Digital Staining
Figure 3 : STEVE interface screenshot. The panel on the left offers a 2D viewer of the 96 z -stacks that compose the 3D image on the right. In the center, the Panel Viewer constitutes a fi ngerprint of the analyzed cell. In the x -axis refractive indexes are reported, while the y -axis shows the gradients. On the bottom (Control Field) the user can chose the color to apply to each cell part in order to produce the 3D reconstruction and modulate its transparency.
“white light” mode can be useful for fi nding a specifi c type of cell in a sparsely populated sample. Once a suitable cell has been found, the user can start an acquisition. STEVE automati- cally verifi es that the microscope is properly aligned and performs calibration steps where necessary. Once running, acquisitions are performed at a rate of 1 second per 3D frame. An explanatory video is also available at the company soſt ware link:
http://nanolive.ch/soſt ware . Digital staining . To label certain parts of the measured cells, a process called digital staining is applied to the acquired data. The Panel Viewer is the central control element for digitally staining a sample. It allows users to define a region in a 2D space defined by the refractive index on one axis and the gradient norm of the refractive index, that is, its spatial variation, on the other. Stains are represented as rectangular areas in the Panel Viewer and can be modified by the user. Changes to stains are shown in real time in the 2D (and 3D) view. To further accelerate the digital staining process, STEVE includes a “stain painter,” with which users can “paint” a part of a cell they want to stain, and STEVE will automatically define the corresponding region in the Panel Viewer. This enables stains to be defined on regions with similar structural features.
Reconstruction and processing . To compute a 3D refractive index distribution from the holograms recorded in the microscope, STEVE relies on routines from the fi eld of optical diff raction tomography [ 1 , 2 ]. Optimized for speed and implemented to run on graphics processing units (GPUs), the 3D reconstruction runs in real time using aff ordable, consumer-grade graphics hardware.
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Figure 4: Living mouse fi broblastic reticular cell obtained with the 3D Cell Explorer. Nucleus is marked with violet, nucleolus in gold, membrane in pink, and lipid vesicles in green. Scale bar = 10 µm.
Beyond reconstruction, STEVE uses the GPU to perform image enhancement steps based on complex deconvolution. Using this process, image aberrations, usually only avoidable using expensive optical components, can be subtracted numerically, enhancing image quality and resolution. Such aberrations normally distort the light wave front and induce
www.microscopy-today.com • 2015 July
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