![]() There are a number of tools and functions to help you. Now start optimizing the Cell- and Signal properties (6) to quantify the number of PLA signals and nuclei in your selected image. Select one of the images in the database and push the Preview button (5). Image data where different channels and z-slices are stored in separate tiff-files can be imported with the Special TIFF import (2). jpg color images, containing both the signal and nuclei channel, are also imported with this function. Import the original microscope file formats if available. Start by importing the images to be analyzed (1). Furthermore, the results overlay can be added to the exported images (12). Images can be exported with split color channels or as RGB images. The Single_cell_results file contains the total number of signals per cell, and the Summary_of_results file contains the total number of signals and nuclei for each analyzed image. The number of detected PLA signals and nuclei is exported into two text files. All images in the project database will be analyzed with the selected settings.įollow the progress of the analysis on the progress bars (3).Ĭlick the Export result button to export the analyzed data into text files that can be opened and further analyzed with Excel (11). When the outlines and white circles correspond well to the content of the original microscope image, push Analyze (10). The green outlines represent the user defined cytoplasm size. PLA signals marked with white circles and nuclei outlined in yellow are quantified at analysis. Push Preview (9) to refresh and get a new correct preview of the result overlay. The complete list of functions can be found in the Help file under the Help menu.Įach time a setting is changed that is not automatically visualized on the computer screen the Preview- and Analyze buttons will be highlighted in pink. Now start optimizing the Cell- and Signal properties (8) to quantify the number of PLA signals and nuclei in your selected image. Select one of the images in the database and push the Preview button (7). Images marked with “OK” do not contain merged signals. Duolink ImageTool can not correctly quantify regions of merged signals, only separable signals are quantified. These images should be visually verified for analytical quality. Images containing merged signal regions will be marked by an “X”. Image data where different channels and z-slices are stored in separate tiff-files can be imported with the Special TIFF import (2).įollow the import progress on the progress bars (3).Ī project database is created and all images imported to the database are listed (4).Ĭhange the channel properties for the signal- and nuclei channels if they are not correctly defined (5).Ĭheck image quality (6). Start by importing the images to be analyzed. Cell detection is easily verified by visual inspection of the cell outlines. If nuclei are densely packed or tissue sections are thick, single cell measurements may be inaccurate. For images taken with fluorescence microscopy, a quality control function displays a warning if signals are merged. ![]() Duolink ImageTool can not quantify regions of merged signals only separable signals can be counted. If the primary antibodies are not properly titrated, the density of signals may be so high that the signals merge. However, you may acquire images with a single focal plane as long as all images to be compared are acquired with similar focus within the sample. To detect all PLA signals it is thus necessary to acquire images at several focal planes through the thickness of the sample. If images are acquired in a single focal plane, several signals can be either above or below the current focus. The result from a Duolink experiment is typically a number of discrete PLA signals in various locations of the studied cell- or tissue samples. The PLA signal is recognized as a spot an individual signal is of sub-micrometer size.
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