Zeiss Lightsheet Z.1 Operating Manual

Contents

CHAPTER 4 - SYSTEM OPERATIONCarl Zeiss Left Tool Area and Hardware Control Tools Lightsheet Z.194 000000-1790-528 02/20133.3.7 Processing – Linear UnmixingLicensing Instrument standard optional  LSM ELYRA Lightsheet Z.1Tool accepts lambda stacks and multi-channel image data (single images and multi-dimensional image data). Linear Unmixing was developed for processing LSM data.Successful processing of multi-channel images obtained with other systems ispotentially possible as well (provided this input meets the requirements for unmixing).See full description of tool for details.The Linear Unmixing processing tool permitsextracting the emission of single fluorescence dyes(e.g. GFP only, YFP only etc.) from stronglyoverlapping multi-fluorescence data acquired inmulti-channel images and so-called "Lambdastacks" (only available in LSM imaging mode).In brief, with the knowledge of the spectralcharacteristic of individual components within amulti-component sample, even heavily overlappingindividual spectral characteristics can bemathematically extracted from an experimentalmulti-channel data. This method is a strictly pixel-by-pixel image analysis procedure.Experimentally, fluorescence spectra of mono-labeled samples are acquired and stored in theSpectra Database as an external reference. Then amulti-channel image or Lambda stack (availableonly in LSM imaging mode) from the multi-labeled sample is acquired.Finally, the individual components are mathematically extracted using the information from the referencespectra. Up to ten different reference signals can be fed into the least-square-fit based algorithm toproduce a 10-channel multi-fluorescence stack without any partial overlap between the channels.If no mono-labeled samples are available, the references can be obtained by the following methods:a) Interactively by user-selection of regions in the image where only one fluorescence dye is present(only available in the Unmixing View tab).b) Automatically by software analyses of what the individual spectral signatures are. This processingfunction is called "Multi-channel Unmixing" or "Automatic Component Extraction" (ACE). Note: insome cases, spectrally acquired images are not appropriate for ACE or "Multi-channel Unmixing"and linear unmixing can lead to wrong results.• To open the Linear unmixing tool, click on Linear unmixing in the Processing tool list under theProcessing tab (Fig. 140).Fig. 140 Processing – Linear unmixing