A novel method to analyse in vivo the physiological state and cell viability of phototrophic microorganisms by confocal laser scanning microscopy using a dual laser

荧光 共焦 激光器 微生物 光养 显微镜 生物物理学 自体荧光 荧光显微镜 显微镜 共焦显微镜 光学 材料科学 光合作用 生物 植物 细菌 物理 遗传学
作者
Laia Millach,Aleix Obiol,Antonio Solé,Isabel Esteve
出处
期刊:Journal of Microscopy [Wiley]
卷期号:268 (1): 53-65 被引量:9
标识
DOI:10.1111/jmi.12586
摘要

Summary Phototrophic microorganisms are very abundant in extreme environments, where are subjected to frequent and strong changes in environmental parameters. Nevertheless, little is known about the physiological effects of these changing environmental conditions on viability of these microorganisms, which are difficult to grow in solid media and have the tendency to form aggregates. For that reason, it is essential to develop methodologies that provide data in short time consuming, in vivo and with minimal manipulating the samples, in response to distinct stress conditions. In this paper, we present a novel method using Confocal Laser Scanning Microscopy and a Dual Laser (CLSM‐DL) for determining the cell viability of phototrophic microorganisms without the need of either staining or additional use of image treating software. In order to differentiate viable and nonviable Scenedesmus sp. DE2009 cells, a sequential scan in two different channels was carried out from each same xyz optical section. On the one hand, photosynthetic pigments fluorescence signal (living cells) was recorded at the red channel (625‐ to 785‐nm fluorescence emission) exciting the samples with a 561‐nm laser diode, and an acousto‐optic tunable filter (AOTF) of 20%. On the other hand, nonphotosynthetic autofluorescence signal (dead cells) was recorded at the green channel (500‐ to 585‐nm fluorescence emission) using a 405‐nm UV laser, an AOTF of 15%. Both types of fluorescence signatures were captured with a hybrid detector. The validation of the CLSM‐DL method was performed with SYTOX green fluorochrome and electron microscopic techniques, and it was also applied for studying the response of distinct light intensities, salinity doses and exposure times on a consortium of Scenedesmus sp. DE2009.
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