ScanG16-example: ---------------- A few images take at 2400dpi resolution with an Epson 4870 scanner and microplate covers as cell plankton holders. The samples were digitized using the pro version of Vuescan with all parameters set in a way that RAW scanner signal is recorder in the images (no gamma correction, nor any other possible transformation of the original pictures; see the \configs subdirectory of the Vuescan version installed with Zoo/PhytoImage: \bin\Vuescan\configs). To load a configuration in Vuescan, use the menu File -> Load options... The Epson scanners 4870/4990 use a special lighting unit and have the technical particularity to change the focusing point 1 to 1.5 mm above the scanner glass when they are used in transparency mode (this feature was designed by the Epson engineers to allow putting the negative film or slides well above the scan glass to avoid Newton annealing effect when such a media is pressed on the glass). Here, this allows us to use a Petri dish on top of the scanner glass and still got the focusing point at a correct location. After various trials, we noted that transparent covers used with microplates (like those used in microbiology for microplate robot analyzers) are particularly suitables. They are made in transparent polystyren, a very clear material, but also rather fragile. You should replace these digitizing cells very often. A quick note: take care of safety rules at work and patents that could prohibit you to use a commercial scanner to digitize biological samples in seawater (the scanner is not waterproof; a dedicated device exists for such a task: the Zooscan, see http://www.zooscan.com). Images provided are two scans of a single sample of neretic plankton out off Tulear, Madagascar. In order to increase the number of large particles scanned, the sample is first fractionated, using a 500µm mesh (fraction A contains the larger particles retained by the mesh, and fraction B contains the smaller particles). Dilution of both fractions is different ('SubPart' field in the 'Import_Scan16G.zie'). For fraction A, 34% of all the large organisms in the sample was used, and only 14.6% for fraction B. Subsampling was done by volumetry method, see 'SubMethod' field). Finally, the scans contain 73% ('CellPart=0.73') of the particles in the subsamples, that is, 27% of the particles are outside the image area and/or are touching its border, and are eliminated from the analysis. This value is determined by scanning the whole area of the cell on a few dozen of images, by editing these images with Photoshop to eliminate the edge of the cell on the picture and by finally comparing countings on the whole, edited, cell area to the countings on a version cropped at final 10000x6000 dimension in the center of the cell. In the present case, there are no replicate images for the sample ('Replicates=1') and 2 +/- 0.2 m^3 of seawater was sampled by the net ('VolIni=2' and 'VolPrec=0.2') All the entries in the '[Subsample]' section of the metadata are important to allow quantitative determination of abundances, biomasses or size spectra per volume of seawater. You should first inspect the 'Import_Scan16G.zie' file (.zie stands for Zoo/PhytoImage Extensions). This file specifies all the information required to 'import' images (here, in the present case, there is not really an "importation" of the images, since the initial format is directly usable. There is just a compilation of .zim files according to data provided in the 'Import_ScanG16.zie' specifications). The key section in the 'Import_ScanG16.zie' file is the '[Map]'. This section contains all the instructions to sequencially import many images and build their respective .zim metadata files. The whole content of the 'Import_ScanG16.zie' file, except the '[Map]' section is a template for each .zim file. Each line of the '[Map]' section contains an instruction, which is one of two types: 1) A line starting with '->' changes the value of the corresponding metadata entry in the template. For instance, "->SubPart=0.340" change the value of the 'SubPart' entry to 0.340 for all subsequents .zim files to be created. 2) The name of an original file, followed by an equal sign and the number, or number.subnumber of the image in the sequence for the given sample. Note that, to avoid redondancy, it is possible to define a common pattern for the name of the initial file (entry 'FilenamePattern' in the'[Import]' section. The pattern represents the common string for all images, and with '<>' indicating where the variable part is located in this string. For instance, if all your images are Scan_XXXX.tif, it is possible to specify 'FilenamePattern=Scan_<>.tif', and you must only place the corresponding XXXX in the '[Map]' entry. For instance, '2007-06-03.a=1' if the variable part is '2007-06-03.a' in the name of the image. Use '' for the placeholder to specify a sequence of numbers with leading zeros. Use, for instance '<4>' for a sequence of '0001', '0002', '0003', ..., '0010', ..., '0100', ..., '1000', ..., etc. (four digits), otherwise the sofware will compile a sequence without leading zeros like: '1', '2', '3', ..., '10', ..., '100', ..., '1000', ... Note also the possibility to define different sections for '[Fraction]' and '[Subsample]'. In the present case, 'Import_ScanG16.zie' uses this possibility to define different values for '[Fraction]', depending if it is fraction A or B. The mechanism is the following one: 1) Create a pattern that defines how to extract the fraction identification from the name of the image. This pattern follows special conventions named "regular expressions". If you don't know regular expressions, just use the one provided in the examples and take care to follow similar conventions. In the present case, the expression "^.*[+]([A|B$]).*$" extracts 'A' or 'B' from the name of the image. Depending on this value, the final section '[Fraction]' introduced in the corresponding .zim file will be the one defined under '[Fraction_A]' or '[Fraction_B]', respectively, in the template. 2) Create a different template for each separate fraction or subsample, by naming them '[Fraction_XXXX]' or '[Subsample_XXXX]'. An alternate possibility is to provide the variable part of the metadata under the form of a table. For a large number of samples to import at once, it is probably more convenient. See 'MacroG16-example', for instance. To run this example: -------------------- - Download and unzip "ScanG16-example.zip" in your 'ZooPhytoImage Examples' directory, or anywhere you like to place it. - Start Zoo/PhytoImage and import the data (second button, select 'Zooimage import extensions' file type at the bottom of the dialog box and then, select the 'Import_ScanG16.zie' file). The import mechanism compiles the .zim files (metadata) for each fraction of the sample. - Once files are ready, they can be analyzed (third button, switch to ImageJ). In ImageJ, select (Plugins -> ZooPhytoImage -> Scanner Gray16). Select one of the .zim files you just created and watch the analysis live on screen. You can examine the mask and other temporary files in the '_work' subdirectory. - Once the analysis is done, you can close ImageJ and switch back to the Zoo/PhytoImage assistant. Click on the fourth button to finalize the .zid file corresponding to this analysis. You should end up with one .zid file (Zoo/PhytoImage Data) plus all the original files moved to the '_raw' subdirectory. What next...: ------------- The .zid files are key files in Zoo/PhytoImage. They contain everything you need for manual and automatic identification of the particles and for the calculation of summary statistics (abundances, biomasses, size spectra, etc.). At this point, you can archive (on DVD, external hard disks, etc.) the original data that are now in the '_raw' subdirectory to free space on your machine, and safely work with the .zid files only for the rest of your analyses. With only one sample, you cannot do much more, but you are supposed to collect more data, then to train a classifier, check its performances and finally use it to identify particles in your samples. For examples with more data, switch to the 'ScanG16-train&data' example.