Department of Engineering, University Roma Tre, Rome, Italy.
Center for Microscopy - Microanalysis and Information Processing, University Politehnica of Bucharest, Bucharest, Romania.
J Biophotonics. 2020 Sep;13(9):e202000097. doi: 10.1002/jbio.202000097. Epub 2020 Jun 23.
Super-resolution microscopy techniques can provide answers to still pending questions on prokaryotic organisms but are yet to be used at their full potential for this purpose. To address this, we evaluate the ability of the rhodamine-like KK114 dye to label various types of bacteria, to enable imaging of fine structural details with stimulated emission depletion microscopy (STED). We assessed fluorescent labeling with KK114 for eleven Gram-positive and Gram-negative bacterial species and observed that this contrast agent binds to their cell membranes. Significant differences in the labeling outputs were noticed across the tested bacterial species, but importantly, KK114-staining allowed the observation of subtle nanometric cell details in some cases. For example, a helix pattern resembling a cytoskeleton arrangement was detected in Bacillus subtilis. Furthermore, we found that KK114 easily penetrates the membrane of bacterial microorganism that lost their viability, which can be useful to discriminate between living and dead cells.
超分辨率显微镜技术可以为原核生物中仍悬而未决的问题提供答案，但尚未充分发挥其在这方面的潜力。为了解决这个问题，我们评估了类似罗丹明的 KK114 染料标记各种类型细菌的能力，以实现用受激发射损耗显微镜（STED）对精细结构细节进行成像。我们评估了 KK114 对 11 种革兰氏阳性和革兰氏阴性细菌的荧光标记，观察到该对比剂与它们的细胞膜结合。在测试的细菌物种中，观察到标记输出存在显著差异，但重要的是，KK114 染色在某些情况下允许观察到细微的纳米级细胞细节。例如，在枯草芽孢杆菌中检测到类似于细胞骨架排列的螺旋模式。此外，我们发现 KK114 很容易穿透失去活力的细菌微生物的细胞膜，这可用于区分活细胞和死细胞。
