Software-Based Improvement of Image Information
and 4) was selected for further processing. Tis single throm- bocyte was cropped out again and presented in an 80×80 pixel square showing a 16.8×16.8 μm area. It is clear that fine details were lost in this cropped image (Figure 5a). By zooming and re- interpolation with Photozoom Pro (S-spline algorithm) followed by successive focus stacking (carried out with Picolay and Com- bine) fundamental improvements of image quality were achieved so that all the pseudopodia can be clearly seen (Figure 5b). Con- tours can be further accentuated by the Sobel operation imple- mented in the deconvolution soſtware Fitswork (Figure 5c). Different morphological types of pseudopodia could be
Figure 3: Activated thrombocytes taken from a platelet concentrate on a coverslip preparation. Images collected with an oil immersion 100×/1.32- 0.60, dark-field objective and extracted from a video clip. Specimen area is 140×103 μm; size of the original image: 667×491 pixels, arrow: thrombocyte of interest (photograph taken by Dr. M. J. Kraus, University of Koblenz, Germany). Image width=140 μm.
images resulting from this zooming are super- imposed to a focus stack. In the case that this stack is affected with over- and under-exposed zones, duplicates may be made based on differ- ent brightness in order to be rendered with HDR techniques. The resulting image reconstruction can be rendered further with the Sobel operator and other standard techniques if necessary.
Variant 2: All single-shot images selected for focus stacking are firstly superimposed to a focus stack. Te resulting image characterized by optimized distinctness is zoomed based on the S-spline algorithm. Te remaining procedures can be car- ried out as described in Variant
1.Note that even in flat or thin specimens that appear completely in focus, focus stacking can lead to a higher level of distinctness when several images or duplicates of one original image are superimposed by use of appropriate stacking soſtware.
Results In
an original still image
(667×491 pixels) extracted from a video clip (Figure 3), circa 50 throm- bocytes were captured in the speci- men area (140×103 μm). Figure 4 shows the right part of the image cropped out and digitally inverted. In the inverted grayscale image (Figure 4, right) small pseudopodia were seen at higher contrast when compared against
the bright back-
ground. A “thrombocyte of interest” (marked by a red arrow in Figures 3
14
distinguished in the reconstructed images: sausage-shaped (Figure 6a), spike-shaped (Figure 6b), with discoid apices (Figure 6c), and loop-shaped (Figure. 6d). Tese fine structural nuances were only resolved in a clear and distinct manner by the multi-step post-processing described. By digital inversion of the grayscale images, the fine tails of pseudopodia could be further accentuated (Figure 7).
Discussion Te various morphological types of pseudopodia observed following digital reconstructions may lead to several
Figure 4: Section from Figure 3 showing the right part. (a) Original grayscale image, (b) digi- tal inversion. The arrow indicates the thrombocyte of interest. Image width=77 µm.
Figure 5: Close-up view of a single thrombocyte cropped from Figure 3. The size of the original section was 80×80 pixels, and the specimen area imaged was 16.8×16.8 μm. (a) Resized version was 1700×1700 pixels; (b) multi-step post-processing with Photozoom, Picolay, and Combine; (c) edge detection with Sobel operator. Image widths=16.8 µm.
www.microscopy-today.com • 2019 July
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68
