Real-time, high-resolution metabolic characterization of live bacteria using label-free optical metabolic imaging.

Metabolic imaging is critical for understanding cellular functions beyond morphology, offering significant insights into various biological processes and disease states. Label-free optical imaging techniques stand out by providing high-resolution, molecularly specific, and/or non-invasive assessments of metabolic activity without relying on exogenous contrast agents. This review discusses the key photon-tissue interactions—absorption, emission, and scattering—that underpin label-free optical imaging modalities for interrogating tissue’s metabolic activities at various scales. Specifically, photoacoustic imaging (PAI) leverages absorption-based contrasts such as hemoglobin oxygenation and glucose concentrations to quantify metabolic dynamics. Emission-based techniques, including two-photon fluorescence (TPF) and fluorescence lifetime imaging microscopy (FLIM), exploit intrinsic fluorophores like nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) to assess cellular energy metabolism. Interferometric methods, particularly optical coherence tomography (OCT), provide insights into tissue morphological changes. Second harmonic generation (SHG) detects extracellular matrix components such as the collagen network. Molecular vibrational imaging methods, such as stimulated Raman scattering (SRS) microscopy, visualizes spatial heterogeneity of molecular compositions. Recent clinical translations of these methods highlight their growing roles in oncology, neurology, and dermatology, underscoring their potential in early disease diagnosis and monitoring therapeutic responses. Despite challenges such as depth limitations, advancements like wavefront engineering and optical clearing techniques promise to enhance imaging penetration and clinical applicability, paving the way for broader adoption of label-free optical metabolic imaging in both research and clinical settings.

The discovery of extracellular vesicles (EVs) in all bodily fluids is rapidly increasing the fundamental knowledge of disease mechanisms and the ways in which cells communicate systemically to facilitate tumor progression and metastasis. The type, content, and magnitude of tumor-associated EVs have been linked to tumor invasiveness both in-vitro and in-vivo. In this study we utilized a novel label-free multimodal multiphoton optical imaging method to detect and characterize isolated urinary EVs and classify their optical signatures. Blinded analysis conducted on urinary EVs isolated from 4 healthy dogs and 19 dogs diagnosed with multiple types of cancer showed different optical signatures; cancer-associated urinary EVs were found to have significantly higher NADPH concentrations in comparison to those isolated from healthy dogs. These results suggest a potential label-free optical methodology to detect and characterize EVs by their optical signatures, which can be utilized as possible diagnostic and prognostic biomarkers for cancer. Citation Format: Ronit Barkalifa, Sixian You, Haohua Tu, Alison Masyr, Rebecca Kamerer, Marina Marjanovic, Jaena Park, Kimberly Selting, Stephen Boppart. Label-free optical imaging and characterization of cancer-associated urinary extracellular vesicles: Implications for biomarker discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1936.

.Significance: Early detection of epithelial cancers and precancers/neoplasia in the presence of benign lesions is challenging due to the lack of robust in vivo imaging and biopsy guidance techniques. Label-free nonlinear optical microscopy (NLOM) has shown promise for optical biopsy through the detection of cellular and extracellular signatures of neoplasia. Although in vivo microscopy techniques continue to be developed, the surface area imaged in microscopy is limited by the field of view. FDA-approved widefield fluorescence (WF) imaging systems that capture autofluorescence signatures of neoplasia provide molecular information at large fields of view, which may complement the cytologic and architectural information provided by NLOM.Aim: A multimodal imaging approach with high-sensitivity WF and high-resolution NLOM was investigated to identify and distinguish image-based features of neoplasia from normal and benign lesions.Approach:In vivo label-free WF imaging and NLOM was performed in preclinical hamster models of oral neoplasia and inflammation. Analyses of WF imaging, NLOM imaging, and dual modality (WF combined with NLOM) were performed.Results: WF imaging showed increased red-to-green autofluorescence ratio in neoplasia compared to inflammation and normal oral mucosa (). In vivo assessment of the mucosal tissue with NLOM revealed subsurface cytologic (nuclear pleomorphism) and architectural (remodeling of extracellular matrix) atypia in histologically confirmed neoplastic tissue, which were not observed in inflammation or normal mucosa. Univariate and multivariate statistical analysis of macroscopic and microscopic image-based features indicated improved performance (94% sensitivity and 97% specificity) of a multiscale approach over WF alone, even in the presence of benign lesions (inflammation), a common confounding factor in diagnostics.Conclusions: A multimodal imaging approach integrating strengths from WF and NLOM may be beneficial in identifying oral neoplasia. Our study could guide future studies on human oral neoplasia to further evaluate merits and limitations of multimodal workflows and inform the development of multiscale clinical imaging systems.

Application of optical imaging in developmental biology marks an exciting frontier in biomedical optics. Optical resolution and imaging depth allow for investigation of growing embryos at subcellular, cellular, and whole organism levels, while the complexity and variety of embryonic processes set multiple challenges stimulating the development of various live dynamic embryonic imaging approaches. Among other optical methods, label-free optical techniques attract an increasing interest as they allow investigation of developmental mechanisms without application of exogenous markers or fluorescent reporters. There has been a boost in development of label-free optical imaging techniques for studying embryonic development in animal models over the last decade, which revealed new information about early development and created new areas for investigation. Here, we review the recent progress in label-free optical embryonic imaging, discuss specific applications, and comment on future developments at the interface of photonics, engineering, and developmental biology.

Label-free optical imaging and sensing for quality control of stem cell manufacturing

Histochemistry is a microscopy-based technology widely used to visualize the molecular distribution in biological tissue. Recent developments in label-free optical imaging has demonstrated the potential to replace the conventional histochemical labels/markers (fluorescent antibodies, organic dyes, nucleic acid probes, and other contrast agents) with diverse optical interactions to generate histochemical contrasts, allowing "virtual" histochemistry in three spatial dimensions without preparing a microscope slide (i.e. labor-intensive sample preparation). However, the histochemical information in a label-free optical image has often been rather limited due to the difficulty in simultaneously generating multiple histochemical contrasts with strict spatial co-registration. Here, in the first part (Part I) of this two-part series study, we develop a technique of slide-free virtual histochemistry based on label-free multimodal multiphoton microscopy, and simultaneously generate up to four histochemical contrasts from in vivo animal and ex vivo human tissue. To enable this functionality, we construct and demonstrate a robust fiber-based laser source for clinical translation and phenotype a wide variety of vital cells in unperturbed mammary tissue.

Many conventional methods to assess engineered tissue morphology and viability are destructive techniques with limited utility for tissue constructs intended for implantation in patients. Sterile label-free optical molecular imaging methods analyzed tissue endogenous fluorophores without staining, noninvasively and quantitatively assessing engineered tissue, in lieu of destructive assessment methods. The objective of this study is to further investigate label-free optical metrics and their correlation with destructive methods. Tissue-engineered constructs (n = 33 constructs) fabricated with primary human oral keratinocytes (n = 10 patients) under control, thermal stress, and rapamycin treatment manufacturing conditions exhibited a range of tissue viability states, as evaluated by quantitative histology scoring, WST-1 assay, Ki-67 immunostaining imaging, and label-free optical molecular imaging methods. Both histology sections of fixed tissues and cross-sectioned label-free optical images of living tissues provided quantitative spatially selective information on local tissue morphology, but optical methods noninvasively characterized both local tissue morphology and cellular viability at the same living tissue site. Furthermore, optical metrics noninvasively assessed living tissue viability with a statistical significance consistent with the destructive tissue assays WST-1 and histology. Over the range of cell viability states created experimentally, optical metrics noninvasively and quantitatively characterized living tissue viability and correlated with the destructive WST-1 tissue assay. By providing, under sterile conditions, noninvasive metrics that were comparable with conventional destructive tissue assays, label-free optical molecular imaging has the potential to monitor and assess engineered tissue construct viability before surgical implantation.

<div>Abstract<p>Patient-derived cancer organoids (PDCO) are a valuable model to recapitulate human disease in culture, with important implications for drug development. However, current methods for rapidly and reproducibly assessing PDCOs are limited. Label-free imaging methods are a promising tool to measure organoid-level heterogeneity and rapidly screen drug responses in PDCOs. This study aimed to assess and predict PDCO response to treatments based on mutational profiles using label-free wide-field optical redox imaging (WF ORI). WF ORI provided organoid-level measurements of treatment response without labels or additional reagents by measuring the autofluorescence intensity of the metabolic coenzymes NAD(P)H and FAD, and the optical redox ratio, defined as the fluorescence intensity of [NAD(P)H/NAD(P)H + FAD], was used to measure the oxidation–reduction state of PDCOs. Development of leading-edge analysis tools helped maximize the sensitivity and reproducibility of treatment response measurements using WF ORI in colorectal cancer PDCOs. Leading-edge analysis improved sensitivity to redox changes in treated PDCOs. Additionally, WF ORI resolved FOLFOX (5-fluorouracil + oxaliplatin) treatment effects across all PDCOs better than two-photon ORI, with an approximately threefold increase in effect size. WF ORI distinguished metabolic differences based on driver mutations in colorectal cancer PDCOs, differentiating between <i>KRAS + PIK3CA</i> double-mutant PDCOs and wild-type PDCOs with 80% accuracy and identified treatment-resistant mutations in mixed PDCO cultures. Overall, WF ORI enables rapid, sensitive, and reproducible measurements of treatment response and heterogeneity in colorectal PDCOs that will affect patient management, clinical trials, and preclinical drug development.</p>Significance:<p>Label-free wide-field optical redox imaging is an accessible tool for monitoring changes in metabolism in patient-derived cancer organoids to reproducibly assess treatment response and heterogeneity in clinically relevant treatment studies.</p></div>

Label-free nonlinear optical microscopy was employed to monitor primary human cells in tissue-engineered constructs. Novel quantitative analysis techniques were developed that successfully distinguished viable from non-viable engineered tissues.

Label-free nonlinear optical microscopy has become a powerful tool for biomedical research. However, the possible photodamage risk hinders further clinical applications. To reduce these adverse effects, we constructed a new platform of simultaneous label-free autofluorescence multi-harmonic (SLAM) microscopy, featuring four-channel multimodal imaging, inline photodamage monitoring, and pulse repetition-rate tuning. Using a large-core birefringent photonic crystal fiber for spectral broadening and a prism compressor for pulse pre-chirping, this system allows users to independently adjust pulse width, repetition rate, and energy, which is useful for optimizing imaging conditions towards no/minimal photodamage. It demonstrates label-free multichannel imaging at one excitation pulse per image pixel and thus paves the way for improving the imaging speed by a faster optical scanner with a low risk of nonlinear photodamage. Moreover, the system grants users the flexibility to autonomously fine-tune repetition rate, pulse width, and average power, free from interference, ensuring the discovery of optimal imaging conditions with high SNR and minimal phototoxicity across various applications. The combination of a stable laser source, independently tunable ultrashort pulse, photodamage monitoring features, and a compact design makes this new system a robust, powerful, and user-friendly imaging platform.

Significance:Human pluripotent stem-cell–derived cardiomyocytes (hPSC-CMs) are a powerful tool for drug discovery, and metabolic changes are associated with their long-term culture and maturation. However, the lack of technologies to monitor hPSC-derived cardiomyocyte metabolism during long-term culture presents a major technical bottleneck.Aim:Efforts to monitor in vitro metabolic maturation of hPSC-CMs are limited by traditional assessment methods, which are generally time-consuming, destructive to samples, and lack single-cell resolution. We report a rapid, noninvasive imaging-based method to monitor hPSC-CM metabolism throughout extended culture (90+ days).Approach:Label-free optical metabolic imaging (OMI) of autofluorescent metabolic coenzymes was performed at multiple time points during the extended culture maturation process. In addition, OMI monitored hPSC-CMs grown on substrates with varying stiffness and on cardiomyocytes derived from induced pluripotent stem cells associated with cardiac arrhythmia. OMI was paired with immunofluorescence to validate structural maturation.Results:Single-cell OMI can identify metabolic changes during cardiomyocyte maturation through extended in vitro culturing. It can also detect metabolic differences induced by substrates of varying stiffnesses, can distinguish diseased from normal cell lines, and is sensitive to patient-level metabolic heterogeneity.Conclusions:Our results demonstrate that label-free OMI can be used to monitor metabolic changes in hPSC-CMs under varying culture conditions in a rapid, non-destructive manner with single-cell resolution, providing insight into metabolic transitions arising from time in culture, culture conditions, or disease states.

SignificanceCellular 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.AimWe 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.ApproachWe 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.ResultsWe 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.ConclusionWe 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 morphology and molecular study of the penguin brain are crucial to define its survival in the extreme conditions of Antarctica. The present study focusses on extracting different optical parameters of the penguin brain using label-free optical imaging and spectroscopic techniques. In label-free optical imaging, we have used quantitative phase imaging, which provides morphological information about the neurons in brain tissue, giving the quantitative phase value of 5 to 20 radians corresponding to the 8 µm tissue section. In label-free spectroscopic techniques, we have used autofluorescence and Raman spectroscopy. Autofluorescence spectroscopy provides molecular information about nicotinamide dinucleotide, flavins, lipofuscins, and porphyrins in the brain’s spectral range of 420 nm to 700 nm. Raman spectroscopy provides multiple peaks associated with different molecules in the brain; among them, few signals are observed at approximately 1305 cm−1, 1448 cm−1, and 1661 cm−1, which correspond to vibrational modes indicative of vibrational features within lipids and protein structures, as well as the presence of amide groups within brain tissue constituents. All these techniques provide the microscopic and molecular fingerprint of the penguin brain, which can be useful for understanding penguin’s anatomical, physiological, and social behavior.

Intraoperative imaging in surgical oncology can provide information about the tumor microenvironment as well as information about the tumor margin. Visualizing microstructural features and molecular and functional dynamics may provide important diagnostic and prognostic information, especially when obtained in real-time at the point-of-procedure. A majority of current intraoperative optical techniques are based on the use of the labels, such as fluorescent dyes. However, these exogenous agents disrupt the natural microenvironment, perturb biological processes, and alter the endogenous optical signatures that cells and the microenvironment can provide. Portable nonlinear imaging systems have enabled intraoperative imaging for real-time detection and diagnosis of tissue. We review the development of a label-free multimodal nonlinear optical imaging technique that was adapted into a portable imaging system for intraoperative optical assessment of resected human breast tissue. New developments have applied this technology to assessing needle-biopsy specimens. Needle-biopsy procedures most always precede surgical resection and serve as the first sampling of suspicious masses for diagnosis. We demonstrate the diagnostic feasibility of imaging core needle-biopsy specimens during veterinary cancer surgeries. This intraoperative label-free multimodal nonlinear optical imaging technique can potentially provide a powerful tool to assist in cancer diagnosis at the point-of-procedure.

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.