بنام خداوند بخشنده مهربان

1. بنام خداوند بخشنده مهربان

2. Flow Cytometry Principles & practice of “Fluorescence Spectroscopy in Biological Diagnosis & Research” M.Farhadi.M.D

3. Definitions • Flow Cytometry • Measuring properties of cells in flow • Flow Sorting • Sorting (separating) cells based on properties measured in flow • Also called Fluorescence-Activated Cell Sorting (FACS)

4. Flow Cytometry • Flow Cytometry is the technological process that allows for the individual measurements of cell fluorescence and light scattering. This process is performed at rates of thousands of cells per second. • This information can be used to individually sort or separate subpopulations of cells.

5. History • Flow cytometry developed from microscopy. Thus Leeuwenhoek is often cited in any discussion regarding it’s history. • F.T. Gucker (1947)build the first apparatus for detecting bacteria in a LAMINAR SHEATH stream of air. • L. Kamentsky (IBM Labs), and M. Fulwyler (Los Alamos Nat. Lab.) experimented with fluidic switching and electrostatic cell sorters respectively. Both described cell sorters in 1965. • M. Fulwyler utilized Pulse Height Analyzers to accumulate distributions from a Coulter counter. This feature allowed him to apply statistical analysis to samples analyzed by flow.

6. Basics of Flow Cytometry • Cells in suspension • flow in single-file through • an illuminated volume where they • scatter light and emit fluorescence • that is collected, filtered and • converted to digital values • that are stored on a computer Fluidics Optics Electronics

7. Fluidics • Need to have cells in suspension flow in single file through an illuminated volume • In most instruments, accomplished by injecting sample into a sheath fluid as it passes through a small (50-300 µm) orifice

8. Flow Cell Injector Tip Sheath fluid Fluorescence signals Focused laser beam

9. Fluidics • When conditions are right, sample fluid flows in a central core that does not mix with the sheath fluid • This is termed Laminar flow

10. Fluidics • The introduction of a large volume into a small volume in such a way that it becomes “focused” along an axis is called Hydrodynamic Focusing

11. Fluidics - Differential Pressure System • Use air (or other gas) to pressurize sample and sheath containers • Use pressure regulators to control pressure on each container separately

12. Fluidics - Differential Pressure System • Sheath pressure will set the sheath volume flow rate (assuming sample flow is negligible) • Difference in pressure between sample and sheath will control sample volume flow rate • Control is not absolute - changes in friction cause changes in sample volume flow rate

13. Fluidics - Differential Pressure System C. Göttlinger, B. Mechtold, and A. Radbruch

14. Fluidics - Particle Orientation and Deformation “a: Native human erythrocytes near the margin of the core stream of a short tube (orifice). The cells are uniformly oriented and elongated by the hydrodynamic forces of the inlet flow. b: In the turbulent flow near the tube wall, the cells are deformed and disoriented in a very individual way. v>3 m/s.” V. Kachel, et al. - MLM Chapt. 3

15. Fluidics - Flow Chambers Flow through cuvette (sense in quartz) H.B. Steen - MLM Chapt. 2

16. Injector Tip Sheath fluid Fluorescence signals Focused laser beam Flow Cell

17. Optics • Need to have a light source focused on the same point where cells have been focused (the illumination volume) • Two types of light sources • Lasers • Arc-lamps

18. Optics - Light Sources • Lasers • can provide a single wavelength of light (a laser line) or (more rarely) a mixture of wavelengths • can provide from milliwatts to watts of light • can be inexpensive, air-cooled units or expensive, water-cooled units • provide coherent light

19. Optics - Light Sources • Arc-lamps • provide mixture of wavelengths that must be filtered to select desired wavelengths • provide milliwatts of light • inexpensive, air-cooled units • provide incoherent light

20. Optics - Forward Scatter Channel • When a laser light source is used, the amount of light scattered in the forward direction (along the same axis that the laser light is traveling) is detected in the forward scatter channel • The intensity of forward scatter is proportional to the size, shape and optical homogeneity of cells (or other particles)

21. Laser FALS Sensor Forward Angle Light Scatter

22. Optics - Side Scatter Channel • When a laser light source is used, the amount of light scattered to the side (perpendicular to the axis that the laser light is traveling) is detected in the side or 90o scatter channel • The intensity of side scatter is proportional to the size, shape and optical homogeneity of cells (or other particles)

23. Laser FALS Sensor 90LS Sensor 90 Degree Light Scatter

24. Optics - Light Scatter • Forward scatter tends to be more sensitive to surface properties of particles (e.g., cell ruffling) than side scatter • can be used to distinguish live from dead cells • Side scatter tends to be more sensitive to inclusions within cells than forward scatter • can be used to distinguish granulated cells from non-granulated cells

25. FALS Sensor Fluorescence Fluorescence detector (PMT3, PMT4 etc.) Fluorescence Detectors Laser

26. Light Scattering, 2 Parameter Histogram Bigger Apoptotic Cells Bigger Cells 90 degree Light Scatter Dead Cells More Granular Y Axis X Axis Live Cells Forward Light Scatter (FLS)

27. 1 Parameter Histogram Positive Negative Brighter Count Dimmer 6 4 1 1 2 3 4 6 7 150 160 170 .. 190 Channel Number Fluorescence picked up from the FITC PMT

28. 2 Parameter Histogram Single Positive PI Population Double Positive Population PE FL Negative Population Single Positive FITC Population FITC FL

29. Optics - Filter Properties • Long pass filters transmit wavelengths above a cut-on wavelength • Short pass filters transmit wavelengths below a cut-off wavelength • Band pass filters transmit wavelengths in a narrow range around a specified wavelength • Band width can be specified

30. Optical Filters

31. Standard Long Pass Filters 520 nm Long Pass Filter Light Source Transmitted Light >520 nm Light Standard Short Pass Filters 575 nm Short Pass Filter Light Source Transmitted Light <575 nm Light

32. Standard Band Pass Filters 630 nm BandPass Filter White Light Source Transmitted Light 620 -640 nm Light

33. Optics - Filter Properties • When a filter is placed at a 45o angle to a light source, light which would have been transmitted by that filter is still transmitted but light that would have been blocked is reflected (at a 90o angle) • Used this way, a filter is called a dichroic filter or dichroic mirror

34. Dichroic Filter/Mirror Filter placed at 45o Light Source Transmitted Light Reflected light

35. Optics - Filter Layout • To simultaneously measure more than one scatter or fluorescence from each cell, we typically use multiple channels (multiple detectors) • Design of multiple channel layout must consider • spectral properties of fluorochromes being used • proper order of filters and mirrors

36. 350 457 488 514 610 632 300 nm 400 nm 500 nm 600 nm 700 nm Common Laser Lines PE-TR Conj. Texas Red PI Ethidium PE FITC cis-Parinaric acid

37. Compensation • A very important function of the electronics system is to perform compensation • There is some overlap between the colors emitted by different fluorescent markers, therefore mathematical compensation is used to reduce overlapping results http://www.bdbiosciences.com

38. Example Channel Layout for Laser-based Flow Cytometry PMT 4 PMT Dichroic 3 Filters Flow cell PMT 2 Bandpass Filters PMT 1 Laser

39. Optics - Detectors • Two common detector types • Photodiode • used for strong signals when saturation is a potential problem (e.g., forward scatter detector) • Photomultiplier tube (PMT) • more sensitive than photodiode but can be destroyed by exposure to too much light

40. Photomultipliers and Photodiodes • These are the two types of detectors that convert photons into electrical signals • They control sensitivity by adjusting voltage http://www.bdbiosciences.com

41. Summary of Part 1 • Cells in suspension • flow in single-file through • an illuminated volume where they • scatter light and emit fluorescence • that is collected, filtered and • converted to digital values • that are stored on a computer Fluidics Optics Electronics

42. Typical Research Cytometer (Coulter 753) (1980s) $200-300,000 Detectors Lasers Fluidics Computers Laser Power Supply

43. Typical Clinical Cytometer Detector & Mechanical Computer System Fluidics $90-120,000

44. Clinical Applications OfFlow Cytometric Analysis FlowCytometric(immunophenotypic) Classification Of Leukemias

45. CD # = cluster designation number Immunophenotyping CD2 CD4

46. NK B T Eosinophils Neutrophils Lymphocyte Immunophenotyping Peripheral White Blood Cells CD45+ Monocytes Granulocytes Lymphocytes Monocytes CD3+ CD3- CD16+ CD56+ Basophils CD3- CD19+ T Helper T Cytotoxic CD3+ CD4+ CD3+ CD8+

47. Immunophenotyping 100 1000 10 1 .1 Log FITC

48. CD4/CD8 Quadstats 1 2 45% 2% 3 4 27% 26% .1 1 10 100 1000 Log FITC Fluorescence (CD8)

49. Light Scatter Gating Side Scatter Projection Neutrophils Scale 1000 200 100 50 40 Monocytes 30 20 15 Lymphocytes 8 200 400 600 800 1000 0 90 Degree Scatter

50. The Cell Cycle M G2 G1 G0 S Quiescent cells