Fluorescence Images
Figure 11: A z-stack of tumor spheroids (height: 100 μm) was acquired with ZEISS Celldiscoverer 7 and a 5×/0.35 objective with 2 × tube lens. Nuclear counterstain (blue), and green and red fluorescence. Top: From left to right: maximum intensity projection of 3-color fluorescence image after constrained Iterative deconvolution in ZEISS ZEN; single plane of a WF stack (green channel) with profile line; single plane of a WF stack, background corrected with the rolling ball method; single plane of a WF stack, deblurred using nearest-neighbors method in ZEISS ZEN; single plane of a WF stack, deconvolved using constrained iterative method in ZEISS ZEN. Approximate processing times of full 3-channel z-stack (relative values): BG-correction (<1 sec), deblurring (1 sec), deconvolution (∼10 sec). Bottom: Intensity values of profile lines shown for all four processed images. Image courtesy of R. Buschow, Max Planck Institute for Molecular Genetics, Berlin.
Table 2: Typical suitability (relative to each other in terms of effort and quality) of clearing methods for WF images. More points mean better suitability.
Image/
Experiment Preview
Unsharp Masking
Background Subtraction Nearest-neighbors
3D Deconvolution (DCV) Optical Sectioning*
Optical Sectioning + 3D DCV ••
••• •• •
•• •
Segmentation, Simple Analysis**
•
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Conference Presentation
(Talk or Poster) ••
••• •••
••••
* Apotome-like structured illumination, single- or multi-point laser scanning. ** For example, cell counting, nuclei or whole cell segmentation, area measurements. *** For example, co-localization analysis, 3D particle tracking, protein fine localization, intensity measurements.
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Advanced Image
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