Paulo's Microscope & Data tips

  The issue : When scanning an area of tissue with the confocal microscope, many users decide to use the tiling (aka mosaic) option. This can sometimes lead to a visible and sometimes pronounced tiling artifact (aka "football field effect", due to the appearance of visible routed lines in the image) in one or more channels. In other occasions, these artifacts are not easily visible in each individual plane of the z-stack, but it becomes strong when performing a max intensity projection, like in the example in the figures under. The main reason for tiling artifacts is uneven illumination of the field of view. I promise to write a longer post about the reasons later. The main issue of the present post is to find ways to prevent or correct tiling artifacts. There are many strategies to avoid or correct tiling artifacts, and the one mentioned in this post is just one of them.   Fig. 1: example of a single plane (plane 35) in a 59 plane z-stack, tiled 3x5. Notice that it is al...

 The issue : in some of my code writing process, sometimes it is useful to see the naked content of a proprietary binary file in order to get an overview of how that file format is structured. I therefore want an easy and fast way to take a look at it.  Solution : The most recent versions of Windows (7 to 10) have easy solutions to do it in the PowerShell. You can read how to do it in the following link here . These solutions can also produce a text file (txt) with the hexadecimal translation side-by-side with an ASCII interpretation. 

 The issue : I have been writing some scripts in Matlab that permit the direct import of the contour and marker data from the delineations users draw with Neurolucida 360 software. For that purpose, I have been using the option of saving the Neurolucida delineation files in the XML format. Neurolucida 360 permits the user to save their data in several different formats, among them the native binary format (DAT), a standard XML format and an ASC (ASCII type) format. I use XML format because of good documentation (links here and here ) provided by MBF. However, Neurolucida 360 saves by default the tracing data in their native binary (DAT) format, so most users will (un)consciously primarily save their data in that format. MBF does not give the key for deciphering this format directly, but attempts were made to reverse engineer the key (link here  and here ). There is also code available for converting between different tracing formats ( NLMorphologyConverter ). Unfortunately, t...

  The issue: I often write scripts in Matlab to batch image processing tasks, and for that purpose I need to load confocal or axioscan Zeiss czi format image files. I use an open source package called Bio-formats . However, I frequently get two kinds of problems when using this library in Matlab: a) working memory problems (Matlabs Java memory capacity gets filled when the files are in the order of the GB size); and b) stitching problems. This post will only focus on the second issue (I will write later a separate post for the first). The problem is that, for a long time, when I tried to open czi image files of confocal images that were tiled, the bio-formats pack would open the tiles individually, but it would not stitch them as a single image.     Diagnose procedure:  I tried to download different versions of the bio-formats library, because according to internet forums like forum.image.sc , the latest version of bio-formats had that problem fixed. The v...

  The issue : One of the regular users of Neurolucida 360 has noticed that some image files produced by the Zeiss LSM 880 (confocal microscope using ZEN black software) do not open. This happens rarely, but she had five examples of medium sized (tens to hundreds of MB sized, mosaics with multiple z-levels) files that NL360 could not open.  Diagnose procedure : I tried to open the same files in Fiji and by using Matlabs bformats pack (v7.0.0) and in both cases, I got error messages concerning corrupted data bytes. Nevertheless it was possible to open those files in ZEN blue lite version. That made me hypothesize that there are a few corrupted data bytes in the file, but that ZEN lite can bypass those data points. That lead me to the think that if I tried to save those files again using the "Save as" menu, the files would be saved with the corrected bytes version, and that would make the files whole again. Upon testing this procedure, the files were fixed and could open normal...