What is Emission Fingerprinting?
Emission Fingerprinting is an innovative method for the recording, analysis and separation of emission signals in multifluorescence imaging. Consisting of a 3-step-procedure, it permits to separate even fluorochomes with widely overlapping emission spectra such as the fluorescent proteins CFP, GFP and YFP. Thus, Emission Fingerprinting greatly enhances the the number and choice of fluorescent dyes that may be used in a multi-fluorescence experiment. Dyes that were so far impossible to identify in multi-fluorescence experiments are now separated with the click of a button.
Step 1: Acquire Complete Fluorescence Emission with Lambda Stacks
First, acquire a Lambda Stack to record the spectral signature of your specimen.
As these spectrally resolved images are recorded simultaneously, this step is completed in minimum time, which not only is friendly to your delicate specimens but also reliably captures fast dynamic processes.
Step 2: Determine Reference Spectra with Mean-of-ROI
Next, open the Unmixing View of the image to determine the reference spectra either with drawing tools or using the Automatic Component Extraction function. The resulting spectra then can be stored for further experiments in the Spectra Database of the LSM 710.
Step 3: Perform Linear Unmixing
Finally, the Linear Unmixing function separates the mixed signals pixel by pixel by means of intelligent algorithms, using the entire emission spectrum of each of the fluorescent markers in your specimen.
As a result, even greatly overlapping emission spectra, as e.g. those of GFP and FITC, are safely separated; broadband autofluorescence is reliably eliminated. New experimental approches become possible, strongly auto-fluorescent samples can now also be used for fluorescent labelling.