TPC-Program for the automated Identification and Classification of stained biological Particles in Microscopic Images
Function of the “Pollen Classification Systems”:
The program operates in three steps: The digital images of microscopical maginficated aroprot-stained biological particles (microorganisms) are transformed to a fix format of 1028 x 1024 pixel and the colours are converted into inverse grey values. A routine called “Segmentierer” scans these processed pictures for regions, that correspond in size and colour intensity to the particles that are searched. Numerous parameters of the selected image segments are analysed and classified according to defined criteria. This way it is possible to identify particles for example as distinct kinds of pollen. The intensity of the staining allows to additionally evaluate the physiological state of these pollen. The different Analyse-Modi include the various settings of the “Segmentierer” and the criteria which have been chosen according to the size and the staining pattern of the particles, the required magnification and the quality of the images used.

On this item the program can be adapted to the Country-Version of your Windows System, if not recognized automatically by the system as German or English. This is important regarding the decimal point used in values.
1. Opening Screen - The Singe File Modus
1.1 Index of Program Components
Additionally to the picture analysis several additional functions are implanted
1.2 Load Image: Digital picture in format .bmp, .jpg, .tif, or .tga are loaded from a directory: resolution is calculated to 1280 x 1024 pixel, full size about 3,5 Mb.
1.3 Previes Screen: The loaded picture is show in black and white inverted. After analysis the segments are indicated by coloured circles. The colours correspond to the mode used: Analyse 1 – blue, Analyse 2 – yellow, Analyse 3 - green
1.4 Analyse: According to the kind of the particles (expected pollen, yeasts) the mode Analyse 1, Analyse 2 or Analyse 3 will be chosen and the operation of program started.
1.5 Display of Analysis Mode applied and Picture Loaded
1.6 Statistic graph of particle colour intensity or their number in given classes
1.7 Save Image of the Preview Screen and Annotate segment numbers therein
1.8 Display of Analyze Operation: The proceeding of the image analysis is indicated by a bar and the cursor changes to the “wait mode”.
1.9 Select Class: The indicated number of segments correlated to each class can be chosen for separate indication on the Preview Screen.
1.10 Output: All operations during a program run are listed here. The picture name with complete path is shown The value of the average light of the image- before and after normation for full scale black to white is given. For proper analysis it should be more than 200.
The loaded set of parameters for analysis is registered after pushing the appropriate button.
Finishing the analysis, the coordinates, the sequential number and the classification of the selected segments are listed, followed by a summary of segments per each class.
1.11 Save Output - Clear Output: The output can be saved as a txt-file. To generate a separate output file for an analysis, the previous data can be discarded.
1.12 Exit Program - Full Screen/Windows Screen:
Closing the program - Widening the frame to full display or leaving the bottom bar available.
2. Sequential Image Analysis

2.1 Multi File Item: Several images can be selected to be sequentially loaded and analysed by the PCS program.
2.2 Choose directory: From a distinct directory images can be selected for analysis.
2.3 Image type to Select: Only images of distinct format(s) are analysed
2.4 Choose Analyse: According to the kind of expected particles - like in the Single File Modus - the Analyse 1, Analyse 2 or Analyse 3 with its appropriate segment size and parameter classification is activated and applied to all images selected.
2.5 Start Program: Press the “Start” button and the progress of the program run is indicated on the Single File frame.
2.6 Activate Images for Analysis: By double click the control hook of an image can be inactivated or reset.
Right mouse click gives access to Select all, Select no or Invert Selection.
3. Statistic Diagrams:

3.1 The button „Refresh statistic (Intensity) and gives a new diagram of all segments according to their Average Intensity after scale of x-axis has be modified.
3.2 „ Refresh statistic (Class)“ produces a new graph of the distribution of the segments to the defined classes.
3.3 Clear all bars to start with a diagram of one data set.
4. Table of Results

Results summarizes in a table the values associated with the segments from the pictures of a complete program run.
It contains the name of the image plus number of the segment, its position within the image, the analyse mode used and the final classification.
The properties of the conture of each single segment follow: The parameters are perimeter, area, area per perimeter (about half radius), deviation from circle, average curving and average intensity
4.1 Save Results: These table may be stored as a CSV file.
4.2 To generate a separate table, the content can be removed by the Clear button.
5. File Manager to administer the images take from the different slides
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