Zeiss LSM 510 Meta Confocal and Zeiss LSM 510 Meta Offline Workstation
Spectral analysis
Using the meta detector and the lambda acquisition mode, you can collect the fluorescent emission in a lambda stack that sorts the emission output into a series of small wavelength bands. After defining a ROI (region of interest within the sample), a graph plotting the pixel intensity versus the mean wavelength of each band is generated. This graph can then be compared to standard reference spectra (from emission fingerprinting) to determine if the emission matches the desired fluorophore or represents non-specific emission.
 | Rat retinal wholemount. Retinal ganglion cells were infected with rAAV/GFP by intravitreal injection. GFP expression is evaluated using the lambda acquisition mode and the meta detector. Note that the cell on the right is visible in the first few frames with maximum emission at about 520nm (white arrowhead). The cell on the left has emission at every wavelength, which is indicative of autofluorescence (red arrow). Interestingly, these cells both appeared green by wide field microscopy and were similarly detectable using the 488nm laser on a regular PMT, demonstrating the importance of evaluating the spectral characteristics of the cells of interest. Image courtesy of Mary Ellen Pease and Harry Quigley. |
 | Rat retinal wholemount, same field. Lambda analysis allows one to select a region(s) of interest in a lambda stack to perform a spectral analysis. In this field, the emission of the background and the other two cells present is graphed. Note the classic GFP emission signature from the cell on the right (ROI 1) and the broad, non-specific emission of the cell on the left (ROI 2). |
