filter set selection
DESCRIPTION
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 PresentationTRANSCRIPT
![Page 1: Filter Set Selection](https://reader031.vdocuments.us/reader031/viewer/2022032207/56814198550346895dad7c94/html5/thumbnails/1.jpg)
Filter Set Selection
![Page 2: Filter Set Selection](https://reader031.vdocuments.us/reader031/viewer/2022032207/56814198550346895dad7c94/html5/thumbnails/2.jpg)
Filter Set Selection
• It is best to follow this presentation with a Fluorophore excitation/emission maxima chart to hand.
![Page 3: Filter Set Selection](https://reader031.vdocuments.us/reader031/viewer/2022032207/56814198550346895dad7c94/html5/thumbnails/3.jpg)
Preliminary
• The MRC series has two dichroics• They are numbered 1 and 2
![Page 4: Filter Set Selection](https://reader031.vdocuments.us/reader031/viewer/2022032207/56814198550346895dad7c94/html5/thumbnails/4.jpg)
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
![Page 5: Filter Set Selection](https://reader031.vdocuments.us/reader031/viewer/2022032207/56814198550346895dad7c94/html5/thumbnails/5.jpg)
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.
![Page 6: Filter Set Selection](https://reader031.vdocuments.us/reader031/viewer/2022032207/56814198550346895dad7c94/html5/thumbnails/6.jpg)
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
![Page 7: Filter Set Selection](https://reader031.vdocuments.us/reader031/viewer/2022032207/56814198550346895dad7c94/html5/thumbnails/7.jpg)
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.
![Page 8: Filter Set Selection](https://reader031.vdocuments.us/reader031/viewer/2022032207/56814198550346895dad7c94/html5/thumbnails/8.jpg)
To select the Correct Filter Set
• First identify the fluorescent dye used in your sample
![Page 9: Filter Set Selection](https://reader031.vdocuments.us/reader031/viewer/2022032207/56814198550346895dad7c94/html5/thumbnails/9.jpg)
Spectra
• The excitation and emission wavelengths of fluorophores are not single wavelengths
• They have spectra
![Page 10: Filter Set Selection](https://reader031.vdocuments.us/reader031/viewer/2022032207/56814198550346895dad7c94/html5/thumbnails/10.jpg)
An example. Imaging CY3
![Page 11: Filter Set Selection](https://reader031.vdocuments.us/reader031/viewer/2022032207/56814198550346895dad7c94/html5/thumbnails/11.jpg)
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
![Page 12: Filter Set Selection](https://reader031.vdocuments.us/reader031/viewer/2022032207/56814198550346895dad7c94/html5/thumbnails/12.jpg)
An example. Imaging CY3• To successfully image CY3, first
look to the left hand side of the chart.
• Locate CY3 on the chart.
![Page 13: Filter Set Selection](https://reader031.vdocuments.us/reader031/viewer/2022032207/56814198550346895dad7c94/html5/thumbnails/13.jpg)
Excitation Filter Selection
• This shows the excitation maximum
![Page 14: Filter Set Selection](https://reader031.vdocuments.us/reader031/viewer/2022032207/56814198550346895dad7c94/html5/thumbnails/14.jpg)
Excitation Filter Selection
• Identify the laser line which is closest to and (if possible) has a shorter wavelength than the dye excitation maximum.
![Page 15: Filter Set Selection](https://reader031.vdocuments.us/reader031/viewer/2022032207/56814198550346895dad7c94/html5/thumbnails/15.jpg)
Excitation Filter Selection• Project the chosen laser line
horizontally across the chart to the RIGHT hand side
![Page 16: Filter Set Selection](https://reader031.vdocuments.us/reader031/viewer/2022032207/56814198550346895dad7c94/html5/thumbnails/16.jpg)
Excitation Filter Selection• Note the dichroic number 1 it
intercepts.• This determines which laser
excitation filter and dichroic number 1 are best
![Page 17: Filter Set Selection](https://reader031.vdocuments.us/reader031/viewer/2022032207/56814198550346895dad7c94/html5/thumbnails/17.jpg)
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
![Page 18: Filter Set Selection](https://reader031.vdocuments.us/reader031/viewer/2022032207/56814198550346895dad7c94/html5/thumbnails/18.jpg)
Emission Filter Selection
• Continue the line horizontally to the right of the chart
• Note the emission filter and the dichroic number 2 it intercepts
![Page 19: Filter Set Selection](https://reader031.vdocuments.us/reader031/viewer/2022032207/56814198550346895dad7c94/html5/thumbnails/19.jpg)
Emission Filter Selection
• Ensure the emission filter is NOT intercepted by the chosen laser line
![Page 20: Filter Set Selection](https://reader031.vdocuments.us/reader031/viewer/2022032207/56814198550346895dad7c94/html5/thumbnails/20.jpg)
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
![Page 21: Filter Set Selection](https://reader031.vdocuments.us/reader031/viewer/2022032207/56814198550346895dad7c94/html5/thumbnails/21.jpg)
Conclusion
• Once these conditions are met, you have successfully chosen the best filter set for your sample