Reduction in DNA Synthesis During Two-Photon Microscopy of Intrinsic NAD(P)H 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 mitochondrial 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 10(53) photon2 cm-4 s-1 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.

Mitigating Phototoxicity during Multiphoton Microscopy of Live Drosophila Embryos in the 1.0–1.2 µm Wavelength Range

“…This is the case in LLLT, but also in certain cases during multiphoton microscopy [33], [50]. However larger amounts of perturbation can exceed the repair capacity of the cell, and toxic effects gradually take place: increase in intracellular calcium concentration [15], [23], [39], membrane depolarisation [51], ROS diffusion inside the cytoplasm [14], destruction of nuclear membranes [37], breakdown of DNA synthesis [20], [49] and finally apoptotic cell death [37].…”

“…Different molecules may be primarily involved depending on the wavelength, but a following step generally involves the creation of ROS and the subsequent rise of oxidative stress [38]. This stress is then transducted differently depending on the amount of the perturbation [15], [49]: if perturbation is limited, repair and protection mechanisms take place that induce cell proliferation. This is the case in LLLT, but also in certain cases during multiphoton microscopy [33], [50].…”

Mitochondrial Redox Imaging for Cancer Diagnostic and Therapeutic Studies

“…It has been shown that these signals in cells/tissues mainly have origins in mitochondria, 63 , 64 and the measurement of NADH, Fp and redox ratios, i.e., Fp/(Fp+NADH) or NADH/(Fp+NADH) provides an index of mitochondrial metabolic states. 15,16,27,28 The fluorescence spectra of NADH and oxidized flavoproteins (Fp) in suspensions of isolated mitochondria are shown in Fig.…”

Metabolic Imaging Using Two-Photon Excited NADH Intensity and Fluorescence Lifetime Imaging

“…We have previously employed two-photon excited fluorescence intensity-based metabolic imaging techniques in monolayer cultures (Indig et al, 2000; Nichols et al, 2005; Tiede and Nichols, 2006), multicell tumor spheroids (Nichols and Webb, 1998), and the organ of Corti of intact but excised murine cochlea (Tiede et al, 2007). Our goal has been to develop and evaluate techniques for non-invasively measuring cellular metabolism so we can better study, understand and ultimately foster the development of better treatments for a wide range of diseases.…”