In vivo spectrally unmixed multi-photon imaging of longitudinal axon-glia changes in injured spinal white matter.

Understanding the sequence of cellular responses and their contributions to pathomorphogical changes in spinal white matter injuries is a prerequisite for developing efficient therapeutic strategies for spinal cord injury (SCI) as well as neurodegenerative and inflammatory diseases of the spinal cord such as amyotrophic lateral sclerosis and multiple sclerosis. We have developed several types of surgical procedures suitable for acute one-time and chronic recurrent in vivo multiphoton microscopy of spinal white matter [1]. Sophisticated surgical procedures were combined with transgenic mouse technology to image spinal tissue labeled with up to four fluorescent proteins (FPs) in axons, astrocytes, microglia, and blood vessels. To clearly separate the simultaneously excited FPs, spectral unmixing including iterative procedures was performed after imaging the diversely labeled spinal white matter with a custom-made 4-channel two-photon laser-scanning microscope. In our longitudinal multicellular studies of injured spinal white matter, we imaged axonal dynamics and invasion of microglia and astrocytes for a time course of over 200 days after SCI. Our methods offer ideal platforms for investigating acute and chronic cellular dynamics, cell-cell interactions, and metabolite fluctuations in health and disease as well as pharmacological manipulations in vivo.

Fluorescence lifetime imaging of human pancreatic lipase activity using a novel probe for early diagnosis of severe acute pancreatitis.

Severe Acute Pancreatitis, a serious condition caused by factors such as gallstones and chronic excessive alcohol consumption, with a very high mortality rate. Human pancreatic lipase (hPL) is a key digestive enzyme and abnormal activity levels of this enzyme are important indicators for diagnosing and monitoring pancreatic diseases. A fluorescent probe, LPP, has been developed to monitor the activity of hPL, especially in cases of SAP. The probe is based on cyanine isoindole derivatives, in vitro experiments confirmed the high specificity and sensitivity of the probe, with a detection limit of 0.012 U/mL, reactions completed within 10 min, and effective monitoring of pancreatic lipase activity in various biological samples. The stability and low cytotoxicity of LPP make it suitable for clinical applications, providing new tools and perspectives for the research and treatment of pancreatic diseases and related metabolic abnormalities. In addition, the change in fluorescence lifetime after the reaction of the probe with lipase allows for fluorescence lifetime imaging (FLIM), effectively monitoring the dynamic changes of hPL and enabling early diagnosis and monitoring of pancreatitis. This research not only enhances the understanding of pancreatic lipase activity detection but also has the potential to improve the diagnostics and treatment of pancreatitis.

A portable photoacoustic microscopy and ultrasound system for rectal cancer imaging.

Photoacoustic microscopy offers functional information regarding tissue vasculature while ultrasound characterizes tissue structure. Combining these two modalities provides novel clinical applications including response assessment among rectal cancer patients undergoing therapy. We have previously demonstrated the capabilities of a co-registered photoacoustic and ultrasound device in vivo, but multiple challenges limited broad adoption. In this paper, we report significant improvements in an acoustic resolution photoacoustic microscopy and ultrasound (ARPAM/US) system characterized by simulation and phantom study, focusing on resolution, optical coupling, and signal characteristics. In turn, higher in-probe optical coupling efficiency, higher signal-to-noise ratio, higher data throughput, and better stability with minimal maintenance requirements were all accomplished. We applied the system to 19 ex vivo resected colorectal cancer samples and found significantly different signals between normal, cancer, and post-treatment tumor tissues. Finally, we report initial results of the first in vivo imaging study.

Near-infrared in vivo imaging system for dynamic visualization of lung-colonizing bacteria in mouse pneumonia.

In vivo imaging of bacterial infection models enables noninvasive and temporal analysis of individuals, enhancing our understanding of infectious disease pathogenesis. Conventional in vivo imaging methods for bacterial infection models involve the insertion of the bacterial luciferase LuxCDABE into the bacterial genome, followed by imaging using an expensive ultrasensitive charge-coupled device (CCD) camera. However, issues such as limited light penetration into the body and lack of versatility have been encountered. We focused on near-infrared (NIR) light, which penetrates the body effectively, and attempted to establish an in vivo imaging method to evaluate the number of lung-colonizing bacteria during the course of bacterial pneumonia. This was achieved by employing a novel versatile system that combines plasmid-expressing firefly luciferase bacteria, NIR substrate, and an inexpensive, scientific complementary metal-oxide semiconductor (sCMOS) camera. The D-luciferin derivative "TokeOni," capable of emitting NIR bioluminescence, was utilized in a mouse lung infection model of Acinetobacter baumannii, an opportunistic pathogen that causes pneumonia and is a concern due to drug resistance. TokeOni exhibited the highest sensitivity in detecting bacteria colonizing the mouse lungs compared with other detection systems such as LuxCDABE, enabling the monitoring of changes in bacterial numbers over time and the assessment of antimicrobial agent efficacy. Additionally, it was effective in detecting A. baumannii clinical isolates and Klebsiella pneumoniae. The results of this study are expected to be used in the analysis of animal models of infectious diseases for assessing the efficacy of therapeutic agents and understanding disease pathogenesis. IMPORTANCE: Conventional animal models of infectious diseases have traditionally relied upon average assessments involving numerous individuals, meaning they do not directly reflect changes in the pathology of an individual. Moreover, in recent years, ethical concerns have resulted in the demand to reduce the number of animals used in such models. Although in vivo imaging offers an effective approach for longitudinally evaluating the pathogenesis of infectious diseases in individual animals, a standardized method has not yet been established. To our knowledge, this study is the first to develop a highly versatile in vivo pulmonary bacterial quantification system utilizing near-infrared luminescence, plasmid-mediated expression of firefly luciferase in bacteria, and a scientific complementary metal-oxide semiconductor camera. Our research holds promise as a useful tool for assessing the efficacy of therapeutic drugs and pathogenesis of infectious diseases.

Epilepsy insights revealed by intravital functional optical imaging.

Despite an abundance of pharmacologic and surgical epilepsy treatments, there remain millions of patients suffering from poorly controlled seizures. One approach to closing this treatment gap may be found through a deeper mechanistic understanding of the network alterations that underly this aberrant activity. Functional optical imaging in vertebrate models provides powerful advantages to this end, enabling the spatiotemporal acquisition of individual neuron activity patterns across multiple seizures. This coupled with the advent of genetically encoded indicators, be them for specific ions, neurotransmitters or voltage, grants researchers unparalleled access to the intact nervous system. Here, we will review how in vivo functional optical imaging in various vertebrate seizure models has advanced our knowledge of seizure dynamics, principally seizure initiation, propagation and termination.

Tailoring near-infrared amyloid-ß probes with high-affinity and low background based on CN and amphipathic regulatory strategies and in vivo imaging of AD mice.

Amyloid-ß (Aß) species (Aß fibrils and Aß plaques), as one of the typical pathological markers of Alzheimer's disease (AD), plays a crucial role in AD diagnosis. Currently, some near-infrared I (NIR I) Aß probes have been reported in AD diagnosis. However, they still face challenges such as strong background interference and the lack of effective probe design. In this study, we propose molecular design strategy that incorporates CN group and amphiphilic modulation to synthesize a series of amphiphilic NIR I Aß probes, surpassing the commercial probe ThT and ThS. Theoretical calculations indicate that these probes exhibit stronger interaction with amino acid residues in the cavities of Aß. Notably, the probes containing CN group display the ability of binding two distinct sites of Aß, which dramatically enhanced the affinity to Aß species. Furthermore, these probes exhibit minimal fluorescence in aqueous solution and offer ultra-high signal-to-noise ratio (SNR) for in vitro labeling, even in wash-free samples. Finally, the optimal probe DM-V2CN-PYC3 was utilized for in vivo imaging of AD mice, demonstrating its rapid penetration through the blood-brain barrier and labelling to Aß species. Moreover, it enabled long-term monitoring for a duration of 120 min. These results highlight the enhanced affinity and superior performance of the designed NIR I Aß probe for AD diagnosis. The molecular design strategy of CN and amphiphilic modulation presents a promising avenue for the development Aß probes with low background in vivo/in vitro imaging for Aß species.

In Vivo Imaging Techniques for the Human Scalp: A Systematic Review of the Literature.

OBJECTIVE: Scalp inflammation and alopecia are distressing conditions for which patients regularly present to dermatology. Although some diagnoses can be made clinically, others require biopsy, which carries the risk of pain, infection, bleeding, and scarring. This review examines the existing literature regarding noninvasive in vivo imaging techniques and their evidence and utility in evaluating scalp pathology, with a focus on the diagnostics of hair conditions. METHODS: A systematic literature search was conducted using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines without timeframe restrictions. The PubMed and Clarivate (Web of Science) databases were searched using the terms ("imaging" OR "in-vivo imaging" OR "non-invasive imaging" OR "non-invasive in vivo imaging" "imaging," "in-vivo imaging) AND ("human scalp disorders" OR "scalp" OR "hair loss" OR "alopecia"). Peer-reviewed randomized control trials (RCTs), prospective studies, retrospective studies, and case series or reports discussing in vivo imaging of the scalp published before 2022 were selected. RESULTS: Forty-two studies were included and discussed; modalities included laser devices (n = 27), ultrasound (US) (n = 13), infrared thermography (n = 1), skin capacitance imaging (SCI), and ultraviolet light-enhanced visualization (ULEV) (n = 1). The most common laser devices used were reflectance confocal microscopy (RCM), multiphoton microscopy (MPM), and optical coherence tomography (OCT). US techniques included high-frequency US (HFUS) and US biomicroscopy (UBM). CONCLUSION: Quality imaging of the scalp in the setting of alopecic, neoplastic, and inflammatory diseases is highly sought after. Many of these noninvasive imaging techniques show promise, each with individual advantages and disadvantages in imaging-specific conditions. Ultimately, noninvasive imaging techniques may be used to optimize patient management and minimize morbidity associated with scalp biopsies.

Transplanted Murine Tumours SPECT Imaging with 99mTc Delivered with an Artificial Recombinant Protein.

99mTc is a well-known radionuclide that is widely used and readily available for SPECT/CT (Single-Photon Emission Computed Tomography) diagnosis. However, commercial isotope carriers are not specific enough to tumours, rapidly clear from the bloodstream, and are not safe. To overcome these limitations, we suggest immunologically compatible recombinant proteins containing a combination of metal binding sites as 99mTc chelators and several different tumour-specific ligands for early detection of tumours. E1b protein containing metal-binding centres and tumour-specific ligands targeting integrin αvß3 and nucleolin, as well as a short Cys-rich sequence, was artificially constructed. It was produced in E. coli, purified by metal-chelate chromatography, and used to obtain a complex with 99mTc. This was administered intravenously to healthy Balb/C mice at an activity dose of about 80 MBq per mouse, and the biodistribution was studied by SPECT/CT for 24 h. Free sodium 99mTc-pertechnetate at the same dose was used as a reference. The selectivity of 99mTc-E1b and the kinetics of isotope retention in tumours were then investigated in experiments in C57Bl/6 and Balb/C mice with subcutaneously transplanted lung carcinoma (LLC) or mammary adenocarcinoma (Ca755, EMT6, or 4T1). The radionuclide distribution ratio in tumour and adjacent normal tissue (T/N) steadily increased over 24 h, reaching 15.7 ± 4.2 for EMT6, 16.5 ± 3.8 for Ca755, 6.7 ± 4.2 for LLC, and 7.5 ± 3.1 for 4T1.

Real-time in vivo confocal laser endomicroscopic imaging of equine endometrium: Preliminary observations and feasibility study.

Endometrial health is vital for the reproductive efficiency of broodmares and accurate diagnostic testing is crucial for directing the best treatment options and outcomes. Confocal laser endomicroscopy (CLE) is an endoscopic technique for obtaining in-vivo, real-time microscopic imaging of tissues using a fiber optic probe. CLE relies on induced tissue fluorescence and fluorescein sodium, given intravenously, is the contrast agent most used in human medicine. This study aimed to determine the feasibility of CLE for imaging equine endometrium and determine a standard dose of fluorescein sodium to achieve optimal cellular imaging. In-vivo CLE was performed on 44 mares, and the images were compared with routine histopathological analysis of endometrial biopsies. No adverse reactions occurred after IV fluorescein sodium administration and a dose of 4 mg/kg was established (0.04 mL/kg of 10 % fluorescein sodium solution) to achieve optimal image contrast. CLE enabled multiple regions of the endometrium to be assessed quickly. Distinct tissue architecture patterns could be appreciated using CLE, and the luminal epithelium could be assessed for integrity (ulceration) and exocytosed inflammatory cells. Endometrial gland distribution, density, shape, and epithelial height were evaluated. Blood vessels were clearly outlined, and inflammatory cells and fibrosis were discernable within the interstitium. Image quality varied between mares, and the stage of oestrous cycle may have been a factor of influence. This novel imaging modality enables collection of "virtual" biopsies and facilitates critical assessment of multiple regions of the uterus compared with the standard histopathologic assessment of a single random tissue biopsy.

Cortical Acetylcholine Response to Deep Brain Stimulation of the Basal Forebrain.

Background: Deep brain stimulation (DBS), the direct electrical stimulation of neuronal tissue in the basal forebrain to enhance release of the neurotransmitter acetylcholine, is under consideration as a method to improve executive function in patients with dementia. While some small studies indicate a positive response in the clinical setting, the relationship between DBS and acetylcholine pharmacokinetics is incompletely understood. Objective: We examined the cortical acetylcholine response to different stimulation parameters of the basal forebrain. Methods: 2-photon imaging was combined with deep brain stimulation. Stimulating electrodes were implanted in the subpallidal basal forebrain, and the ipsilateral somatosensory cortex was imaged. Acetylcholine activity was determined using the GRABACh-3.0 muscarinic acetylcholine receptor sensor, and blood vessels were imaged with Texas red. Results: Experiments manipulating pulse train frequency demonstrated that integrated acetylcholine induced fluorescence was insensitive to frequency, and that peak levels were achieved with frequencies from 60 to 130 Hz. Altering pulse train length indicated that longer stimulation resulted in higher peaks and more activation with sublinear summation. The acetylcholinesterase inhibitor donepezil increased the peak response to 10s of stimulation at 60Hz, and the integrated response increased 57% with the 2 mg/kg dose, and 126% with the 4 mg/kg dose. Acetylcholine levels returned to baseline with a time constant of 14 to 18 seconds in all experiments. Conclusions: These data demonstrate that acetylcholine receptor activation is insensitive to frequency between 60 and 130 Hz. High peak responses are achieved with up to 900 pulses. Donepezil increases total acetylcholine receptor activation associated with DBS but did not change temporal kinetics. The long time constants observed in the cerebral cortex add to the evidence supporting volume in addition to synaptic transmission.

Progress in Structural and Functional In Vivo Imaging of Microglia and Their Application in Health and Disease.

The first line of defense for the central nervous system (CNS) against injury or disease is provided by microglia. Microglia were long believed to stay in a dormant/resting state, reacting only to injury or disease. This view changed dramatically with the development of modern imaging techniques that allowed the study of microglial behavior in the intact brain over time, to reveal the dynamic nature of their responses. Over the past two decades, in vivo imaging using multiphoton microscopy has revealed numerous new functions of microglia in the developing, adult, aged, injured, and diseased CNS. As the most dynamic cells in the brain, microglia continuously contact all structures and cell types, such as glial and vascular cells, neuronal cell bodies, axons, dendrites, and dendritic spines, and are believed to play a central role in sculpting neuronal networks throughout life. Following trauma, or in neurodegenerative or neuroinflammatory diseases, microglial responses range from protective to harmful, underscoring the need to better understand their diverse roles and states in different pathological conditions. In this chapter, we introduce multiphoton microscopy and discuss recent advances in structural and functional imaging technologies that have expanded our toolbox to study microglial states and behaviors in new ways and depths. We also discuss relevant mouse models available for in vivo imaging studies of microglia and review how such studies are constantly refining our understanding of the multifaceted role of microglia in the healthy and diseased CNS.

FoxO transcription factors regulate urea cycle through Ass1.

When amino acids are plentiful in the diet, the liver upregulates most enzymes responsible for amino acid degradation. In particular, the activity of urea cycle enzymes increases in response to high-protein diets to facilitate the excretion of excess nitrogen. KLF15 has been established as a critical regulator of amino acid catabolism including ureagenesis and we have recently identified FoxO transcription factors as an important upstream regulator of KLF15 in the liver. Therefore, we explored the role of FoxOs in amino acid metabolism under high-protein diet. Our findings revealed that the concentrations of two urea cycle-related amino acids, arginine and ornithine, were significantly altered by FoxOs knockdown. Additionally, using KLF15 knockout mice and an in vivo Ad-luc analytical system, we confirmed that FoxOs directly regulate hepatic Ass1 expression under high-protein intake independently from KLF15. Moreover, ChIP analysis showed that the high-protein diet increased FoxOs DNA binding without altering the nuclear protein amount. Therefore, FoxOs play a direct role in regulating ureagenesis via a KLF15-independent pathway in response to high-protein intake.

Establishment of a visualized mouse orthotopic xenograft model of nasopharyngeal carcinoma.

Mouse orthotopic xenograft tumor models are commonly employed in studies investigating the mechanisms underlying the development and progression of tumors and their preclinical treatment. However, the unavailability of mature and visualized orthotopic xenograft models of nasopharyngeal carcinoma limits the development of treatment strategies for this cancer. The aim of this study was to provide a simple and reliable method for building an orthotopic xenograft model of nasopharyngeal carcinoma. Human nasopharyngeal carcinoma (C666-1-luc) cells, stably expressing the firefly luciferase gene, were injected subcutaneously into the right axilla of BALB/C nude mice. Four weeks later, the resulting subcutaneous tumors were cut into small blocks and grafted into the nasopharynx of immunodeficient BALB/C nude mice to induce tumor formation. Tumor growth was monitored by bioluminescence imaging and small animal magnetic resonance imaging (MRI). The expression of histological and immunological antigens associated with orthotopic xenograft nasopharyngeal carcinoma was analyzed by tissue section analysis and immunohistochemistry (IHC). A visualized orthotopic xenograft nasopharyngeal carcinoma model was successfully developed in this study. Luminescence signal detection, micro-MRI, and hematoxylin and eosin staining revealed the successful growth of tumors in the nasopharynx of the nude mice. Moreover, IHC analysis detected cytokeratin (CK), CK5/6, P40, and P63 expression in the orthotopic tumors, consistent with the reported expression of these antigens in human nasopharyngeal tumors. This study established a reproducible, visual, and less lethal orthotopic xenograft model of nasopharyngeal carcinoma, providing a platform for preclinical research.

In vivo detection of endogenous toxic phenolic compounds of intestine.

Phenol and p-cresol are two common toxic small molecules related to various diseases. Existing reports confirmed that high L-tyrosine in the daily diet can increase the concentration of phenolic compounds in blood and urine. L-tyrosine is a common component of protein-rich foods. Some anaerobic bacteria in the gut can convert non-toxic l-tyrosine into these two toxic phenolic compounds, phenol and p-cresol. Existing methods have been constructed for measuring the concentration of phenolic compound in feces. However, there is still a lack of direct visual evidence to measure the phenolic compounds in the intestine. In this study, we aimed to construct a whole-cell biosensor for phenolic compounds detection based on the dmpR, the regulator from the phenol metabolism cluster. The commensal bacterium Citrobacter amalonaticus PS01 was selected and used as the chassis. Compared with the biosensor based on ECN1917, the biosensor PS01[dmpR] could better implant into the mouse gut through gavage and showed a higher sensitive to phenolic compound. And the concentration of phenolic compounds in the intestines could be observed with the help of in vivo imaging system using PS01[dmpR]. This paper demonstrated endogenous phenol synthesis in the gut and the strategy of using commensal bacteria to construct whole-cell biosensors for detecting small molecule compounds in the intestines.

Light-Driven Microrobots for Targeted Drug Delivery.

As a new micromanipulation tool with the advantages of small size, flexible movement and easy manipulation, light-driven microrobots have a wide range of prospects in biomedical fields such as drug targeting and cell manipulation. Recently, microrobots have been controlled in various ways, and light field has become a research hotspot by its advantages of noncontact manipulation, precise localization, fast response, and biocompatibility. It utilizes the force or deformation generated by the light field to precisely control the microrobot, and combines with the drug release technology to realize the targeted drug application. Therefore, this paper provides an overview of light-driven microrobots with drug targeting to provide new ideas for the manipulation of microrobots. Here, this paper briefly categorizes the driving mechanisms and materials of light-driven microrobots, which mainly include photothermal, photochemical, and biological. Then, typical designs of light-driven microrobots with different driving mechanisms and control strategies for multiple physical fields are summarized. Finally, the applications of microrobots in the fields of drug targeting and bioimaging are presented as well as the future prospects of light-driven microrobots in the biomedical field are demonstrated.

Current advances in the development of bioluminescent probes toward spatiotemporal trans-scale imaging.

Bioluminescence imaging has recently attracted great attention as a highly sensitive and non-invasive analytical method. However, weak signal and low chemical stability of the luciferin are conventional drawbacks of bioluminescence imaging. In this review article, we describe the recent progress on the development and applications of bioluminescent probes for overcoming the aforementioned limitations, thereby enabling spatiotemporal trans-scale imaging. The detailed molecular design for manipulation of their luminescent properties and functions enabled a variety of applications, including in vivo deep tissue imaging, long-term imaging, and chemical sensor.

Hard X-ray imaging and tomography at the Biomedical Imaging and Therapy beamlines of Canadian Light Source.

The Biomedical Imaging and Therapy facility of the Canadian Light Source comprises two beamlines, which together cover a wide X-ray energy range from 13 keV up to 140 keV. The beamlines were designed with a focus on synchrotron applications in preclinical imaging and veterinary science as well as microbeam radiation therapy. While these remain a major part of the activities of both beamlines, a number of recent upgrades have enhanced the versatility and performance of the beamlines, particularly for high-resolution microtomography experiments. As a result, the user community has been quickly expanding to include researchers in advanced materials, batteries, fuel cells, agriculture, and environmental studies. This article summarizes the beam properties, describes the endstations together with the detector pool, and presents several application cases of the various X-ray imaging techniques available to users.

Half-Curcumin-Based Chemiluminescence Probes and Their Applications in Detecting Quasi-Stable Oxidized Proteins.

Numerous methods have been reported for detecting ROS/RNS in vitro and in vivo; however, detecting methods for the secondary products of the reactive oxygen species (ROS)/reactive nitrogen species (RNS) reactions, particularly quasi-stable oxidized products, have been much less explored. In this report, we observed that half-curcumins could generate chemiluminescence (CL). In contrast to other chemiluminescence scaffolds, the distinguishing feature of a half-curcumin is the formation of a carbanion intermediate of its acetylacetone moiety, opening unique avenues for applications. In this study, we designed a series of half-curcumins CRANAD-Xs and found that CRANAD-164 could be used to detect quasi-stable oxidized proteins (QSOP) in vivo and in patient serum samples. We illustrated that CRANAD-164 could be used to monitor the responses of taurine, an amino acid with newly reported anti-aging capacity, in an inflammatory mouse model. Remarkably, we further demonstrated that the QSOP levels were much higher in the disease serum samples, including Alzheimer's disease (AD), compared to the samples from healthy controls. Moreover, our results revealed that the sera chemiluminescence intensities were higher in aged healthy controls compared to young healthy subjects, suggesting that CRANAD-164 can be used to monitor the increase of QSOP during aging.

Live Imaging of the Shoot Apical Meristem of Intact, Soil-Grown, Flowering Arabidopsis Plants.

All aerial organs in plants originate from the shoot apical meristem, a specialized tissue at the tip of a plant, enclosing a few stem cells. Understanding developmental dynamics within this tissue in relation to internal and external stimuli is of crucial importance. Imaging the meristem at the cellular level beyond very early stages requires the apex to be detached from the plant body, a procedure that does not allow studies in living, intact plants over longer periods. This protocol describes a new confocal microscopy method with the potential to image the shoot apical meristem of an intact, soil-grown, flowering Arabidopsis plant over several days. The setup opens new avenues to study apical stem cells, their interconnection with the whole plant, and their responses to environmental stimuli. Key features • Novel dissection and imaging method of the shoot apical meristem of Arabidopsis. • Procedure performed with intact, soil-grown, flowering plants. • Possibility of long-term live imaging of the shoot apical meristem. • Protocol can be adapted to different plant species.

Multispectral label-free in vivo cellular imaging of human retinal pigment epithelium using adaptive optics fluorescence lifetime ophthalmoscopy improves feasibility for low emission analysis and increases sensitivity for detecting changes with age and eccentricity.

Significance: Adaptive optics fluorescence lifetime ophthalmoscopy (AOFLIO) provides a label-free approach to observe functional and molecular changes at cellular scale in vivo. Adding multispectral capabilities improves interpretation of lifetime fluctuations due to individual fluorophores in the retinal pigment epithelium (RPE). Aim: To quantify the cellular-scale changes in autofluorescence with age and eccentricity due to variations in lipofuscin, melanin, and melanolipofuscin in RPE using multispectral AOFLIO. Approach: AOFLIO was performed on six subjects at seven eccentricities. Four imaging channels ( λ ex / λ em ) were used: 473/SSC, 473/LSC, 532/LSC, and 765/NIR. Cells were segmented and the timing signals of each pixel in a cell were combined into a single histogram, which were then used to compute the lifetime and phasor parameters. An ANOVA was performed to investigate eccentricity and spectral effects on each parameter. Results: A repeatability analysis revealed < 11.8 % change in lifetime parameters in repeat visits for 532/LSC. The 765/NIR and 532/LSC had eccentricity and age effects similar to previous reports. The 473/LSC had a change in eccentricity with mean lifetime and a phasor component. Both the 473/LSC and 473/SSC had changes in eccentricity in the short lifetime component and its relative contribution. The 473/SSC had no trend in eccentricity in phasor. The comparison across the four channels showed differences in lifetime and phasor parameters. Conclusions: Multispectral AOFLIO can provide a more comprehensive picture of changes with age and eccentricity. These results indicate that cell segmentation has the potential to allow investigations in low-photon scenarios such as in older or diseased subjects with the co-capture of an NIR channel (such as 765/NIR) with the desired spectral channel. This work represents the first multispectral, cellular-scale fluorescence lifetime comparison in vivo in the human RPE and may be a useful method for tracking diseases.