microscopy i fluorescence confocal multiphoton flow cytometry by luis filgueira
TRANSCRIPT
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Microscopy IFluorescence
ConfocalMultiphoton
Flow CytometryBy
Luis Filgueira
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For Wednesday TutorialPlease
Activate you PHEME accountIf not yet done
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For next week’s labsplease
put your name down on list at the white board
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Fluorescence
Three-stage process
1. ExitationPhoton of energy
hEX
2. Exitation-stateLifetime
(1-10x10-9 seconds)
3. EmissionPhoton of energy
hEM
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Difference in energy= hEX – hEM
Differerence in vave length
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Exitation and Emision
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polyaromatic hydrocarbons heterocycles
Fluorophore = fluorescent dye
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Fluorophore = fluorescent dye
Binding specifically to biochemical structures
ProteinDNARNA
LipidsCaKH+
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Fluorophore = fluorescent dye
Fluorescence labelled macromolecules
AntibodiesLipids
NucleotidesOligo-DNAOligo-RNA
PeptidesLectines
Phalloidin
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Fluorophore = fluorescent dye
Detection of Organelles
NucleusGolgi
LysosomesRER
MitochondriaCytoskeletonMembranes
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Fluorophore = fluorescent dye
Enzymes and Enzyme Substrates and Cell Function
GlycosidasesPhosphatases
Proteases/PeptidasesPeroxidases
LipasesMembrane potential
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Applications for fluorescent dyes
Microscopy
Flow Cytometry
Molecular Biology
Spectrofluorometric Assays
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Molecular BiologyGel-Electrophoresis
Real time/quantitative RT-PCR
DNA RNA Protein
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Spectrofluorometric Assays
Cell ViabilityCytotoxicity
Enzymatic AssaysCa+ Measurement
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Flow Cytometry
Cell Cycle Analysis
Cell Viability
Cytotoxicity
Quantification of
Surface marker expression
Intracellular Protein expression
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Microscopes
Axial Invert
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Fluorescence Microscopy
Mercury Vapor lamp = Arc Lamp
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Filters
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Confocal Microscopy
Ar UV 351, 364nmHeCd 442nmAr 457nm, 488nm, 514nmArKr 488nmKr 568nmHeNe 633nm
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Comparison
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Confocal Microscopyoptical sectioning
<>electronic reconstruction
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Axial optical sectioning of the object is possible in two ways.Either by- direct acquisition of x/z- or y/z-optical cross sections, or - calculation of vertical optical sections using the normal x/y-image stack (as shown here).
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Rotation and volume rendering.Rotation of the image stack and projection into one plane allows viewing the object from different angles.
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Stereoscopic imaging.The combination of a left and a right image either as- stereo pairs or- red/green anaglyphsallows easy visualisation of 3D-structures!Use red/green glasses for stereoscopic viewing of this red/green anaglyph.
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Multi-photonFluorescenceMicroscopy
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Flow Cytometry
Measurement and quantification of optical properties of particles in suspension
Beads
BacteriaCells
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Charging electrode
Sample injection tube
Sheat tube
Vent tubeLaser light
Deflection plates
Collection tubes
Flow Cytometry
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Flow Cytometry
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Flow Cytometry
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Flow Cytometry
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Flow Cytometry
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Flow Cytometry