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- When using laser light sources, filters must have very sharp cutons and
cutoffs since there will be many orders of magnitude more scattered
laser light than fluorescence
- Can specify wavelengths that filter must reject to certain tolerance
(e.g., reject 488 nm light at 10-6 level: only 0.0001% of
incident light at 488 nm gets through)
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- This lecture is intended to describe the nature and function of optical
systems
- It will describe how filters operate
- When filters should be used
- What problems and issues must be taken into consideration
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- Long pass filters transmit wavelengths above a cut-on wavelength
- Short pass filters transmit wavelengths below a cut-off wavelength
- Band pass filters transmit wavelengths in a narrow range around a
specified wavelength
- Band width can be specified
- Neutral Density filter is a nondiscriminant intensity reducing filter
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- When a filter is placed at a 45o angle to a light source,
light which would have been transmitted by that filter is still
transmitted but light that would have been blocked is reflected (at a 90o
angle)
- Used this way, a filter is called a dichroic filter or dichroic mirror
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- Diffraction essentially describes a departure from theoretical geometric
optics
- Thus a sharp objet casts an alternating shadow of light and dark
“patterns” because of interference
- Diffraction is the component that limits resolution
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- Small amounts of incident light are reflected at the interface between two material of different RI
- Thickness of the material will alter the constructive or destructive
interference patterns - increasing or decreasing certain wavelengths
- Optical filters can thus be created that “interfere” with the normal
transmission of light
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- Interference filters: Dichroic, Dielectric, reflective
filters…….reflect the unwanted wavelengths
- Absorptive filters: Colour glass filters…..absorb the unwanted
wavelengths
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- They are composed of transparent glass or quartz substrate on which
multiple thin layers of dielectric material, sometimes separated by
spacer layers .
- Permit great selectivity.
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- They used to direct light in different spectral region to different
detectors.
- They are interference filters , long pass or short pass.
- "dichroic" Di- is Greek for two, and -chroic is Greek for
color - from Greek dikhroos, bicolored
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- Constructive and destructive interference occurs between reflections
from various layers
- Transmission determined by :
- thickness of the dielectric layers
- number of these layers
- angle of incidence light on the filters
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- Such as coloured glass filters which absorb unwanted light.
- Consist of dye molecules uniformly suspended in glass or plastic.
- Remove much more of the unwanted light than do the interference filters
- Will often fluoresce (not good!)
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- Bandpass filters: characterized by there
- T max and (the Full Width at Half Maximum) FWHM
- Notch filters are band pass filters in the upside down position
- Long pass and Short pass filters: characterized by their T max and
cuton, cutoff wavelength.
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- 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.
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- Lower blocking properties
- Reduced passing properties
- Their reflecting and passing properties are not absolute, this should be
considered while dealing with multiple wavelengths
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- They are inexpensive.
- They have very good blocking properties.
- They have very good transmission properties.
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- 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.
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- Attenuation of the light without discrimination of the wavelength.
- N.D filters could be reflective or absorptive type.
- They are partially silvered mirrors.
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- 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%
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- 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
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- 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)
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- The thicker the glass the less light transmitted.
- Problems with glass - UV light will not pass
- In UV light system use minimum optics.
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- 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
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- 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.
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- 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
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- To obtain acceptable blocking of the light outside the pass band, most
interference filters incorporate some absorptive elements as well as
dielectric layers
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- 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
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- 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.
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- 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)
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- Fiber optics and other optical waveguides operate by total internal
reflection
- problems with stray light, low NA of fibers, thus low sensitivity, light
collection difficult
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- At the conclusion of this lecture the student should understand:
- Field stops and obscuration bars are necessary in systems where air or
round capillaries are used
- Appropriate optical filters must be placed in combinations
- Filters degrade over time and should be checked
- The least number of filters should be used in a system
- Forward angle scatter is frequently collected using a diode detector
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