1. Lasers in medicine: blood and blood flow in the tissues Today’s talk... 1.Introduction to blood and blood flow 2.Laser Flow-Cytometry (LFC) of blood... diagnostics 3.Laser Doppler blood Flow measurement (LDF)... imaging & clinical measurement

2. Bench-top automated Laser Flow Cytometer for routine blood sample analysis (from Sysmex-Toa)

3. The optics of a Laser Flow Cytometer

4. Light Scatter Cells pass through a flow cell on which a laser of light is focused Cells cause light scatter which is detected by photo detectors Low-angle light scatter measures cell volume Structure inside the cell is measured by forward high-angle scatter

5. The heart of a Laser Flow Cytometer is the hydrodynamicaly focussed flow cell... but why do we need such sophistication?

7. Measuring blood cells using Laser Flow Cytometry Basic principles: 1.Blood sample is diluted 1000’s of times in isotonic solution 2.Very stable fluid flow makes cells pass one-by-one through a laser beam 3.The laser beam shape may be a spot or a slit spot for diffraction mode (size only) slit for slit-scanning mode (size [and shape?] )

8. Measuring blood cells using Laser Flow Cytometry Very stable fluid flow makes cells pass one-by-one through a laser beam

12. Measuring blood cells using Laser Flow Cytometry Very stable fluid flow makes cells pass one-by-one through a laser beam … diffraction scatter models 1. side 2. backwards 3. forwards narrow wide

13. Measuring blood cells using Laser Flow Cytometry Scatter: forwards narrow angle Fraunhofer diffraction pattern described by Bessel function We see this as Fraunhofer rings Ring-spacing gives approximate size

14. Measuring blood cells using Laser Flow Cytometry Scatter: forwards narrow angle Fraunhofer diffraction pattern described by Bessel function

15. Measuring blood cells using Laser Flow Cytometry Scatter: forwards narrow angle Fraunhofer diffraction pattern described by Bessel function

16. Measuring blood cells using Laser Flow Cytometry Slit-scanning: Also called obscuration scanning the light is obscured … The bigger the cell, the greater the obscuration ….. the detector sees lees light

17. Measuring blood cells using Laser Flow Cytometry Slit-scanning: Also called obscuration scanning the light is obscured … Analysis of the obscuration signal (amplitude and time) gives us shape information eg: long and thin ? short and fat ?

22. Laser Flow Cytometer: differential white cell analysis uses a 2D plot bio-chemicals (antibodies) tagged with fluorescent markers ‘stain’ the white cells characteristically and are detected from the side wrt axial path size signal intensity (fluorescence)

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