Microscopy I Fluorescence Confocal Multiphoton Flow Cytometry By Luis Filgueira.

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