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