microscopy i fluorescence confocal multiphoton flow cytometry by luis filgueira
TRANSCRIPT
Microscopy IFluorescence
ConfocalMultiphoton
Flow CytometryBy
Luis Filgueira
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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
Difference in energy= hEX – hEM
Differerence in vave length
Exitation and Emision
polyaromatic hydrocarbons heterocycles
Fluorophore = fluorescent dye
Fluorophore = fluorescent dye
Binding specifically to biochemical structures
ProteinDNARNA
LipidsCaKH+
Fluorophore = fluorescent dye
Fluorescence labelled macromolecules
AntibodiesLipids
NucleotidesOligo-DNAOligo-RNA
PeptidesLectines
Phalloidin
Fluorophore = fluorescent dye
Detection of Organelles
NucleusGolgi
LysosomesRER
MitochondriaCytoskeletonMembranes
Fluorophore = fluorescent dye
Enzymes and Enzyme Substrates and Cell Function
GlycosidasesPhosphatases
Proteases/PeptidasesPeroxidases
LipasesMembrane potential
Applications for fluorescent dyes
Microscopy
Flow Cytometry
Molecular Biology
Spectrofluorometric Assays
Molecular BiologyGel-Electrophoresis
Real time/quantitative RT-PCR
DNA RNA Protein
Spectrofluorometric Assays
Cell ViabilityCytotoxicity
Enzymatic AssaysCa+ Measurement
Flow Cytometry
Cell Cycle Analysis
Cell Viability
Cytotoxicity
Quantification of
Surface marker expression
Intracellular Protein expression
Microscopes
Axial Invert
Fluorescence Microscopy
Mercury Vapor lamp = Arc Lamp
Filters
Confocal Microscopy
Ar UV 351, 364nmHeCd 442nmAr 457nm, 488nm, 514nmArKr 488nmKr 568nmHeNe 633nm
Comparison
Confocal Microscopyoptical sectioning
<>electronic reconstruction
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).
Rotation and volume rendering.Rotation of the image stack and projection into one plane allows viewing the object from different angles.
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.
Multi-photonFluorescenceMicroscopy
Flow Cytometry
Measurement and quantification of optical properties of particles in suspension
Beads
BacteriaCells
Charging electrode
Sample injection tube
Sheat tube
Vent tubeLaser light
Deflection plates
Collection tubes
Flow Cytometry
Flow Cytometry
Flow Cytometry
Flow Cytometry
Flow Cytometry
Flow Cytometry