Intrinsic fluorescence and redox changes associated with apoptosis of primary human epithelial cells

Abstract

Apoptosis plays a key role in the development and maintenance of human tissues. This process has been studied traditionally in cells that are stained with exogenous fluorophores. These approaches affect cell viability, and thus are ill-suited for in vivo applications. We present an imaging approach that can identify apoptotic cells in living cell populations based on detection and quantification of distinct changes in the intensity and localization of cellular autofluorescence. Specifically, we acquire NAD(P)H, FAD, and redox ratio autofluorescence images of primary keratinocytes following 1, 9, 14, and 18 h of treatment with cisplatin, a known apoptosis-inducing chemotherapy agent. We find that intense autofluorescence combined with a low redox fluorescence ratio is progressively confined to a gradually smaller perinuclear cytoplasmic region with cisplatin treatment. Studies with exogenous nuclear fluorophores demonstrate that these autofluorescence changes occur at early stages of apoptosis. Additional costaining experiments suggest that this strongly autofluorescent, highly metabolically active perinuclear ring represents a subpopulation of mitochondria that are mobilized in response to the apoptotic stimulus and may provide the energy required to execute the final apoptotic steps. Thus, autofluorescence localization changes could serve as a sensitive, noninvasive indicator of early apoptosis in vivo.

Analysis of the metabolic deterioration of ex vivo skin from ischemic necrosis through the imaging of intracellular NAD(P)H by multiphoton tomography and fluorescence lifetime imaging microscopy

“…For example, autofluorescence increases during the early stages of apoptosis in primary human epithelial cells treated with cisplatin. 22 Ischemia has been well described to cause a physical reduction in NAD͑P͒H and a corresponding decrease in autofluorescence in a variety of cells and tissues. 23,45,46 The correlation with NADH autofluorescence intensity is related 47 to the physical concentration of the NADH.…”

“…[20][21][22][23] Recently, fluorescence lifetime imaging microscopy ͑FLIM͒ analysis of intracellular NAD͑P͒H has been used to monitor metabolic activity in healthy and diseased tissue. [24][25][26][27] NAD͑P͒H refers to the summation of the molecules NADH and NADPH.…”

Endogenous optical biomarkers of normal and human papillomavirus immortalized epithelial cells

“…[31][32][33][34] Thus, we sought to examine the presence of differences in intensity or localization of these fluorophores between normal and HPV-immortalized cells. Representative examples of normal HFKs and HKc/DR cell images acquired at optimal excitation/ emission wavelengths for detecting NADH (365/450 nm) and FAD (450/525 nm) fluorescence are shown in Figure 2.…”

Probe pressure effects on human skin diffuse reflectance and fluorescence spectroscopy measurements

“…NADH and FAD are byproducts of metabolic activities, and significant changes in NADH and FAD fluorescence could serve as a noninvasive indicator of tumor growth in vivo. 6,7 DRS in human tissue is commonly analyzed using a physically-based [8][9][10][11][12][13] or empirically-based models. 9,14 A model-based analysis of DRS provides quantitative measures in terms of wavelength-dependent reduced scattering (μ s ) and absorption coefficient (μ a ), representative of the physiological structure and activity of the sampled site.…”