Autofluorescence Lifetime Research Articles

Abstract 5616: Optical metabolic imaging of treatment-naive human NSCLC to determine long-term outcome

Abstract Lung cancer remains the leading cause of cancer deaths in the US and worldwide.​ The five-year survival rate of patients with non-small cell lung carcinoma (NSCLC) remains significantly low given that over half present with locally advanced or metastatic disease at time of diagnosis, and experience tumor recurrence following therapeutic intervention.​Current evaluation techniques to assess treatment response are lacking, given they are implemented several weeks after treatment completion and are solely based on anatomical changes in tumor size, forgoing other criteria such as functional or metabolic changes.​ There is a critical need to identify markers early on following diagnosis that are indicative of patient long-term outcome. Two photon microscopy (TPM) techniques are well-suited to provide non-invasive high-resolution information on cell metabolism within three-dimensional tissue. Specifically, two-photon excited fluorescence imaging of autofluorescent cofactors, nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), can be used to metabolically characterize cancerous tissue. The goal of this study is to utilize the optical redox ratio (ORR) of FAD/[NADH+FAD] autofluorescence and NADH fluorescence lifetime decay to identify measurable differences in optical metabolic endpoints of human NSCLC that are indicative of their long-term outcome. Twenty-nine NSCLC specimens were obtained from the Lung Cancer Biospecimen Resource Network. They were resected from the lung prior to the patient receiving therapy. Clinical detail reports were used to evaluate the follow-up data of each patient and classify them into groups that reflected their eventual outcomes: responder (N=21), non-responder (N=18), non-metastatic (N=25), and metastatic (N=14). TPM was used to determine the ORR for each sample. NADH lifetime images were collected and fit to a biexponential model to separate the short (A1) and long (A2) components of free and bound NADH. We observed a significantly higher optical redox ratio for tumors within the metastatic group compared to non-metastatic. ​This is consistent with previous findings from our lab that revealed increases in the ORR with increasing metastatic potential in cells, as well as other studies reporting a decrease in NADH concentration in metastatic cancers.​ Additionally, a significantly shorter mean NADH lifetime in the metastatic tumor group was revealed when compared to non-metastatic. This stands in agreement with other studies highlighting decreases in the average lifetime of NADH in metastatic cells compared to non-metastatic. These results demonstrate the feasibility of using optical imaging of autofluorescence and lifetimes of metabolic cofactors to characterize treatment-naïve primary NSCLC tumors and determine differences in their optical metabolic endpoints that are indicative of their long-term outcome. Citation Format: Paola Monterroso Diaz. Optical metabolic imaging of treatment-naive human NSCLC to determine long-term outcome. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5616.

Electronic Energy Migration in Microtubules.

The repeating arrangement of tubulin dimers confers great mechanical strength to microtubules, which are used as scaffolds for intracellular macromolecular transport in cells and exploited in biohybrid devices. The crystalline order in a microtubule, with lattice constants short enough to allow energy transfer between amino acid chromophores, is similar to synthetic structures designed for light harvesting. After photoexcitation, can these amino acid chromophores transfer excitation energy along the microtubule like a natural or artificial light-harvesting system? Here, we use tryptophan autofluorescence lifetimes to probe energy hopping between aromatic residues in tubulin and microtubules. By studying how the quencher concentration alters tryptophan autofluorescence lifetimes, we demonstrate that electronic energy can diffuse over 6.6 nm in microtubules. We discover that while diffusion lengths are influenced by tubulin polymerization state (free tubulin versus tubulin in the microtubule lattice), they are not significantly altered by the average number of protofilaments (13 versus 14). We also demonstrate that the presence of the anesthetics etomidate and isoflurane reduce exciton diffusion. Energy transport as explained by conventional Förster theory (accommodating for interactions between tryptophan and tyrosine residues) does not sufficiently explain our observations. Our studies indicate that microtubules are, unexpectedly, effective light harvesters.

Fluorescence lifetime distribution in phakic and pseudophakic healthy eyes.

To investigate the influence of the lens status and to describe fundus autofluorescence lifetimes (FLT) in a large cohort of healthy eyes across a wide age range. FLT data were acquired from healthy phakic and pseudophakic eyes using fluorescence lifetime imaging ophthalmoscopy (FLIO). Retinal autofluorescence was excited with a 473 nm laser and emitted autofluorescence was detected in a short and a long spectral channel (SSC: 498-560 nm; LSC: 560-720 nm). 141 healthy eyes from 141 participants (56 ± 18 years) were included. The shortest mean FLTs were measured within the macular center, followed by the temporal inner and outer ETDRS (Early Treatment Diabetic Retinopathy Study) grid segments, and the remaining areas of the inner and the outer ETDRS ring. In phakic participants (81%), mean, short and long FLTs correlated with the age (SSC: r2 = 0.54; LSC: r2 = 0.7; both p<0.0001) with an increase of about 33 ps in the SSC resp. 28 ps in the LSC per decade. In pseudophakic subjects (19%), mean FLTs only correlated with age in the long spectral channel (r2 = 0.44; p = 0.0002) but not in the short spectral channel (r2 = 0.066; p = 0.2). Fundus autofluorescence lifetimes are age dependent. FLTs in the SSC are more susceptible to lens opacities but less dependent on age changes, whereas FLTs in the LSC are largely independent of the lens status but display a higher degree of age dependency. ClinicalTrials.gov NCT01981148.

Medical femtosecond laser

Medical femtosecond laser devices are used in dermatology for non-linear high-resolution imaging to obtain non-invasive and label-free optical skin biopsies (multiphoton tomography) as well as in ophthalmology for refractive corneal surgery and cataract surgery. Applications of commercial certified multiphoton tomographs include early detection of skin cancer within minutes by two-photon autofluorescence imaging of coenzymes and melanin and second harmonic imaging of collagen as well as by testing the efficacy of pharmaceutical and cosmetical products. Goals are (i) to reduce the number of physically taken human skin biopsies in hospitals and research institutions, (ii) to optimize personalized medicine, and (iii) to reduce animal studies in pharmacy. Current diagnostic tools in dermatology include surface microscopy with a dermatoscope and ultrasound but have poor resolution. Optical coherence tomography and confocal reflectance microscopy have better resolution but provide limited information based on changes of the intratissue refractive index. Multiphoton tomography provides the best resolution of all clinical imaging methods and offer functional imaging such as optical metabolic imaging based on autofluorescence lifetime imaging. Goals of femtosecond laser eye treatment are (i) the replacement of mechanical microkeratomes for corneal flap generation, (ii) the replacement of the UV nanosecond excimer laser for stroma removal, and (iii) to replace, in part, the scalpel in the surgery of cataracts and other eye diseases. So far, millions of eye treatments have been conducted around the world. The major disadvantage of current certified medical femtosecond laser devices is the high price compared with the standard mechanical and optical medical devices.

Fluorescence Lifetime and Spectral Characteristics of Subretinal Drusenoid Deposits and Their Predictive Value for Progression of Age-Related Macular Degeneration.

To measure fundus autofluorescence (FAF) lifetimes and peak emission wavelengths (PEW) of subretinal drusenoid deposits (SDD) in age-related macular degeneration (AMD) and their development over time. Fluorescence lifetime imaging ophthalmoscopy (FLIO) was performed in 30 eyes with optical coherence tomography (OCT)-confirmed early or intermediate AMD and SDD. Contrasts of mean lifetimes in short- (SSC) and long-wavelength channels (LSC), PEW, and relative fluorescence intensity were determined as differences of the respective measures at individual SDD and their environment. Measurements were made at baseline and at follow-up intervals 1 (13-36 months) and 2 (37-72 months), respectively. Of 423 SDD found at baseline, 259, 47, and 117 were hypoautofluorescent, isoautofluorescent, and hyperautofluorescent, respectively. FAF lifetimes of SDD were significantly longer than those of their environment by 14.5 ps (SSC, 95% confidence interval [CI], 13.3-15.7 ps) and 3.9 ps (LSC, 3.1-4.7 ps). PEW was shorter by 1.53 nm (1.07-1.98 nm, all contrasts P < 0.001) with higher contrasts for hyperfluorescent SDD. Over follow-up, SDD tended to hyperautofluorescence (relative intensities increased by 3.4% [95% CI, 2.9%-4.1%; P < 0.001] in follow-up 2). Hyperautofluorescence was associated with disruption of the ellipsoid zone on OCT. Disease progression to late-stage AMD was associated with higher lifetime contrast in SSC (15.9ps [14.2-17.6 ps] vs. 11.7 ps [9.9-13.5 ps], P < 0.001) at baseline. SDD show longer FAF lifetimes and shorter PEW than their environments. A high lifetime contrast of SDD in SSC might predict disease progression to late-stage AMD.

Fluorescence microscopic approach for detection of two different modes of breast cancer cell death induced by nanosecond pulsed electric field

Application of nanosecond pulsed electric field (nsPEF) to living systems is a fast-growing research area with great potential for cancer treatment. Here, we have demonstrated a cellular-level approach applying nsPEF having a pulse width of 50 ns and a frequency of 2 kHz to induce the change in intracellular function and dynamics for two subtypes of breast cancerous cells, that is, estrogen and progesterone-positive (ER+ and PR+) and human epidermal growth factor 2 (HER2) negative MCF-7 cells and highly aggressive drug resistance triple-negative MDA-MB-231 cells. Microscopic monitoring of the field effects in real-time or before and after the application of nsPEF indicated that nsPEF induced different field effects on intracellular function and dynamics in MCF-7 and MDA-MB-231 cells and activated two different modes of cell death, that is, apoptosis in MCF-7 cells and necrosis (or necroptosis) in MDA-MB-231 cells. These two different modes of nsPEF-induced cell death were confirmed based on the following microscopic measurements: 1) autofluorescence intensity and lifetime imaging of nicotinamide adenine dinucleotide (NADH); 2) production of reactive oxygen species (ROS); 3) change in mitochondrial membrane potential; 4) morphological alternation of cells; 5) imaging of phosphatidyl serine (PS) externalization by Annexin V; 6) cell viability. The present finding of nanosecond pulsed electric field-induced apoptosis and necrosis (or necroptosis) in subtypes of breast cancerous cells provides valuable information that the mechanism of field-induced cell death depends on the subtype of cancerous cells. It will be helpful for further development and optimization of field-induced cancer therapy.

Prolonged Lifetimes of Histologic Autofluorescence in Ectopic Retinal Pigment Epithelium in Age-Related Macular Degeneration.

The purpose of this study was to investigate histologic autofluorescence lifetimes and spectra of retinal pigment epithelium (RPE) on the transition from normal aging to RPE activation and migration in age-related macular degeneration (AMD). Autofluorescence lifetimes and spectra of 9 donor eyes were analyzed in cryosections by means of 2-photon excited fluorescence at 960 nm. Spectra were detected at 483 to 665 nm. Lifetimes were measured using time-correlated single photon counting in 2 spectral channels: 500 to 550 nm (short-wavelength spectral channel [SSC]) and 550 to 700 nm (long-wavelength spectral channel [LSC]). Fluorescence decays over time were approximated by a series of three exponential functions. The amplitude-weighted mean fluorescence lifetime was determined. Markers for retinoid activity (RPE65) and immune function (CD68) were immunolocalized in selected neighboring sections. We identified 9 RPE morphology phenotypes resulting in 399 regions of interest (ROIs) for spectral and 497 ROIs for lifetime measurements. RPE dysmorphia results in a shorter wavelength peak of spectral emission: normal aging versus RPE migrated into the retina (intraELM) = 601.7 (9.5) nm versus 581.6 (7.3) nm, P < 0.001, whereas autofluorescence lifetimes increase: normal aging versus intraELM: SSC 180 (44) picosecond (ps) versus 320 (86) ps, P < 0.001; and LSC 250 (55) ps versus 441 (76) ps, P < 0.001. Ectopic RPE within the neurosensory retina is strongly CD68 positive and RPE65 negative. In the process of RPE degeneration, comprising different steps of dysmorphia and migration, lengthening of autofluorescence lifetimes and a hypsochromic shift of emission spectra can be observed. These autofluorescence changes might provide early biomarkers for AMD progression and contribute to our understanding of RPE-driven pathology.

Deep learning-assisted co-registration of full-spectral autofluorescence lifetime microscopic images with H&E-stained histology images

Autofluorescence lifetime images reveal unique characteristics of endogenous fluorescence in biological samples. Comprehensive understanding and clinical diagnosis rely on co-registration with the gold standard, histology images, which is extremely challenging due to the difference of both images. Here, we show an unsupervised image-to-image translation network that significantly improves the success of the co-registration using a conventional optimisation-based regression network, applicable to autofluorescence lifetime images at different emission wavelengths. A preliminary blind comparison by experienced researchers shows the superiority of our method on co-registration. The results also indicate that the approach is applicable to various image formats, like fluorescence in-tensity images. With the registration, stitching outcomes illustrate the distinct differences of the spectral lifetime across an unstained tissue, enabling macro-level rapid visual identification of lung cancer and cellular-level characterisation of cell variants and common types. The approach could be effortlessly extended to lifetime images beyond this range and other staining technologies.

Changes in drusen-associated autofluorescence over time observed by fluorescence lifetime imaging ophthalmoscopy in age-related macular degeneration.

To observe fundus autofluorescence (FAF) lifetimes and peak emission wavelength (PEW) of drusen with respect to the pathology of the overlying RPE in the follow-up of AMD-patients. Forty eyes of 38 patients (age: 75.1± 7.1 years) with intermediate AMD were included. FAF lifetimes and PEW were recorded by fluorescence lifetime imaging ophthalmoscopy (FLIO). Twenty-six eyes had a follow-up investigation between months 12 and 36, and 10 at months 37-72. AMD progression was retrieved from color fundus photography (CFP) and OCT. Drusen were classified with respect to changes in the overlying RPE into groups no, questionable or faint, and apparent hyperpigmentation based on CFP. Among the 210 hyperautofluorescent drusen found at baseline, those with hyperpigmentation had longer lifetimes and shorter PEW than those without. Drusen without hyperpigmentation had shorter lifetimes and PEW than neighboring RPE (all p< 0.001) at baseline, but drusen lifetimes increased, and PEW shortened further over follow-up. Eyes, showing AMD progression, had significantly longer FAF lifetimes at baseline than non-progressing eyes: 282 ± 102 ps versus 245 ± 98 ps, p< 0.001 and 365 ± 44 ps vs. 336 ± 48 ps, p= 0.025 for short and long wavelength FLIO channel, respectively. Depending on hyperpigmentation properties, drusen show lifetimes and PEW different from that of adjacent RPE which change over the natural history of AMD. This difference and change, however, might reflect progressive dysmorphia of the RPE rather than representing fluorescence of drusen material itself. Nevertheless, the observed FAF changes could make FLIO a useful tool for the early detection of AMD progression risk.

Computational macroscopic lifetime imaging and concentration unmixing of autofluorescence.

Single-pixel computational imaging can leverage highly sensitive detectors that concurrently acquire data across spectral and temporal domains. For molecular imaging, such methodology enables to collect rich intensity and lifetime multiplexed fluorescence datasets. Herein we report on the application of a single-pixel structured light-based platform for macroscopic imaging of tissue autofluorescence. The super-continuum visible excitation and hyperspectral single-pixel detection allow for parallel characterization of autofluorescence intensity and lifetime. Furthermore, we exploit a deep learning based data processing pipeline, to perform autofluorescence unmixing while yielding the autofluorophores' concentrations. The full scheme (setup and processing) is validated in silico and in vitro with clinically relevant autofluorophores flavin adenine dinucleotide, riboflavin, and protoporphyrin. The presented results demonstrate the potential of the methodology for macroscopically quantifying the intensity and lifetime of autofluorophores, with higher specificity for cases of mixed emissions, which are ubiquitous in autofluorescence and multiplexed in vivo imaging.

Modulation of calcium signaling by nanosecond electric pulses and cell death through apoptosis in A549 lung cancerous cells

Calcium ion (Ca2+), which serves as one of the important signaling agents, is imperative to control normal cell function. The altered Ca2+ signaling is known to play a crucial role in the resistance of cancerous cells against apoptosis. Herein, nanosecond pulsed electric field (nsPEF) was applied to non-small lung cancerous cells A549, and Ca2+ mobilization along with mitochondria-mediated apoptotic cell death that accompanies morphological changes was investigated. In the presence of nsPEF, intracellular mobilization of Ca2+ ions through the efflux from endoplasmic reticulum storage was dependent on the pulse-width of the applied field. The field-induced Ca2+ mobilization has shown to correlate with the superoxide anion (O2−) generation, which probably occurred in mitochondria utilizing the nicotinamide adenine dinucleotide (NADH), as supported by the field-induced change in autofluorescence intensity and lifetime of NADH. Mitochondrial dysfunction and O2− generation, which promoted apoptotic cell death, were also induced by nsPEF with strong correlation with intracellular Ca2+ ions.

A novel method for determining murine skeletal muscle fiber type using autofluorescence lifetimes.

This work describes a simple way to identify fiber types in living muscles by fluorescence lifetime imaging microscopy (FLIM). We quantified the mean values of lifetimes τ1 and τ2 derived from a two-exponential fit in freshly dissected mouse flexor digitorum brevis (FDB) and soleus muscles. While τ1 values changed following a bimodal behavior between muscles, the distribution of τ2 is shifted to higher values in FDB. To understand the origin of this difference, we obtained maps of autofluorescence lifetimes of flavin mononucleotide and dinucleotide (FMN/FAD) in cryosections, where excitation was set at 440 nm and emission at a bandwidth of between 500 and 570 nm, and paired them with immunofluorescence images of myosin heavy chain isoforms, which allowed identification of fiber types. In soleus,τ2was 3.16 ns for type I (SD 0.11, 97 fibers), 3.45 ns for IIA (0.10, 69), and 3.46 ns for IIX (0.12, 65). In FDB muscle,τ2was 3.17 ns for type I (0.08, 22), 3.46 ns for IIA (0.16, 48), and 3.66 ns for IIX (0.15, 43). Fromτ2distributions, it follows that an FDB fiber withτ2> 3.3 ns is expected to be of type II, and of type I otherwise. This simple classification method has first and second kind errors estimated at 0.02 and 0.10, which can be lowered by reducing the threshold for identification of type I and increasing it for type II. Lifetime maps of autofluorescence, therefore, constitute a tool to identify fiber types that, for being practical, fast, and noninvasive, can be applied in living tissue without compromising other experimental interventions.

Tracking the binding of multi-functional fluorescent tags for Alzheimer's disease using quantitative multiphoton microscopy.

A recent theranostic approach to address Alzheimer's disease (AD) utilizes multifunctional targets that both tag and negate the toxicity of AD biomarkers. These compounds, which emit fluorescence with both an activation and a spectral shift in the presence of Aβ, were previously characterized with traditional fluorescence imaging for binary characterization. However, these multifunctional compounds have broad and dynamic emission spectra that are dependent on factors such as the local environment, presence of Aβ deposits, etc. Since quantitative multiphoton microscopy is sensitive to the binding dynamics of molecules, we characterized the performance of two such compounds, LS-4 and ZY-12-OMe, using Simultaneous Label-free Autofluorescence Multi-harmonic (SLAM) microscopy and Fast Optical Coherence, Autofluorescence Lifetime imaging and Second harmonic generation (FOCALS) microscopy. This study shows that the combination of quantitative multiphoton imaging with multifunctional tags for AD offers new insights into the interaction of these tags with AD biomarkers and the theranostic mechanisms.

Discrimination of cancerous from benign pigmented skin lesions based on multispectral autofluorescence lifetime imaging dermoscopy and machine learning.

.SignificanceAccurate early diagnosis of malignant skin lesions is critical in providing adequate and timely treatment; unfortunately, initial clinical evaluation of similar-looking benign and malignant skin lesions can result in missed diagnosis of malignant lesions and unnecessary biopsy of benign ones.AimTo develop and validate a label-free and objective image-guided strategy for the clinical evaluation of suspicious pigmented skin lesions based on multispectral autofluorescence lifetime imaging (maFLIM) dermoscopy.ApproachWe tested the hypothesis that maFLIM-derived autofluorescence global features can be used in machine-learning (ML) models to discriminate malignant from benign pigmented skin lesions. Clinical widefield maFLIM dermoscopy imaging of 41 benign and 19 malignant pigmented skin lesions from 30 patients were acquired prior to tissue biopsy sampling. Three different pools of global image-level maFLIM features were extracted: multispectral intensity, time-domain biexponential, and frequency-domain phasor features. The classification potential of each feature pool to discriminate benign versus malignant pigmented skin lesions was evaluated by training quadratic discriminant analysis (QDA) classification models and applying a leave-one-patient-out cross-validation strategy.ResultsClassification performance estimates obtained after unbiased feature selection were as follows: 68% sensitivity and 80% specificity with the phasor feature pool, 84% sensitivity, and 71% specificity with the biexponential feature pool, and 84% sensitivity and 32% specificity with the intensity feature pool. Ensemble combinations of QDA models trained with phasor and biexponential features yielded sensitivity of 84% and specificity of 90%, outperforming all other models considered.ConclusionsSimple classification ML models based on time-resolved (biexponential and phasor) autofluorescence global features extracted from maFLIM dermoscopy images have the potential to provide objective discrimination of malignant from benign pigmented lesions. ML-assisted maFLIM dermoscopy could potentially assist with the clinical evaluation of suspicious lesions and the identification of those patients benefiting the most from biopsy examination.

Clinical label-free endoscopic imaging of biochemical and metabolic autofluorescence biomarkers of benign, precancerous, and cancerous oral lesions.

Early detection is critical for improving the survival rate and quality of life of oral cancer patients; unfortunately, dysplastic and early-stage cancerous oral lesions are often difficult to distinguish from oral benign lesions during standard clinical oral examination. Therefore, there is a critical need for novel clinical technologies that would enable reliable oral cancer screening. The autofluorescence properties of the oral epithelial tissue provide quantitative information about morphological, biochemical, and metabolic tissue and cellular alterations accompanying carcinogenesis. This study aimed to identify novel biochemical and metabolic autofluorescence biomarkers of oral dysplasia and cancer that could be clinically imaged using novel multispectral autofluorescence lifetime imaging (maFLIM) endoscopy technologies. In vivo maFLIM clinical endoscopic images of benign, precancerous, and cancerous lesions from 67 patients were acquired using a novel maFLIM endoscope. Widefield maFLIM feature maps were generated, and statistical analyses were applied to identify maFLIM features providing contrast between dysplastic/cancerous vs. benign oral lesions. A total of 14 spectral and time-resolved maFLIM features were found to provide contrast between dysplastic/cancerous vs. benign oral lesions, representing novel biochemical and metabolic autofluorescence biomarkers of oral epithelial dysplasia and cancer. To the best of our knowledge, this is the first demonstration of clinical widefield maFLIM endoscopic imaging of novel biochemical and metabolic autofluorescence biomarkers of oral dysplasia and cancer, supporting the potential of maFLIM endoscopy for early detection of oral cancer.

In situ autofluorescence lifetime assay of a photoreceptor stimulus response in mouse retina and human retinal organoids.

Photoreceptors are the key functional cell types responsible for the initiation of vision in the retina. Phototransduction involves isomerization and conversion of vitamin A compounds, known as retinoids, and their recycling through the visual cycle. We demonstrate a functional readout of the visual cycle in photoreceptors within stem cell-derived retinal organoids and mouse retinal explants based on spectral and lifetime changes in autofluorescence of the visual cycle retinoids after exposure to light or chemical stimuli. We also apply a simultaneous two- and three-photon excitation method that provides specific signals and increases contrast between these retinoids, allowing for reliable detection of their presence and conversion within photoreceptors. This multiphoton imaging technique resolves the slow dynamics of visual cycle reactions and can enable high-throughput functional screening of retinal tissues and organoid cultures with single-cell resolution.

Label-Free Imaging to Track Reprogramming of Human Somatic Cells.

The process of reprogramming patient samples to human-induced pluripotent stem cells (iPSCs) is stochastic, asynchronous, and inefficient, leading to a heterogeneous population of cells. In this study, we track the reprogramming status of patient-derived erythroid progenitor cells (EPCs) at the single-cell level during reprogramming with label-free live-cell imaging of cellular metabolism and nuclear morphometry to identify high-quality iPSCs. EPCs isolated from human peripheral blood of three donors were used for our proof-of-principle study. We found distinct patterns of autofluorescence lifetime for the reduced form of nicotinamide adenine dinucleotide (phosphate) and flavin adenine dinucleotide during reprogramming. Random forest models classified iPSCs with ∼95% accuracy, which enabled the successful isolation of iPSC lines from reprogramming cultures. Reprogramming trajectories resolved at the single-cell level indicated significant reprogramming heterogeneity along different branches of cell states. This combination of micropatterning, autofluorescence imaging, and machine learning provides a unique, real-time, and nondestructive method to assess the quality of iPSCs in a biomanufacturing process, which could have downstream impacts in regenerative medicine, cell/gene therapy, and disease modeling.

Label-free metabolic and structural profiling of dynamic biological samples using multimodal optical microscopy with sensorless adaptive optics

Label-free optical microscopy has matured as a noninvasive tool for biological imaging; yet, it is criticized for its lack of specificity, slow acquisition and processing times, and weak and noisy optical signals that lead to inaccuracies in quantification. We introduce FOCALS (Fast Optical Coherence, Autofluorescence Lifetime imaging, and Second harmonic generation) microscopy capable of generating NAD(P)H fluorescence lifetime, second harmonic generation (SHG), and polarization-sensitive optical coherence microscopy (OCM) images simultaneously. Multimodal imaging generates quantitative metabolic and morphological profiles of biological samples in vitro, ex vivo, and in vivo. Fast analog detection of fluorescence lifetime and real-time processing on a graphical processing unit enables longitudinal imaging of biological dynamics. We detail the effect of optical aberrations on the accuracy of FLIM beyond the context of undistorting image features. To compensate for the sample-induced aberrations, we implemented a closed-loop single-shot sensorless adaptive optics solution, which uses computational adaptive optics of OCM for wavefront estimation within 2 s and improves the quality of quantitative fluorescence imaging in thick tissues. Multimodal imaging with complementary contrasts improves the specificity and enables multidimensional quantification of the optical signatures in vitro, ex vivo, and in vivo, fast acquisition and real-time processing improve imaging speed by 4–40 × while maintaining enough signal for quantitative nonlinear microscopy, and adaptive optics improves the overall versatility, which enable FOCALS microscopy to overcome the limits of traditional label-free imaging techniques.

A Rapid Method for Detecting Microplastics Based on Fluorescence Lifetime Imaging Technology (FLIM).

With the increasing use and release of plastic products, microplastics have rapidly accumulated in ecological environments. When microplastics enter the food chain, they cause serious harm to organisms and humans. Microplastics pollution has become a growing concern worldwide; however, there is still no standardized method for rapidly and accurately detecting microplastics. In this work, we used fluorescence lifetime imaging technology to detect four kinds of Nile red-stained and unstained microplastics, and the unique phasor fingerprints of different microplastics were obtained by phasor analysis. Tracing the corresponding pixels of the “fingerprint” in the fluorescence lifetime image allowed for the quick and intuitive identification of different microplastics and their location distributions in a mixed sample. In our work, compared with staining the four microplastics with a fluorescent dye, using the phasor “fingerprint library” formed by the autofluorescence lifetimes of the microplastics was more easily distinguished than microplastics in the mixed samples. The feasibility of this method was further tested by adding three single substances—SiO2, chitin and decabromodiphenyl ethane (DBDPE), and surface sediments to simulate interferent in the environment, and the results providing potential applications for the identification and analysis of microplastics in complex environments.

Assessment of Murine Colon Inflammation Using Intraluminal Fluorescence Lifetime Imaging.

Inflammatory bowel disease (IBD) is typically diagnosed by exclusion years after its onset. Current diagnostic methods are indirect, destructive, or target overt disease. Screening strategies that can detect low-grade inflammation in the colon would improve patient prognosis and alleviate associated healthcare costs. Here, we test the feasibility of fluorescence lifetime imaging (FLIm) to detect inflammation from thick tissue in a non-destructive and label-free approach based on tissue autofluorescence. A pulse sampling FLIm instrument with 355 nm excitation was coupled to a rotating side-viewing endoscopic probe for high speed (10 mm/s) intraluminal imaging of the entire mucosal surface (50–80 mm) of freshly excised mice colons. Current results demonstrate that tissue autofluorescence lifetime was sensitive to the colon anatomy and the colonocyte layer. Moreover, mice under DSS-induced colitis and 5-ASA treatments showed changes in lifetime values that were qualitatively related to inflammatory markers consistent with alterations in epithelial bioenergetics (switch between -oxidation and aerobic glycolysis) and physical structure (colon length). This study demonstrates the ability of intraluminal FLIm to image mucosal lifetime changes in response to inflammatory treatments and supports the development of FLIm as an in vivo imaging technique for monitoring the onset, progression, and treatment of inflammatory diseases.