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