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 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.
- NSF-PAR ID:
- 10370886
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Journal of Biophotonics
- Volume:
- 15
- Issue:
- 9
- ISSN:
- 1864-063X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract STUDY QUESTION Is the combined use of fluorescence lifetime imaging microscopy (FLIM)-based metabolic imaging and second harmonic generation (SHG) spindle imaging a feasible and safe approach for noninvasive embryo assessment?
SUMMARY ANSWER Metabolic imaging can sensitively detect meaningful metabolic changes in embryos, SHG produces high-quality images of spindles and the methods do not significantly impair embryo viability.
WHAT IS KNOWN ALREADY Proper metabolism is essential for embryo viability. Metabolic imaging is a well-tested method for measuring metabolism of cells and tissues, but it is unclear if it is sensitive enough and safe enough for use in embryo assessment.
STUDY DESIGN, SIZE, DURATION This study consisted of time-course experiments and control versus treatment experiments. We monitored the metabolism of 25 mouse oocytes with a noninvasive metabolic imaging system while exposing them to oxamate (cytoplasmic lactate dehydrogenase inhibitor) and rotenone (mitochondrial oxidative phosphorylation inhibitor) in series. Mouse embryos (n = 39) were measured every 2 h from the one-cell stage to blastocyst in order to characterize metabolic changes occurring during pre-implantation development. To assess the safety of FLIM illumination, n = 144 illuminated embryos were implanted into n = 12 mice, and n = 108 nonilluminated embryos were implanted into n = 9 mice.
PARTICIPANTS/MATERIALS, SETTING, METHODS Experiments were performed in mouse embryos and oocytes. Samples were monitored with noninvasive, FLIM-based metabolic imaging of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) autofluorescence. Between NADH cytoplasm, NADH mitochondria and FAD mitochondria, a single metabolic measurement produces up to 12 quantitative parameters for characterizing the metabolic state of an embryo. For safety experiments, live birth rates and pup weights (mean ± SEM) were used as endpoints. For all test conditions, the level of significance was set at P < 0.05.
MAIN RESULTS AND THE ROLE OF CHANCE Measured FLIM parameters were highly sensitive to metabolic changes due to both metabolic perturbations and embryo development. For oocytes, metabolic parameter values were compared before and after exposure to oxamate and rotenone. The metabolic measurements provided a basis for complete separation of the data sets. For embryos, metabolic parameter values were compared between the first division and morula stages, morula and blastocyst and first division and blastocyst. The metabolic measurements again completely separated the data sets. Exposure of embryos to excessive illumination dosages (24 measurements) had no significant effect on live birth rate (5.1 ± 0.94 pups/mouse for illuminated group; 5.7 ± 1.74 pups/mouse for control group) or pup weights (1.88 ± 0.10 g for illuminated group; 1.89 ± 0.11 g for control group).
LIMITATIONS, REASONS FOR CAUTION The study was performed using a mouse model, so conclusions concerning sensitivity and safety may not generalize to human embryos. A limitation of the live birth data is also that although cages were routinely monitored, we could not preclude that some runt pups may have been eaten.
WIDER IMPLICATIONS OF THE FINDINGS Promising proof-of-concept results demonstrate that FLIM with SHG provide detailed biological information that may be valuable for the assessment of embryo and oocyte quality. Live birth experiments support the method’s safety, arguing for further studies of the clinical utility of these techniques.
STUDY FUNDING/COMPETING INTEREST(S) Supported by the Blavatnik Biomedical Accelerator Grant at Harvard University and by the Harvard Catalyst/The Harvard Clinical and Translational Science Center (National Institutes of Health Award UL1 TR001102), by NSF grants DMR-0820484 and PFI-TT-1827309 and by NIH grant R01HD092550-01. T.S. was supported by a National Science Foundation Postdoctoral Research Fellowship in Biology grant (1308878). S.F. and S.A. were supported by NSF MRSEC DMR-1420382. Becker and Hickl GmbH sponsored the research with the loaning of equipment for FLIM. T.S. and D.N. are cofounders and shareholders of LuminOva, Inc., and co-hold patents (US20150346100A1 and US20170039415A1) for metabolic imaging methods. D.S. is on the scientific advisory board for Cooper Surgical and has stock options with LuminOva, Inc.
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The development of effective and safe agricultural treatments requires sub-cellular insight of the biochemical effects of treatments in living tissue in real-time. Industry-standard mass spectroscopic imaging lacks real-time
in vivo capability. As an alternative, multiphoton fluorescence lifetime imaging microscopy (MPM-FLIM) allows for 3D sub-cellular quantitative metabolic imaging but is often limited to low frame rates. To resolve relatively fast effects (e.g., photosynthesis inhibiting treatments), high-frame-rate MPM-FLIM is needed. In this paper, we demonstrate and evaluate a high-speed MPM-FLIM system, “Instant FLIM”, as a time-resolved 3D sub-cellular molecular imaging system in highly scattering, living plant tissues. We demonstrate simultaneous imaging of cellular autofluorescence and crystalline agrochemical crystals within plant tissues. We further quantitatively investigate the herbicidal effects of two classes of agricultural herbicide treatments, photosystem II inhibiting herbicide (Basagran) and auxin-based herbicide (Arylex), and successfully demonstrate the capability of the MPM-FLIM system to measure biological changes over a short time with enhanced imaging speed. Results indicate that high-frame-rate 3D MPM-FLIM achieves the required fluorescence lifetime resolution, temporal resolution, and spatial resolution to be a useful tool in basic plant cellular biology research and agricultural treatment development. -
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Abstract Vascularization is an important strategy to overcome diffusion limits and enable the formation of complex, physiologically relevant engineered tissues and organoids. Self‐assembly is a technique to generate in vitro vascular networks, but engineering the necessary network morphology and function remains challenging. Here, autofluorescence multiphoton microscopy (aMPM), a label‐free imaging technique, is used to quantitatively evaluate in vitro vascular network morphology. Vascular networks are generated using human embryonic stem cell–derived endothelial cells and primary human pericytes encapsulated in synthetic poly(ethylene glycol)‐based hydrogels. Two custom‐built bioreactors are used to generate distinct fluid flow patterns during vascular network formation: recirculating flow or continuous flow. aMPM is used to image these 3D vascular networks without the need for fixation, labels, or dyes. Image processing and analysis algorithms are developed to extract quantitative morphological parameters from these label‐free images. It is observed with aMPM that both bioreactors promote formation of vascular networks with lower network anisotropy compared to static conditions, and the continuous flow bioreactor induces more branch points compared to static conditions. Importantly, these results agree with trends observed with immunocytochemistry. These studies demonstrate that aMPM allows label‐free monitoring of vascular network morphology to streamline optimization of growth conditions and provide quality control of engineered tissues.
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Background and Objectives To determine the efficacy of targeted fluorescent biomarkers and multiphoton imaging to characterize early changes in ovarian tissue with the onset of cancer.
Study Design/Materials and Methods A transgenic TgMISIIR‐TAg mouse was used as an animal model for ovarian cancer. Mice were injected with fluorescent dyes to bind to the folate receptor α, matrix metalloproteinases, and integrins. Half of the mice were treated with 4‐vinylcyclohexene diepoxide (VCD) to simulate menopause. Widefield fluorescence imaging (WFI) and multiphoton imaging of the ovaries and oviducts were conducted at 4 and 8 weeks of age. The fluorescence signal magnitude was quantified, and texture features were derived from multiphoton imaging. Linear discriminant analysis was then used to classify mouse groups.
Results Imaging features from both fluorescence imaging and multiphoton imaging show significant changes (
P < 0.01) with age, VCD treatment, and genotype. The classification model is able to classify different groups to accuracies of 75.53%, 69.53%, and 86.76%, for age, VCD treatment, and genotype, respectively. Building a classification model using features from multiple modalities shows marked improvement over individual modalities.Conclusions This study demonstrates that using WFI with targeted biomarkers, and multiphoton imaging with endogenous contrast shows promise for detecting early changes in ovarian tissue with the onset of cancer. The results indicate that multimodal imaging can provide higher sensitivity for classifying tissue types than using single modalities alone. Lasers Surg. Med. © 2020 Wiley Periodicals, Inc.
