash with dH220 2x10 min0 2x10 min 8.Coverslip with Vectashield & view with 8.Coverslip with Vectashield & view with
fluorescence/confocal microscopefluorescence/confocal microscope
Confocal MicroscopeConfocal Microscope
Better resolutionBetter resolution Cells can be live or fixedCells can be live or fixed Serial optical sections can be collectedSerial optical sections can be collected
Laser BeamLaser Beam
Laser goes thru aperture, Laser goes thru aperture, then objective lens; pixel by then objective lens; pixel by pixel scanningpixel scanning
Light is reflected back thru Light is reflected back thru objective lens, beam splitter objective lens, beam splitter allows laser thru, and allows laser thru, and reflects fluorescence reflects fluorescence
To the detector, pic can be To the detector, pic can be viewed on the computerviewed on the computer
Fluorochromes Fluorochromes
FITC: fluorescein isothiocyanate absorption FITC: fluorescein isothiocyanate absorption maximum at 495 nm, 488nm excitation maximum at 495 nm, 488nm excitation wavelengthwavelength
TEXAS RED: 595nm excitation wavelength, TEXAS RED: 595nm excitation wavelength, 615 max absorption, red dye, marks protein615 max absorption, red dye, marks protein
Florescence: Dark GFP 2SCFlorescence: Dark GFP 2SC
Cotyledon 20x DAPI Root tip 40x DAPICotyledon 20x DAPI Root tip 40x DAPI
Florescence: Dark GFP 2SCFlorescence: Dark GFP 2SC
Cotyledon 20x GFP Root tip 40x GFPCotyledon 20x GFP Root tip 40x GFP
Florescence: Dark Wild TypeFlorescence: Dark Wild Type
Cotyledon 20x DAPI Root tip 40x DAPI Cotyledon 20x DAPI Root tip 40x DAPI
Florescence: Wild TypeFlorescence: Wild Type
Cotyledon 20x GFP: chloroplasts Cotyledon 20x GFP: chloroplasts
Confocal: GCNC 19Confocal: GCNC 19
GFP, Mito Tracker, Auto fluorescence GFP, Mito Tracker, Auto fluorescence
Confocal: GCNC 19Confocal: GCNC 19
Animation VideoAnimation Video
Confocal: GFPConfocal: GFP
Animation VideoAnimation Video GFP, Mito Tracker, Auto florescence
GFP 2SC LightGFP 2SC Light
Light Cotyledon 40x Light Cotyledon 40xLight Cotyledon 40x Light Cotyledon 40x
GFP 2SC DarkGFP 2SC Dark
Cotyledon 40x Cotyledon 20xCotyledon 40x Cotyledon 20x
ReferencesReferences
http://micro.magnet.fsu.edu/primer/techniques/http://micro.magnet.fsu.edu/primer/techniques/fluorescence/fluorescenceintro.htmlfluorescence/fluorescenceintro.html
http://micro.magnet.fsu.edu/primer/http://micro.magnet.fsu.edu/primer/lightandcolor/fluorescencehome.htmllightandcolor/fluorescencehome.html
http://www.bdbiosciences.com/pharmingen/http://www.bdbiosciences.com/pharmingen/protocols/Fluorochrome_Absorption.shtmlprotocols/Fluorochrome_Absorption.shtml
http://web.uvic.ca/ail/techniques/epi-http://web.uvic.ca/ail/techniques/epi-fluorescence.htmlfluorescence.html
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