Search

Significance: Deranged metabolism and dysregulated growth factor signaling are closely associated with abnormal levels of proliferation, a recognized hallmark in tumorigenesis. Fluorescence lifetime imaging microscopy (FLIM) of endogenous nicotinamide adenine dinucleotide (NADH), a key metabolic coenzyme, offers a non-invasive, diagnostic indicator of disease progression, and treatment response. The model-independent phasor analysis approach leverages FLIM to rapidly evaluate cancer metabolism in response to targeted therapy. Aim: We combined lifetime and phasor FLIM analysis to evaluate the influence of human epidermal growth factor receptor 2 (HER2) inhibition, a prevalent cancer biomarker, on both nuclear and cytoplasmic NAD(P)H of two squamous cell carcinoma (SCC) cultures. While better established, the standard lifetime analysis approach is relatively slow and potentially subject to intrinsic fitting errors and model assumptions. Phasor FLIM analysis offers a rapid, model-independent alternative, but the sensitivity of the bound NAD(P)H fraction to growth factor signaling must also be firmly established. Approach: Two SCC cultures with low- and high-HER2 expression, were imaged using multiphoton-excited NAD(P)H FLIM, with and without treatment of the HER2 inhibitor AG825. Cells were challenged with mitochondrial inhibition and uncoupling to investigate AG825’s impact on the overall metabolic capacity. Phasor FLIM and lifetime fitting analyses were compared within nuclear and cytoplasmic compartments to investigate epigenetic and metabolic impacts of HER2 inhibition. Results: NAD(P)H fluorescence lifetime and bound fraction consistently decreased following HER2 inhibition in both cell lines. High-HER2 SCC74B cells displayed a more significant response than low-HER2 SCC74A in both techniques. HER2 inhibition induced greater changes in nuclear than cytoplasmic compartments, leading to an increase in NAD(P)H intensity and concentration. Conclusions: The use of both, complementary FLIM analysis techniques together with quantitative fluorescence intensity revealed consistent, quantitative changes in NAD(P)H metabolism associated with inhibition of growth factor signaling in SCC cell lines. HER2 inhibition promoted increased reliance on oxidative phosphorylation in both cell lines.

Melanin is known to provide strong third-harmonic generation (THG) contrast in human skin. With a high concentration in basal cell cytoplasm, THG contrast provided by melanin overshadows other THG sources in human skin studies. For better understanding of the THG signals in keratinocytes without the influence of melanin, an in vivo THG microscopy (THGM) study was first conducted on vitiliginous skin. As a result, the THG-brightness ratio between the melanin-lacking cytoplasm of basal cells and collagen fibers is about 1.106 at the dermal–epidermal junctions of vitiliginous skin, indicating high sensitivity of THGM for the presence of melanin. We further applied the in vivo THGM to assist evaluating the therapeutic outcome from the histopathological point of view for those showed no improvement under narrowband ultraviolet B therapy based on the seven-point Physician Global Assessment score. Our clinical study indicates the high potential of THGM to assist the histopathological assessment of the therapeutic efficacy of vitiligo treatments.

While the nucleus-to-cytoplasmic (N:C) ratio has traditionally been used for assessing cell malignancy, most N:C measurement techniques are time-consuming and performed on thin histological sections, which prohibit assessment of three-dimensional cell structure. A combined ultrahigh frequency ultrasound (US) and photoacoustic (PA) technique was used to assess the size and N:C ratio of cultured cancer cells in three dimensions (3D). The diameters of the cells and their stained nuclei were obtained by fitting the power spectrum of backscattered US pulses and emitted PA waves, respectively, to well-established theoretical models. For comparison, an imaging flow cytometer (IFC) was also used to determine the two-dimensional cell and nucleus sizes from large cell populations using brightfield and fluorescence images, respectively. An N:C ratio was calculated for each cell using the quotient of the measured nucleus diameter and the total cell diameter. The mean N:C ratios calculated using the sound-based approach were 0.68, 0.66, and 0.54 for MCF-7, PC-3, and MDA-MB-231 cells, respectively, and were in good agreement with the corresponding values of 0.68, 0.67, and 0.68 obtained using the IFC. The combined US and PA technique, which assesses cellular N:C ratio in 3D, has potential applications in the detection of circulating tumor cells in liquid biopsies.

A membrane potential change in cells is accompanied with mechanical deformation. This electromechanical response can play a significant role in regulating action potential in neurons and in controlling voltage-gated ion channels. However, measuring this subtle deformation in mammalian cells has been a difficult task. We show a plasmonic imaging method to image mechanical deformation in single cells upon a change in the membrane potential. Using this method, we have studied the electromechanical response in mammalian cells and have observed the local deformation within the cells that are associated with cell–substrate interactions. By analyzing frequency dependence of the response, we have further examined the electromechanical deformation in terms of mechanical properties of cytoplasm and cytoskeleton. We demonstrate a plasmonic imaging approach to quantify the electromechanical responses of single mammalian cells and determine local variability related to cell–substrate interactions.