Label-free optical imaging provides non-invasive, high-speed, high-resolution metabolic characterization of live bacteria with single-cell resolution. Here, we demonstrate the ability of label-free multiphoton autofluorescence microscopy to characterize the fast (between 0 and 30 min) metabolic changes in bacteria in response to antibiotic treatments and observe the cell-to-cell metabolic heterogeneity of planktonic bacteria and biofilms. Results indicate that bacteria exhibit a distinct measurable response to bactericidal treatments within seconds. Furthermore, S. aureus biofilms exhibit metabolic heterogeneity, with local pockets of high metabolic activity. Bacteria in biofilms exhibit altered metabolic profiles compared to planktonic bacteria for all four species examined: S. aureus, P. aeruginosa, M. catarrhalis, and S. pneumoniae. These results shed light on the spatial and temporal metabolic heterogeneity of bacteria and the quantification possibilities using label-free nonlinear optical microscopy.

The dynamics in the mitochondrial structure and function are closely related to cellular health. Traditional fluorescence imaging techniques for observing mitochondria are limited by phototoxicity, photobleaching and staining artifacts. In this study, we propose RedoxSegNet, an AI-enhanced imaging platform that enables label-free segmentation of mitochondria for concurrent morphological and functional analysis without the aid of labeling dyes. Our approach uses high-resolution two-photon excitation fluorescence microscopy, in conjunction with a custom-built conditional diffusion model, to reconstruct mitochondrial features from NAD(P)H autofluorescence images. Subsequent segmentation through post-processing algorithms demonstrates a task-specific performance error of less than 6% on average compared to the mitochondria-stained images. Our trained model effectively extracts mitochondrial features from label-free images, thereby facilitating mapping of mitochondria-specific optical redox ratios. We find that our analysis elucidates metabolic heterogeneity both within and between the organelles. Further validation under mitochondrial stress conditions induced by carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) confirms that RedoxSegNet can capture dynamic mitochondrial fragmentation and heterogeneous metabolic response. Overall, these findings establish our technique as a non-invasive, reliable tool for investigating mitochondrial morpho-functional dynamics in native cellular environments.

Magnetic fields may modulate the cellular reduction-oxidation (redox) state and subsequent redox signaling pathways through quantum spin chemistry in biochemical radical pair reactions. This study applied two-channel, two-photon autofluorescence lifetime microscopy for non-invasive label-free measurements of the metabolic cofactors FAD and NAD(P)H and the redox state in live human non-small lung adenocarcinoma A549 cells. This custom microscope and technique were used to investigate the cellular effects induced by 30-minute exposure to redox modulating chemicals as well as 72-hour exposure to a range of static magnetic fields between 50 μT and 10 mT. The label-free methods showed little sensitivity to the acute chemical exposure in A549 cells, while longer-term magnetic field exposure showed a potential yet non-significant increase in an oxidative stress-linked long lifetime species. A standard H2O2 assay used to validate these responses showed significant sensitivity to chemical redox modulation reactions, and a weak response to effects caused by magnetic fields. Cellular H2O2 was observed to increase then gradually saturate as magnetic field exposure increased. These results demonstrate the potential of label-free microscopy for studying subtle magnetic bioeffects in individual living cells.

Significance:Cellular metabolism plays a central role in health and disease, making its study critical for advancing diagnostics and therapies. Label-free optical metabolic imaging using endogenous fluorescence from reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] and flavin adenine dinucleotide (FAD) provides nondestructive, high-resolution insights into metabolic function and heterogeneity from the sub-cellular to the tissue level. Standardized approaches are essential to ensure reproducibility and comparability across studies. Aim:We aim to establish a consensus framework for the acquisition, calibration, and reporting of microscopic imaging metabolic function assessments based on fluorescence intensity and lifetime measurements of NAD(P)H and FAD. Approach:We present best practices for calibrating, analyzing, and reporting fluorescence intensity-based optical redox ratios and fluorescence lifetime data using multiexponential fitting and phasor analysis. Guidelines for validation experiments and cross-system standardization are provided to improve accuracy and reproducibility. Results:We demonstrate the importance of calibration procedures and normalization strategies for intensity-based optical redox measurements. We highlight needed calibration, signal-to-noise ratio considerations, and the impact of distinct analytical approaches on fluorescence lifetime-based metabolic function metrics. Conclusion:We recommend a consistent, practical framework for reproducible, label-free, optical metabolic imaging, facilitating robust comparisons across studies and supporting the broader adoption of optical metabolic imaging technologies for biomedical research and clinical translation.

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.

Sample health is critical for live-cell fluorescence microscopy and has promoted light-sheet microscopy that restricts its ultraviolet-visible excitation to one plane inside a three-dimensional sample. It is thus intriguing that laser-scanning nonlinear optical microscopy, which similarly restricts its near-infrared excitation, has not broadly enabled gentle label-free molecular imaging. We hypothesize that intense near-infrared excitation induces phototoxicity via linear absorption of intrinsic biomolecules with subsequent triplet buildup, rather than the commonly assumed mechanism of nonlinear absorption. Using a reproducible phototoxicity assay based on the time-lapse elevation of auto-fluorescence (hyper-fluorescence) from a homogeneous tissue model (chicken breast), we provide strong evidence supporting this hypothesis. Our study justifies a simple imaging technique, e.g., rapidly scanned sub-80-fs excitation with full triplet-relaxation, to mitigate this ubiquitous linear-absorption-mediated phototoxicity independent of sample types. The corresponding label-free imaging can track freely moving C. elegans in real-time at an irradiance up to one-half of water optical breakdown.

We demonstrate polarimetric coherent anti-Stokes Raman scattering (PCARS) microscopy for quantitative analysis of collagen orientation. Using polarization-modulated excitation and cross-polarized detection, we extract both chemical and orientational information from intact tissues. We introduce two complementary analyses: the CARS peak polarization ratio (PPR) and Fourier domain polarimetric analysis, providing a robust characterization of collagen organization. This method enables rapid, label-free imaging of a collagen structure with molecular specificity, offering what we believe to be new capabilities for studying collagen remodeling in biological processes.

Broad and safe access to ultrafast laser technology has been hindered by the absence of optical fiber-delivered pulses with tunable central wavelength, pulse repetition rate, and pulse width in the picosecond-femtosecond regime. To address this long-standing obstacle, we developed a reliable accessory for femtosecond ytterbium fiber chirped pulse amplifiers, termed a fiber-optic nonlinear wavelength converter (FNWC), as an adaptive optical source for the emergent field of femtosecond biophotonics. This accessory empowers the fixed-wavelength laser to produce fiber-delivered similar to 20 nJ pulses with central wavelength across 950 to 1150 nm, repetition rate across 1 to 10 MHz, and pulse width across 40 to 400 fs, with a long-term stability of >2000 h. As a prototypical label-free application in biology and medicine, we demonstrate the utility of FNWC in real-time intravital imaging synergistically integrated with modern machine learning and large-scale fluorescence lifetime imaging microscopy.

With the expanding utility of ultrafast sources for interferometric techniques like optical coherence tomography (OCT), we present a Dispersion Compensation Technique for Evident Chromatic Anomalies (DISCOTECA) as a universal solution to correct dispersion mismatch. We report a chromatic anomaly in the propagation of an ultrafast pulse through an interferometer beyond lower-order dispersions that worsens the axial resolution and causes image artifacts. We demonstrate the origin of these artifacts, explain our algorithm for the piecewise reconstruction and correction of the phase mismatch, and present a decision-making guide for interferometry with ultrashort sources. DISCOTECA corrects the artifacts from using ultrashort sources in OCT.

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.

Weak magnetic fields affect a multitude of biological processes including cell metabolism and are hypothesized to be a result of magnetic field-sensitive spin-selective radical-pair reactions. To provide much needed visualization of this process, we demonstrate the use of a custom-built multimodal nonlinear optical imaging system capable of measuring the redox state of cells through multi-photon-excited autofluorescence and autofluorescence lifetime of metabolic cofactors. We demonstrate a custom multi-axis Helmholtz coil system to apply time-varying magnetic fields across the sample during imaging. This imaging platform allows for characterization and optimization of the effects of magnetic fields on live cells and tissues.

Extracellular vesicles (EVs) serve as crucial mediators of cell-to-cell communication in normal physiology as well as in diseased states; they have been largely studied in regard to their role in cancer progression. However, the mechanisms by which their biogenesis and secretion are regulated by metabolic or endocrine factors remain unknown. Here, we delineate a mechanism by which EV secretion is regulated by a cholesterol metabolite, 27-hydroxycholesterol (27HC), where treatment of myeloid immune cells (RAW 264.7 and J774A.1) with 27HC impairs lysosomal homeostasis, leading to shunting of multivesicular bodies (MVBs) away from lysosomal degradation, toward secretion as EVs. This altered lysosomal function is likely caused by mitochondrial dysfunction and subsequent increase in reactive oxygen species (ROS). Interestingly, cotreatment with a mitochondria-targeted antioxidant rescued the lysosomal impairment and attenuated the 27HC-mediated increase in EV secretion. Overall, our findings establish how a cholesterol metabolite regulates EV secretion and paves the way for the development of strategies to regulate cancer progression by controlling EV secretion.