CTAC / ICBR SOP : Setting the Emission Filter for a Luminescence or Fluorescence Scan using the IVIS Software Control Panel

 

Title: Setting the Emission Filter for a Luminescence or Fluorescence Scan using the IVIS Software (Living Image™) Control Panel

Materials Required:

  • Computer
  • Living Image™ software

Purpose:

To detail the use and selection of an emission filter on the software Control Panel to conduct a luminescence or fluorescence scan.

Software component identification:

IVIS Software Control Panel

The fluorescence or luminescence setting toggle is found in section D.

Section D luminescent Section D FLU

To conduct a fluorescent scan, the “Fluorescent” toggle in section D must be checked.

To conduct a luminescent scan, the “Luminescent” toggle in section D must be checked.

The scanning controls comprise the areas noted in GREEN.

Fluorescence Camera Controls

Emission Filter

The emission filter allows the peak energy being emitted from the subject to be isolated from wavelengths outwith the 20nm span of the filter.  This cuts down on any unwanted signaling “noise” or autofluorescence at other wavelengths in the spectrum (signal-to-noise), and increases the fidelity of the data acquired.

The emission filter is used in both fluorescent scans and luminescence scans, as the subject in both cases is emitting photons requiring detection.

The instrument’s Emission Filter setting is a drop down menu, which allows a number of settings.  These include “Open” (all wavelengths), “Block” (no light, used to assess any naturally emitting light if required), and 18 filters with bandwidths of 20 nanometers.

IVIS Emission Filters

The user will select the filter band most appropriate for capturing the most energy from the fluorophore of interest, dependent on its emission curve.  Selection should be made to avoid an emission range that overlaps with the fluorophore’s excitation curve.

CFP Emission plus Filters

The two curves for CFP (Cyan Fluorescent Protein) are shown, the excitation curve is to the left of the emission curve.  While there are 6 filters that cover the emission curve, the first and second are within the peak ranges of the emission curve.  The first filter on the left should be selected.

GFP Emission plus Filters

The two curves for GFP (Green Fluorescent Protein) are shown, the excitation curve is to the left of the emission curve.  While there are 5 filters that cover the emission curve  (from left to right), the first and second are within the peak ranges of the emission curve.  The third filter on the left should be selected. The filters to the right will have little available signal to capture.

DsRed Emission plus Filters

The two curves for DsRed (Discosoma Red Protein) are shown, the excitation curve is to the left of the emission curve.  While there are 6 filters that cover the emission curve  (from left to right), the first four are within the mid to high value ranges of the emission curve.  The fifth filter on the left could be selected, although is also covering part of the excitation curve. The sixth filter, while not capturing the peak energy, would be preferred. The filters to the right will have little available signal to capture.

YFP Emission plus Filters

The two curves for YFP (Yellow Fluorescent Protein) are shown, the excitation curve is to the left of the emission curve.  While there are 3 filters that cover the emission curve  (from left to right), the first and second are within the ranges of the excitation curve, and the third covers peak values of both the excitation and emission curves.  YFP is challenging as the peak values for both excitation and emission are very close. Although not capturing a peak value, the emission filter selected should be the fourth filter on the left. The filters to the right will have little available signal to capture.