1. Filter Set Selection

2. It is best to follow this presentation with a Fluorophore excitation/emission maxima chart to hand.

3. Preliminary The MRC series has two dichroics They are numbered 1 and 2

4. 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

5. 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.

6. 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

7. 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.

8. To select the Correct Filter Set First identify the fluorescent dye used in your sample

9. Spectra The excitation and emission wavelengths of fluorophores are not single wavelengths They have spectra

10. An example. Imaging CY3

11. 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

12. An example. Imaging CY3 To successfully image CY3, first look to the left hand side of the chart. Locate CY3 on the chart.

13. Excitation Filter Selection This shows the excitation maximum

14. Excitation Filter Selection Identify the laser line which is closest to and (if possible) has a shorter wavelength than the dye excitation maximum.

15. Excitation Filter Selection Project the chosen laser line horizontally across the chart to the RIGHT hand side

16. Excitation Filter Selection Note the dichroic number 1 it intercepts. This determines which laser excitation filter and dichroic number 1 are best

17. 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

18. Emission Filter Selection Continue the line horizontally to the right of the chart Note the emission filter and the dichroic number 2 it intercepts

19. Emission Filter Selection Ensure the emission filter is NOT intercepted by the chosen laser line

20. 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

21. Conclusion Once these conditions are met, you have successfully chosen the best filter set for your sample