Drug-induced nephrotoxicity (DIN) is a major cause of drug development failure, yet many cases are detected only late in clinical trials or after approval. Conventional methods to detect DIN often lack sensitivity and specificity, particularly for early or region-specific kidney injury. Here, we evaluate Simultaneous Label-free Autofluorescence Multiharmonic (SLAM) microscopy as a rapid, label-free imaging approach to quantify kidney microstructural and functional metabolic changes associated with DIN. SLAM simultaneously captures endogenous NAD(P)H and FAD autofluorescence, collagen-derived second harmonic generation, and third-harmonic generation from structural interfaces. SLAM images were acquired from the cortex and outer medulla (OM) of rat kidneys after cisplatin dosing at days 1, 6, and 29 post-administration. Imaging revealed region-specific injury, with the OM showing greater sensitivity to DIN than the cortex. The most prominent changes-tubular degeneration and hyaline casts-peaked at day 6, whereas tubular dilation and fibrosis-related features persisted to day 29. Feature-based classification captured these spatial and temporal patterns, achieving higher balanced accuracy in the OM (0.944) than in the cortex (0.860). Key model drivers included granularity and inter-channel correlations, underscoring the value of multi-channel SLAM data. Overall, these results demonstrate the potential of SLAM microscopy for sensitive, region-specific detection and characterization of DIN in preclinical safety studies. SLAM microscopy provides a rapid, label-free way to detect, localize, and classify drug-induced nephrotoxicity with improved sensitivity and regional specificity, potentially strengthening preclinical kidney safety screening and reducing late-stage development failures.

The selection of high-performing cell lines is crucial for biopharmaceutical production but is often time-consuming and labor-intensive. We investigated label-free multimodal nonlinear optical microscopy for non-perturbative profiling of biopharmaceutical cell lines based on their intrinsic molecular contrast. Employing simultaneous label-free autofluorescence multiharmonic (SLAM) microscopy with fluorescence lifetime imaging microscopy (FLIM), we characterized Chinese hamster ovary (CHO) cell lines at early passages (0-2). A machine learning (ML)-assisted analysis pipeline leveraged high-dimensional information to classify single cells into their respective lines. Remarkably, the monoclonal cell line classifiers achieved balanced accuracies exceeding 96.8% as early as passage 2. Correlation features and FLIM modality played pivotal roles in early classification. This integrated optical bioimaging and machine learning approach presents a promising solution to expedite cell line selection process while ensuring identification of high-performing biopharmaceutical cell lines. The techniques have potential for broader single-cell characterization applications in stem cell research, immunology, cancer biology and beyond.

Oxidative stress plays a key role in aging and related diseases, including neurodegeneration, cancer, and organ failure. Copper (Cu), a redox-active metal ion, generates reactive oxygen species (ROS), and its dysregulation contributes to aging. Here, we develop activity-based imaging probes for the sensitive detection of Cu(I) and show that labile hepatic Cu activity increases with age, paralleling a decline in ALDH1A1 activity, a protective hepatic enzyme. We also observe an age-related decrease in hepatic glutathione (GSH) activity through noninvasive photoacoustic imaging. Using these probes, we perform longitudinal studies in aged mice treated with ATN-224, a Cu chelator, and demonstrate that this treatment improves Cu homeostasis and preserves ALDH1A1 activity. Our findings uncover a direct link between Cu dysregulation and aging, providing insights into its role and offering a therapeutic strategy to mitigate its effects.

Intrinsically photosensitive retinal ganglion cells are photoreceptors discovered in the last 20 years. These cells project to the suprachiasmatic nucleus of the brain to drive circadian rhythms, regulated by ambient light levels. The photopigment responsible for photoactivation in these cells, melanopsin, has been shown to exhibit many unique activation features among opsins. Notably, the photopigment can exist in three states dependent on the intensity and spectrum of ambient light, which affects its function. Despite increasing knowledge about these cells and melanopsin, tools that can manipulate their three states, and do so with single-cell precision, are limited. This reduces the extent to which circuit-level phenomena, and studying the implications of melanopsin tri-stability in living systems, can be pursued. In this report, we evoke and modulate calcium transients in live cells and intrinsically photosensitive retinal ganglion cells from isolated retinal tissues following two-photon excitation using near-infrared light pulses. We demonstrate that two-photon activation of melanopsin can successfully stimulate melanopsin-expressing cells with high spatio-temporal precision. Moreover, we demonstrate that the functional tri-stability of the photopigment can be interrogated by multiphoton excitation using spectral-temporal modulation of a broadband, ultrafast laser source.

[This corrects the article PMC10174331.].

Hyperspectral coherent Raman scattering microscopy provides a significant improvement in acquisition time compared to spontaneous Raman scattering yet still suffers from the time required to sweep through individual wavenumbers. To address this, we present the use of a pulse shaper with a 2D spatial light modulator for phase- and amplitude-based shaping of the Stokes beam to create programmable spectrally tailored excitation envelopes. This enables collection of useful spectral information in a more rapid and efficient manner.

The applications of ultrafast optics to biomedical microscopy have expanded rapidly in recent years, including interferometric techniques like optical coherence tomography and microscopy (OCT/OCM). The advances of ultra-high resolution OCT and the inclusion of OCT/OCM in multimodal systems combined with multiphoton microscopy have marked a transition from using pseudo-continuous broadband sources, such as superluminescent diodes, to ultrafast supercontinuum optical sources. We report anomalies in the dispersion profiles of low-coherence ultrafast pulses through long and non-identical arms of a Michelson interferometer that are well beyond group delay or third-order dispersions. This chromatic anomaly worsens the observed axial resolution and causes fringe artifacts in the reconstructed tomograms in OCT/OCM using traditional algorithms. We present DISpersion COmpensation Techniques for Evident Chromatic Anomalies (DISCOTECA) as a universal solution to address the problem of chromatic dispersion mismatch in interferometry, especially with ultrafast sources. First, we demonstrate the origin of these artifacts through the self-phase modulation of ultrafast pulses due to focusing elements in the beam path. Next, we present three solution paradigms for DISCOTECA: optical, optoelectronic, and computational, along with quantitative comparisons to traditional methods to highlight the improvements to the dynamic range and axial profile. We explain the piecewise reconstruction of the phase mismatch between the arms of the spectral-domain interferometer using a modified short-term Fourier transform algorithm inspired by spectroscopic OCT. Finally, we present a decision-making guide for evaluating the utility of DISCOTECA in interferometry and for the artifact-free reconstruction of OCT images using an ultrafast supercontinuum source for biomedical applications.

Fluorescence lifetime imaging microscopy (FLIM) provides valuable insights into molecular interactions and states in complex cellular environments. Conventional FLIM analysis methods struggle with accurate lifetime estimation with low photons-per-pixel (PPP). We propose DeepFLR, a self-supervised deep learning framework for robust FLIM signal restoration with limited photons. By exploiting the spatiotemporal dependencies of FLIM signals, DeepFLR reconstructs the fluorescence decay curves, leading to accurate lifetime estimations using existing lifetime estimation methods. The results demonstrate that DeepFLR enables reliable lifetime estimation with less than 10 PPP for a diverse set of biological samples. The proposed approach significantly reduces the photon budget of FLIM and opens up numerous low-light FLIM applications.

To achieve a comprehensive optical characterization of collagen within its native environment, we present a novel label-free multimodal polarimetric imaging system that integrates polarization-sensitive second-harmonic generation microscopy, polarization-sensitive optical coherence microscopy, and two-photon autofluorescence lifetime imaging. Through simultaneous and co-registered measurements, this multimodal approach captures different collagen signatures by exploring diverse light-collagen interactions across multiple scales. Quantitative information was retrieved at the tissue level, the collagen fibril level, and the molecular level. We believe that this innovative multimodal and quantitative approach has the potential to significantly enhance our fundamental understanding of collagen, especially its intricate roles in human health and disease.