Topical steroids are known for their anti‐inflammatory properties and are commonly prescribed to treat many adverse skin conditions such as eczema and psoriasis. While these treatments are known to be effective, adverse effects including skin atrophy are common. In this study, the progression of these effects is investigated in an
Two-photon fluorescence lifetime imaging microscopy (FLIM) is a widely used technique in biomedical optical imaging. Presently, many two-photon time-domain FLIM setups are limited by long acquisition and postprocessing times that decrease data throughput and inhibit the ability to image fast sub-second processes. Here, we present a versatile two-photon FLIM setup capable of video-rate (up to 25 fps) imaging with graphics processing unit (GPU)-accelerated pixelwise phasor analysis displayed and saved simultaneously with acquisition. The system uses an analog output photomultiplier tube in conjunction with 12-bit digitization at 3.2 GHz to overcome the limited maximum acceptable photon rate associated with the photon counting electronics in many FLIM systems. This allows for higher throughput FLIM acquisition and analysis, and additionally enables the user to assess sample fluorescence lifetime in real-time. We further explore the capabilities of the system to examine the kinetics of Rhodamine B uptake by human breast cancer cells and characterize the effect of pixel dwell time on the reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) autofluorescence lifetime estimation accuracy.
more » « less- NSF-PAR ID:
- 10249221
- Publisher / Repository:
- Optical Society of America
- Date Published:
- Journal Name:
- Biomedical Optics Express
- Volume:
- 12
- Issue:
- 7
- ISSN:
- 2156-7085
- Page Range / eLocation ID:
- Article No. 4003
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract in vivo mouse model using multimodal optical microscopy. Utilizing a system capable of performing two‐photon excitation fluorescence microscopy (TPEF ) of reduced nicotinamide adenine dinucleotide (NADH ) to visualize the epidermal cell layers and second harmonic generation (SHG ) microscopy to identify collagen in the dermis, these processes can be studied at the cellular level. Fluorescence lifetime imaging microscopy (FLIM ) is also utilized to image intracellularNADH levels to obtain molecular information regarding metabolic activity following steroid treatment. In this study, fluticasone propionate (FP )‐treated, mometasone furoate (MF )‐treated and untreated animals were imaged longitudinally using a custom‐built multimodal optical microscope. Prolonged steroid treatment over the course of 21 days is shown to result in a significant increase in mean fluorescence lifetime ofNADH , suggesting a faster rate of maturation of epidermal keratinocytes. Alterations to collagen organization and the structural microenvironment are also observed. These results give insight into the structural and biochemical processes of skin atrophy associated with prolonged steroid treatment. -
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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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Nicotinamide adenine dinucleotide (NAD) is found in all living cells where the oxidized (NAD + ) and reduced (NADH) forms play important roles in many enzymatic reactions. However, little is known about NAD + and NADH conformational changes and kinetics as a function of the cell environment. In the present work, an analytical workflow is utilized to study NAD + and NADH dynamics as a function of the organic content in solution using fluorescence lifetime spectroscopy and in the gas-phase using trapped ion mobility spectrometry coupled to mass spectrometry (TIMS-MS) and infrared multiple photon dissociation (IRMPD) spectroscopy. NAD solution time decay studies showed a two-component distribution, assigned to changes from a “close” to “open” conformation with the increase of the organic content. NAD gas-phase studies using nESI-TIMS-MS displayed two ion mobility bands for NAD + protonated and sodiated species, while four and two ion mobility bands were observed for NADH protonated and sodiated species, respectively. Changes in the mobility profiles were observed for NADH as a function of the starting solution conditions and the time after desolvation, while NAD + profiles showed no dependence. IRMPD spectroscopy of NAD + and NADH protonated species in the 800–1800 and 3200–3700 cm −1 spectral regions showed common and signature bands between the NAD forms. Candidate structures were proposed for NAD + and NADH kinetically trapped intermediates of the protonated and sodiated species, based on their collision cross sections and IR profiles. Results showed that NAD + and NADH species exist in open, stack, and closed conformations and that the driving force for conformational dynamics is hydrogen bonding of the N–H–O and O–H–O forms with ribose rings.more » « less
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Abstract A major challenge in ART is to select high-quality oocytes and embryos. The metabolism of oocytes and embryos has long been linked to their viability, suggesting the potential utility of metabolic measurements to aid in selection. Here, we review recent work on noninvasive metabolic imaging of cumulus cells, oocytes, and embryos. We focus our discussion on fluorescence lifetime imaging microscopy (FLIM) of the autofluorescent coenzymes NAD(P)H and flavine adenine dinucleotide (FAD+), which play central roles in many metabolic pathways. FLIM measurements provide quantitative information on NAD(P)H and FAD+ concentrations and engagement with enzymes, leading to a robust means of characterizing the metabolic state of cells. We argue that FLIM is a promising approach to aid in oocyte and embryo selection.
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Over the last half century, the autofluorescence of the metabolic cofactors NADH (reduced nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide) has been quantified in a variety of cell types and disease states. With the spread of nonlinear optical microscopy techniques in biomedical research, NADH and FAD imaging has offered an attractive solution to noninvasively monitor cell and tissue status and elucidate dynamic changes in cell or tissue metabolism. Various tools and methods to measure the temporal, spectral, and spatial properties of NADH and FAD autofluorescence have been developed. Specifically, an optical redox ratio of cofactor fluorescence intensities and NADH fluorescence lifetime parameters have been used in numerous applications, but significant work remains to mature this technology for understanding dynamic changes in metabolism. This article describes the current understanding of our optical sensitivity to different metabolic pathways and highlights current challenges in the field. Recent progress in addressing these challenges and acquiring more quantitative information in faster and more metabolically relevant formats is also discussed. Expected final online publication date for the Annual Review of Biomedical Engineering, Volume 25 is June 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.more » « less
