Confocal Microscopy of Living Cells

In situ cell nucleus deformation in tendons under tensile load; a morphological analysis using confocal laser microscopy

“…to remove excess stain. Ascorbic acid is a reducing agent commonly used to reduce photodynamic damage to the cells and to prevent the stain from fading during observation [47]. The light sensitivity of the acridine orange stain required that the stained tendons be protected from light until tested [47].…”

Confocal laser scanning microscopy of calcium dynamics in living cells

“…In order to conduct successful calcium imaging studies by CLSM, the dye-loaded cells must remain physiologically viable during imaging (Terasaki and Dailey, 1995). Accordingly, a major problem with vital fluorescent probes in general is their potential for phototoxicity, a phenomenon wherein triplet excited states of the dye combine with molecular oxygen to produce damaging singlet oxygen (Tsien and Waggoner, 1995).…”

“…Accordingly, a major problem with vital fluorescent probes in general is their potential for phototoxicity, a phenomenon wherein triplet excited states of the dye combine with molecular oxygen to produce damaging singlet oxygen (Tsien and Waggoner, 1995). The phototoxic effects of calcium-sensitive fluorophores employed in conventional CLSM can be at least partially diminished by: (1) utilizing lower-power and/or lessfrequent laser illumination (Terasaki and Dailey, 1995); (2) minimizing the amounts of molecular oxygen around the dye-loaded cells (Tsien and Waggoner, 1995); and/or (3) reducing singlet oxygen levels by means of vital scavengers such as crocetin (Tsien and Waggoner, 1995). However, even with such measures, it may turn out the certain cell types are too sensitive for conventional CLSM and may thus require less stressful methods, such as two-photon confocal microscopy, which is discussed further in subsequent sections.…”

Two-Photon Fluorescence Excitation Microscopy to Assess Transscleral Diffusional Pathways in an Isolated Perfused Bovine Eye Model

“…18 Therefore, with TPM, images can be obtained routinely at depths in excess of 250 m, 19,20 compared with confocal microscopy, which typically loses contrast at depths of ϳ75 m. 21 TPM imaging in the neocortex is capable of penetration depths greater than 600 m, 22 and it has been reported that imaging up to a 2 mm depth is possible in brain tissue by using gradient index lenses together with in vivo TPM. 23 It should be noted, however, that the imaging depth is dependent on whether tissue structures (such as thick cell body layers or surface blood vessels) contribute to optical aberrations.…”