Zeiss Lightsheet Z.1 Operating Manual

Contents

CHAPTER 4 - SYSTEM OPERATIONCarl Zeiss Left Tool Area and Hardware Control Tools Lightsheet Z.1144 000000-1790-528 02/2013Fast Iterative: The "Fast Iterative" method is an iterative restoration method that uses only one iterationper convolution step (see Meinel, E.S.: Origins of linear and nonlinear recursive restoration algorithms. J.Opt. Soc. Am, 3 (&), 1986, 787-799). No regularization is used in this case. Due to the fast processingand convergence after just a few iterations, this method is suitable in particular for the processing oflarger time lapse images. The results of the method can quickly lead to good results and remove most ofthe out-of-focus light. They do not create quantitative brightness conditions in the image. If under-sampled images are present, artifact formation may also result.Constrained Iterative: The best image quality is achieved using the iterative maximum likelihoodalgorithm (see Schaefer et al.: "Generalized approach for accelerated maximum likelihood based imagerestoration applied to three-dimensional fluorescence microscopy", J. of Microscopy, Vo. 204, Pt2,November 2001, pp. 99ff.): This algorithm is able to calculate light from various focal planes back to itsplace of origin. Consequently, with this method it is possible to derive the 3D structure from fluorescenceimages with the correct brightness distribution and to visualize optical sections. It is also possible formissing information to be partially restored from neighboring voxels. The spatial resolution can beincreased without artifacts up to a theoretical limit (one voxel). It is essential for Z-Stacks to have beenacquired in accordance with Nyquist. Acquiring sufficient planes above and below the structure ofinterest is also imperative for achieving good results. As this is a complex mathematical method, thecalculation can take longer, depending on the image size.If the checkbox Enable channel selection isactivated, the settings are applied on a channel-specific basis. This allows you to set parameters ina targeted way for each channel. You will see aseparate colored tab for each of the channels(Fig. 231).In the Normalization drop-down list, you canspecify how the data from the resulting image arehandled:Clip: For non-registered and non-fused multiviewimages, this should be used as the default setting.It sets negative values to 0 (black). If the valuesexceed the maximum possible gray value of 65636when the calculation is performed, they are limitedto 65636 (pixel is 100% white).Automatic: Normalizes the output imageautomatically. Not to be used for non-registeredand non-fused multiview images.In this case, the lowest value is 0 and the highestvalue is the maximum possible gray value in theimage (gray value of 65636). The maximumavailable gray value range is always utilized fully inthe resulting imageFig. 231 Processing – Lightsheet Processing.Multiview Processing, Deconvolution,Enable channel selection