Lasers in medicine: blood and blood flow in the tissues Today’s talk ...

1. Lasers in medicine: blood and blood flow in the tissues • Today’s talk ... • Introduction to blood and blood flow • Laser Flow-Cytometry (LFC) of blood ... diagnostics • 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?

6. Measuring blood cells using Laser Flow Cytometry • Basic principles: • Blood sample is diluted 1000’s of times in isotonic solution • Very stable fluid flow makes cells pass one-by-one through a laser beam • 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?] )

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

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

9. 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

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

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

12. 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

13. 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 ?

14. size signal intensity (fluorescence) 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

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