Visualisation of mitochondria in living neurons with single- and two-photon fluorescence laser microscopy

被引:40
作者
Dedov, VN [1 ]
Cox, GC
Roufogalis, BD
机构
[1] Univ Sydney, Dept Pharm, Sydney, NSW 2006, Australia
[2] Univ Sydney, Australian Key Ctr Microscopy & Microanal, Sydney, NSW 2006, Australia
关键词
mitochondria; confocal laser microscopy; two-photon laser microscopy; sensory neurons; JC-1;
D O I
10.1016/S0968-4328(00)00065-2
中图分类号
TH742 [显微镜];
学科分类号
摘要
In this work we investigated the relative merits of conventional single-photon confocal laser scanning fluorescence microscopy (CLSM) and two-photon laser scanning fluorescence microscopy (2p-LSM) for the study of mitochondria in living neurons. Dorsal root ganglion neurons were loaded with the mitochondrion-specific fluorescent dye JC-1, the ratio between red (J-aggregates) and green (monomer) fluorescence of which reflects mitochondrial membrane potential. Cells were illuminated at 488 nm for single-photon excitation or at 870 nm for two-photon excitation. In both modalities we found that mitochondria showed: (i) similar appearance; (ii) similar fluorescence ratio values over both whole cell bodies and individual mitochondria; and (iii) similar responses to mitochondrial uncoupler, which dropped the ratio values by 50%. However, 2p-LSM exhibited several advantages over CLSM: (i) better signal/noise ratio in the green emission channel; (ii) less phototoxicity upon repetitive scanning in the focal plane; and (iii) no significant loss of image quality upon repetitive scans in the z direction. We conclude that, while both techniques enable visualisation of individual mitochondria in living cells, 2p-LSM has significant advantages for physiological work requiring time-lapse experiments or four-dimensional reconstructions of mitochondria. (C) 2001 Elsevier Science Ltd. All rights reserved.
引用
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页码:653 / 660
页数:8
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