Optics - Filter Properties & manipulation of light in flow cytometry

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Slide 18

Fluorescein (FITC)

400 nm

500 nm

600 nm

700 nm

Wavelength

Protein

Excitation

Emission

300 nm 400 nm 500 nm 600 nm 700 nm

Slide 19

Interference filters advantages

They can be used as reflectors in two and three color analysis.

They usually do not themselves produce fluorescence.

They are available in short pass versions.

They are excellent as primary barrier filters.

Slide 20

Interference filters: disadvantages

Lower blocking properties

Reduced passing properties

Their reflecting and passing properties are not absolute, this should be considered while dealing with multiple wavelengths

Slide 21

Absorbance filters: advantages

They are inexpensive.

They have very good blocking properties.

They have very good transmission properties.

Slide 22

Absorbance filters: disadvantages

They can only pass long wavelengths

( hence, can only block short wavelength)

Since they are made of solution of dye and glass, they can themselves produce fluorescence.

Slide 23

Neutral density filters (N.D)

Attenuation of the light without discrimination of the wavelength.

N.D filters could be reflective or absorptive type.

They are partially silvered mirrors.

Slide 24

Beam splitters

Absorptive N.D filters can not be used here; simply because of the heat, they will melt.

Common cover slips can be used as beamsplitters if small portion of the light is wanted, up to 5%

Slide 25

Measuring Filter Properties

Filters must be measured at the angle they are going to be used

filters placed at 90o have different properties when they are placed at 45o

Slide 26

Short pass and long pass filters

T

R

A

N

S

M

I

S

S

I

O

N

WAVELENGTH

SP filter

LP filter

cutoff

cuton

T max

T max

Slide 27

Optical filter evaluation

Slide 28