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Filter Set Selection. Filter Set Selection. It is best to follow this presentation with a Fluorophore excitation/emission maxima chart to hand. Preliminary. The MRC series has two dichroics They are numbered 1 and 2. Preliminary. Dichroic number 1 is to the rear of the scan head - PowerPoint PPT Presentation
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Filter Set Selection
Filter Set Selection
• It is best to follow this presentation with a Fluorophore excitation/emission maxima chart to hand.
Preliminary
• The MRC series has two dichroics• They are numbered 1 and 2
Preliminary• Dichroic number 1 is to the rear of
the scan head• It filters the excitation beam• They are identified with names
such as K1 or I1 etc. Also VHS/BHS
Preliminary• Dichroic number 2 is to the front of
the scan head• It filters the emissions from the
sample• It is identified with a name such as
K2 or I2 etc.
The MRC1000/1024
• The MRC1000/1024 has extra filters• They have excitation filters and
emission filters as well as dichroics• The dichroics are changed manually• The extra filters are computer
controlled
The Radiance
• The Radiance has similar filters to the MRC1000/1024 series
• They are all computer controlled.• No manual filter changes are
necessary• Instead of changing dichroics,
dichroic filter wheels are rotated by computer control.
To select the Correct Filter Set
• First identify the fluorescent dye used in your sample
Spectra
• The excitation and emission wavelengths of fluorophores are not single wavelengths
• They have spectra
An example. Imaging CY3
An example. Imaging CY3• From the spectra, the useful excitation
and emission ranges are seen to be:
• Excitation = 470 - 580 nm • maximum at 550 nm
• Emission = 540 - 660 nm• maximum at 565 nm
An example. Imaging CY3• To successfully image CY3, first
look to the left hand side of the chart.
• Locate CY3 on the chart.
Excitation Filter Selection
• This shows the excitation maximum
Excitation Filter Selection
• Identify the laser line which is closest to and (if possible) has a shorter wavelength than the dye excitation maximum.
Excitation Filter Selection• Project the chosen laser line
horizontally across the chart to the RIGHT hand side
Excitation Filter Selection• Note the dichroic number 1 it
intercepts.• This determines which laser
excitation filter and dichroic number 1 are best
Emission Filter Selection
• From the excitation maximum, follow the solid white line on the chart from left to right
• This will identify your emission maximum
Emission Filter Selection
• Continue the line horizontally to the right of the chart
• Note the emission filter and the dichroic number 2 it intercepts
Emission Filter Selection
• Ensure the emission filter is NOT intercepted by the chosen laser line
Emission Filter Selection
• If it is, you must choose an emission filter which passes longer wavelengths
• Or a laser line which has a shorter wavelength
• Or both
Conclusion
• Once these conditions are met, you have successfully chosen the best filter set for your sample