Reduction in DNA Synthesis During Two‐photon Microscopy of Intrinsic Reduced Nicotinamide Adenine Dinucleotide Fluorescence^¶

Abstract

Two-photon laser scanning microscopy (TPLSM) of endogenous reduced nicotinamide adenine dinucleotide (NAD(P)H) provides important information regarding the cellular metabolic state, When imaging the punctate mitochondria1 fluorescence originating from NAD(P)H in a rat basophilic leukemia (RBL) cell at low laser powers, no morphological changes are evident, and photobleaching is not observed when many images are taken. At higher powers, mitochondrial NAD(P)H fluorescence bleaches rapidly. To assess the limitations of this technique and to quantify the extent of photodamage, we have measured the effect of TPLSM on DNA synthesis. Although previous reports have indicated a threshold power for "safe" two-photon imaging, we find the laser power to be an insufficient indicator of photodamage. A more meaningful metric is a two-photon-absorbed dose that is proportional to the number of absorbed photon pairs. A temporary reduction of DNA synthesis in RBL cells occurs whenever a threshold dose of approximately 2 X photon' cm-4 s-' is exceeded. This threshold is independent of laser intensity when imaging with average powers ranging from 5 to 17 mW at 740 nm. Beyond this threshold, the extent of the reduction is intensity dependent. DNA synthesis returns to control levels after a recovery period of several hours.YPosted on the website

Photobleaching of Reduced Nicotinamide Adenine Dinucleotide and the Development of Highly Fluorescent Lesions in Rat Basophilic Leukemia Cells During Multiphoton Microscopy

“…Previously we have demonstrated that the intrinsic fluorescence signal was appropriately modified by a mitochondrial uncoupler (p‐trifluoromethoxyphenylhydrazone) and inhibitor (sodium cyanide), indicating that the dominant source of intrinsic fluorescence generated during multiphoton microscopy at 740 nm was cellular NADH (20). The response to mitochondrial uncouplers and inhibitors and the punctate nature of the fluorescence distribution, evident in the micrograph shown in Fig.…”

“…But this “live‐dead” assay offers only a coarse indication of biological damage. Previously we have measured a reduction of DNA synthesis in RBL cells imaged under the same conditions used in this current study (20). Changes in the level of DNA synthesis provide a very sensitive indicator of sublethal photodamage because this occurs at much lower photodynamic doses than that required to adversely affect membrane integrity or esterase activity or cause mitochondrial distress (25).…”

“…Two‐photon microscopy. Adherent RBL cells were exposed to intense near‐IR illumination at 740 nm by two‐photon laser scanning microscopy as described previously (20). Blue fluorescence originating approximately 2 to 4 microns above the coverslip within the RBL cell was isolated by a custom‐made blocking filter (HQ450/100, Chroma Technology, Brattleboro, VT) and detected by a photomultiplier tube without descanning.…”

“…Determination of the power‐law dependence of NADH fluorescence. As described previously (20), NADH was the primary source of the initial cellular fluorescence signal. The fluorescence intensity from each cell in the first frame of an image stack was measured and the average initial cellular fluorescence was calculated for the frame.…”

Disruption of the Cox-1 gene slows repair of microscopic lesions in the mouse gastric epithelium

“…This is the mechanism by which photodynamic therapy can kill cancer cells that have selectively absorbed photosensitizing chemicals (2,6,22). Recently, the use of two-photon absorption has been applied to cause focal cell damage (13,20,22,35). Endogenous cytosolic fluorophores, like NAD(P)H, can be visualized by both conventional and two-photon fluorescence imaging, providing insights into cell and tissue bioenergetics (22,24,38,40).…”