Optics - Filter Properties & manipulation of light in flow cytometry

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Optical filter evaluation

Slide 29

Light loss in dichroics

Reducing reliance on the in line arrangement PMTs

Placing a second fluorescence collection lens at 180o from the first one (this is more difficult in most instruments)

Slide 30

Light loss by optics

The thicker the glass the less light transmitted.

Problems with glass - UV light will not pass

In UV light system use minimum optics.

Slide 31

Light loss by optics

Glass can absorb UV light and can fluoresce when illuminated at that wavelength.

For excitation > 450nm, you can use glass filters, < 450nm use quartz or silica filters.

Plastic optical filters are unsatisfactory

Slide 32

Optical filters evaluation

Use a population of appropriately stained particles and identify which filters give the maximum signal.

Spectrofluorometer amd spectrophotometers can be used as tools for assessment of optical filters.

Slide 33

Issue to Note

Problems with filters are more likely due to using the wrong filters

Filters degrade overtime, so they have to be changed eventually

Buy high quality filters, not cheap ones

Slide 34

Hints on filters

To obtain acceptable blocking of the light outside the pass band, most interference filters incorporate some absorptive elements as well as dielectric layers

Slide 35

More hints .

You have to be careful while using short pass filters, specially with short wavelength, because of the transmission ability of these filters for long wavelengths (they behave like notch filters). If you have long red/near IR signals they will pass

Slide 36

In general

Use the least number of filters necessary to reduce signal loss

Absorption result in conversion of light into heat. Thus, laser beams hitting colour glass filters may destroy these filters .

Filters have a finite lifetime.

Slide 37

Practical consideration

In measuring weak fluorescence, we usually use field stop and interference filters behind the field stop to remove the stray light.

The shiny part (mirror side) of the filter should face the light source (collection lens)