Simple Methods to Correlate


Figure 5: (A) and (D) Representative images of DAPI stained cells showing cellular nucleoli in the areas voided of DAPI signal. (B) and (E) BSE-SEM images of the same cells. (C) and (F) FM and BSE-SEM overlays created using MAPS 3.1. Scale bars: A, B, and C: 10 μm; D, E, and F: 7 μm.


images. BSE-SEM images were acquired using 2.5 keV, 0.2 nA, and the DBS detector. Although here we only show the correla- tion between 2D images, the same samples were subjected to 3D FIB-SEM to study the ultrastructural aspects of the nucle- oli region on these cells (unpublished results). In-resin fluorescence preservation and correlation


with BSE-SEM imaging. As described in the Materials and Methods section, the Figure 1C protocol was performed at low temperatures, and the dehydration process proceeded up to 90% ethanol to preserve fluorescence signal from the Cisplatin-TRITC in the mouse pancreas. Keeping 10% water content in the specimen helps preserve the signal of the fluorophore most likely due to the maintenance of a hydra- tion shell around it. Figure 6A shows a representative image


of the fluorescence signal obtained from the LRW sections. Although there is some autofluorescence background present in the tissue, there are specific areas in the specimen show- ing higher signal intensity, more specifically on certain cells located near pancreatic ducts. Figure 6B shows a low-mag- nification SEM image of the same field of view of the image shown in Figure 6A aſter heavy metal staining. Here we used the shape of the plastic section to correlate the images as well as features present in the tissue (that is, ducts). Fig- ure 6C shows a higher-magnification image from a specific region boxed in Figure 6B. One of the cells showing TRITC signal near the duct can be observed. Although we only used mild chemical fixation methods to fix this specimen, cellular membranes and cellular structures were well preserved.


Figure 6: LRW plastic section showing Cisplatin-TRITC fluorescence preservation in mouse pancreas. (A) Pancreatic duct showing fluorescent positive acinar cells. (B) Same area as in (A) acquired by BSE-SEM imaging. The area boxed in black corresponds to an acinar cell showing fluorescence signal. (C) Higher magni- fication of the boxed area in (B) is shown. Secretory granules are identifiable in the acinar cells. N: nucleus, S: stroma, D: duct.


28 www.microscopy-today.com • 2020 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  |  Page 69  |  Page 70  |  Page 71  |  Page 72  |  Page 73  |  Page 74  |  Page 75  |  Page 76  |  Page 77  |  Page 78  |  Page 79  |  Page 80  |  Page 81  |  Page 82  |  Page 83  |  Page 84