Inside the black box

1. Inside the black box

2. fluidics electronics light optics

3. 1) Blocking capillary 2) Depth of focus hoy@cf.ac.uk Flow Cytometry : Problems with early systems. Detector

4. Hydrodynamic focusing V. Kachel, H. Fellner-Feldegg & E. Menke - MLM Chapt. 3

5. Fluidics– Particle Orientation and Deformation V. Kachel, et al. - MLM Chapt. 3

6. Sheath SampleStream Cell The Flow Cell 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. Original from Purdue University Cytometry Laboratories, Modified by R. Duggan

7. Sample differential pressure 10 psi 10 psi 10 psi 10.2 psi 10.4 psi 10.8 psi • If differential is too large, cells will no longer line up single file • Results in wider CV’s and increase in multiple cells passing through the laser at once. No more single cell analysis!

9. What can we measure • Scattered light • Fluorescence Thats it

10. Forward Angle Light Scatter Laser FALS Sensor Intensity is said to be proportional to cellsize

11. 90 Degree Light Scatter Laser FALS Sensor Intensity is said to be proportional to Granularity or internal complexity of the cell 90LS Sensor

12. Lymphozyt Basophiler Monozyt Neutrophiler Eosinophiler FACSAcademy

13. Granulocytes Lymphocytes SSC Monocytes RBCs, Debris, Dead Cells FSC Why Look at FSC v. SSC • Since FSC ~ size and SSC ~ internal structure, a correlated measurement between them can allow for differentiation of cell types in a heterogenous cell population

14. HO O C CO2H How to visualize a specific antibody fluorescein

15. Fluorescence Stokes Shift is 25 nm Fluorescein molecule 520 nm 495 nm Fluorescnece Intensity Wavelength low energy high energy wavelength (nm)

16. 350 457 488 514 610 632 Spectra of Common Fluorochromes Laser Lines (nm) PE-Texas Red Texas Red PI Ethidium PE FITC cis-Paranaric Acid 300 400 500 600 700 Original from Purdue University Cytometry Laboratories, Modified by R. Duggan

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

18. The photoreceptors of a flow cytometer longpass filter bandpass filter

19. Flow Cytometry Optics dissection of fluorescence PMT 4 Dichroic Longpass filters PMT 3 Flow cell PMT 2 Bandpass Filters PMT 1 Laser

21. Signal Detection - PMTsphoto multiplier tube Secondary emission Cathode Anode Amplified Signal Out Photons in End Window Dynodes Voltage

22. 350 457 488 514 610 632 The color blindness of a flow cytometer Laser Lines (nm) PE-Texas Red Texas Red PI Ethidium PE FITC cis-Paranaric Acid 300 400 500 600 700 Original from Purdue University Cytometry Laboratories, Modified by R. Duggan

23. Signal time Laser

24. Signal Laser time

25. Laser Signal time

26. Pulse Width Pulse Area Pulse Height Measurements of the Pulse Voltage Intensity Time

27. A mixed cell population

30. One parameter display (Histogram)

33. Two parameter display (Dot Plot) 320 FL2 480 FL1 FSC SSC FL1 FL2 FL3 FL4 75 42 320 10 10 9 FSC SSC FL1 FL2 FL3 FL4 20 41 7 480 8 8 FSC SSC FL1 FL2 FL3 FL4 20 41 320 480 8 8

34. Two parameter display (Dot Plot) CD45PE CD14 FITC

35. Häufigkeit Parameter2 Parameter1 dot plot Two parameters displayS 3D plot contour plot density plot

36. Data Analysis Concepts Regions A certain characteristic (blond, little, man, woman) Gates Logical combination of characteristics (blond women, little men)

37. Characterizationofsubpopulations Gate Auswertung CD45PE CD14FITC

38. 11-color, 13-parameter flow cytometry: Identification of human naive T cells by phenotype, function, and T-cell receptor diversity S C De Rosa, L A Herzenberg, L A Herzenberg & M Roederer Nature Medicine 7, 245 - 248 (2001).

40. http://www.cyto.purdue.edu/ Jean Paul Robinson