Digital Staining
NA of 0.8, the Abbe limit would be roughly d=0.5*λ /NA=325 nm. Using the correction scheme above, the 3D Cell Explorer can reach 200 nm.
Results Fibroblastic reticular cells .
Figure 5 : Four stills from a video of a mouse fi broblastic reticular cell in the process of dying. Times of the frames are (a) 0 hours, (b) 2.7 h, (c) 7.8 h, and (d) 9.8 h. Scale bar = 10 µm.
T e fi rst prototype of the 3D Cell Explorer was delivered in 2014 to the Laboratory of Lymphatic and Cancer Bioengineering (LLCB) at the École Polytechnique Fédérale de Lausanne in Switzerland. T is group uses the microscope to study how T-cells interact with lymphatic endothelial cells and the morphological changes this interaction involves. Moreover, they want to compare this specifi c interaction with the one between T-cells and other stromal cell types to determine the dynamics of such interactions. Cells were observed over 30 minutes at RT in transparent cell culture media composed of 1×PBS, 1 g/L glucose, and 25 mM HEPES. One image per second was recorded, and the result- ing video (60×speed) shows the interaction of a T-cell (the smaller cell) with a lymphatic endothelial cell (the bigger cell) expressing an antigen ( https://www.youtube. com/watch?v=bWhiOmtdXEU ). Figure 4 shows the 3D recon- struction of a living mouse fi broblastic reticular cell obtained with the 3D Cell Explorer. T e cell nucleus, nucleolus, lipid vesicles, and membrane were stained in contrasting colors. Digital staining can also be accomplished for each frame of a video sequence. Figure 5 shows four stills from a video of a mouse fi broblastic reticular cell in the process of dying.
Comparison with traditional
Figure 6 : Comparison between (a) a fi xed mouse fi broblastic reticular cell (FRC) imaged with a traditional fl uores- cence microscope in 2D and (b) the same cell type with the 3D Cell Explorer in a 3D dataset. Scale bar = 10 µm.
optical distortion in the reconstructed image. However, because these distortions are predictable and tied to a specific set of optical components (mirrors, objective, etc.), they can be accurately measured and calibrated for each model of the 3D Cell Explorer, allowing automatic digital corrections to be applied. Thus, considering green light (520 nm) and an
2015 July •
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methods . T e 3D Cell Explorer can be used for routine cell analysis such as the identifi cation of diff erent cell parts in 3D. Figure 6a shows an example of a
standard immunostaining procedure, which requires expen- sive reagents and long procedures to stain the nucleus and membrane respectively with DAPI and GFP labeled antibody. T e same results that required more than 4 hours of preparation with traditional fl uorescence microscopy were achieved with the 3D Cell Explorer in less than fi ve minutes ( Figure 6b ).
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