Flow cytometry basics fluidics, optics, electronics...

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Reynold Barnett
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1 Title Flow cytometry basics fluidics, optics, electronics... RNDr. Jan Svoboda, Ph.D. Cytometry and Microscopy Core Facility IMB, CAS, v.v.i Vídeňská 1083

2 Fluorescence Fluorescence occurs when a valence electron absorbs light, excites and after short relaxation, returns to its basic state. Fluorescent molecules can be isolated, synthetised and used to label probes. Fluorescently labelled antibodies, DNA probes, Ca 2+ probes, Apoptosis probes, Cell cycle,... E=hc/λ Direct labelling Indirect labelling

Fluorescent molecules can be isolated, synthetised and used to label probes.

3 Excitation and emission spectra E=hc/λ Photon absorption is energy dependant various fluorochromes have various absorption maxima. Photon emission is dependant on the input energy and Stokes shift (the difference between ipnut/output energy).

4 Basic cytometer construction principles Cytometer has three basic systems: Fluidics delivers cells Optics excites andcollects FL Electronics signal processing Cells with fluorescent probe(s) in suspension travel one by one through laser(s). Light scatter in long angle (SSC) andshort angle (FSC) is measured. Fluorescence is measured orthogonally (as SSC).

fluorescent probe(s) in suspension travel one by one through laser(s).

5 Hydrodynamic focusing Sample stream Sheath fluid Obscuration bar. Cells in a thin stream intersect the laser beam (interrogation point) and a light pulse occurs. Cell suspension (sample stream) is carried by sheath fluid. This causes the sample stream to focus into a thin stream with cells lining up one after another (not next to each other).

pulse occurs. Cell suspension (sample stream) is carried by sheath fluid.

6 Hydrodynamic focusing and sample flow rate. Laser Laser High flow rates may hamper resolution.

7 Scattered light pulse and its processing. Threshold Light pulse is gene a functio on of cell siz to scatter light (opti rated e and cal de in time as its ability nsity).

8 Non gaussian light pulse and its processing for extremes in size. Pulse Height Pulse Area Laser beam cell Laser beam cell Diffraction?

9 Doublets and their indetification. Doublet pulse has almost double area and width (compared to height).

10 Excitation and collection of fluorescence. LASERS SS SC Fl luorescen nce FSC FSC is measured in parallel to laser light path. Fluorescence and SSC are colected perpendicularly. l

11 Emitted light isolation optical filters PMT 550 SP 550 LP PMT BandPass (520/35BP) PMT LP, SP and BP dichroic mirrors and filters.

12 Photomultiplier tube (PMT) function limits. Photon knocks out electrons when hitting the photocatode these are then multiplied 2:1 (ideally) on the following dynodes. The signal (pulse height) is being measured on the anode.

13 Fluorescence intensity output scales Fluorescence Log Scatters Lin Scatters Log Fluorescence Lin Relative intensity scale is commonly depicted on the log scale (except for scatters). Every dot represents an abovethreshold event (not necessarily a cell).

commonly depicted on the log scale (except for scatters).

14 Cell sorting Droplet delay droplet charging Piezoel. vibration Analysis Two to six way sorting (partial droplet charge) and single cell cloning. Sort left (+ charge) Sort right ( charge)

15 Sorting parameters Analysis Droplet delay droplet charging Piezoel. vibration Sorting envelope: Droplets/s (frequency) Event count/s (threshold) Droplet delay: Distance of break off point from interrogation point Sorted drops: Deflection envelope cell must be within dfi defined dnumber of drops.

(threshold) Droplet delay: Distance of break off point from interrogation

16 Thank You for Your attention.

Katharina Lückerath (AG Dr. Martin Zörnig) adapted from Dr. Jörg Hildmann BD Biosciences,Customer Service

Introduction into Flow Cytometry Katharina Lückerath (AG Dr. Martin Zörnig) adapted from Dr. Jörg Hildmann BD Biosciences,Customer Service How does a FACS look like? FACSCalibur FACScan What is Flow Cytometry?