Imaging mitochondrial dynamics in human skin reveals depth-dependent hypoxia and malignant potential for diagnosis

Abstract

Active changes in mitochondrial structure and organization facilitate cellular homeostasis. Because aberrant mitochondrial dynamics are implicated in a variety of human diseases, their assessment is potentially useful for diagnosis, therapy, and disease monitoring. Because current techniques for evaluating mitochondrial morphology are invasive or necessitate mitochondria-specific dyes, their clinical translation is limited. We report that mitochondrial dynamics can be monitored in vivo, within intact human skin by relying entirely on endogenous two-photon–excited fluorescence from the reduced metabolic coenzyme nicotinamide adenine dinucleotide (NADH). We established the sensitivity of this approach with in vivo, fast temporal studies of arterial occlusion-reperfusion, which revealed acute changes in the mitochondrial metabolism and dynamics of the lower human epidermal layers. In vitro hypoxic-reperfusion studies validated that the in vivo outcomes were a result of NADH fluorescence changes. To demonstrate the diagnostic potential of this approach, we evaluated healthy and cancerous human skin epithelia. Healthy tissues displayed consistent, depth-dependent morphological and mitochondrial organization patterns that varied with histological stratification and intraepithelial mitochondrial protein expression. In contrast, these consistent patterns were absent in cancerous skin lesions. We exploited these differences to successfully differentiate healthy from cancerous tissues using a predictive classification approach. Collectively, these results demonstrate that our label-free, automated, near real-time assessments of mitochondrial organization—relying solely on endogenous contrast—could be useful for accurate, noninvasive in vivo diagnosis.

Non-destructive two-photon excited fluorescence imaging identifies early nodules in calcific aortic-valve disease

“…75–78 Briefly, the image intensity patterns within the tissue regions, as determined by the Otsu intensity thresholding method described in the previous section, were cloned and randomly positioned in the image background to create a new image without distinct borders and only tissue patterns covering the entire field. Then, we acquired the power spectral density (PSD) of the two-dimensional Fourier transform of this image, which represents the level of autocorrelation of the intensity fluctuations within the image over a range of relevant spatial frequencies.…”

A pilot study with flow mediated skin fluorescence: A novel device to assess microvascular endothelial function in coronary artery disease

“…Additionally, there is compelling evidence for a link between the pathogenesis of CVD and increased mitochondrial dysfunction [16]. Therefore, the possibility of monitoring the oxygen-dependent processes in the mitochondria is crucial in understanding the pathophysiology of CVDs such as CAD [17]. In the presented pilot study, mitochondrial function was evaluated in vivo in humans by measuring an ischemic response via NADH fluorescence recording.…”

Label free monitoring of megakaryocytic development and proplatelet formation in vitro

“…These masks captured the entire cellular area including the nuclei. To isolate intracellular image features attributed to NAD(P)H fluorescence, while also removing dark nuclear and vesicular features, custom digital bandpass filters were employed as described previously [16] that created stricter binary masks (Appendix Fig. …”

“…Using batch-analysis custom software, a mean normalized RR value was computed for each selected cell as the normalized fluorescence intensity contributions from FAD over the sum of the intensity contributions from NAD(P)H and FAD within each respective cellular ROI. Time-resolved fluorescence decay data were analyzed by the phasor approach [19] as described previously [16,20,21]. Briefly, sine and cosine transforms were applied to decay profiles at each pixel and an instrument response function calibration was applied to the resulting phasors based on an umbelliferone standard.…”