Fluorescence Lifetime Imaging Research Articles

Layer-controllable synthesis, lattice structural Determination, and fluorescence lifetime imaging of (HA)2(MA)n-1PbnI3n+1 Quasi-2D organic-inorganic perovskite microsheets

Hybrid quasi-two-dimensional (quasi-2D) perovskites have attracted great interest due to their excellent photovoltaic and light-emitting properties, which enable the rapid development of high-performance perovskite-based solar cells, light-emitting diodes, and lasers. Although many efforts have been made to improve the quality and stability of organic-inorganic perovskites, the controlled synthesis of high-quality layered perovskites remains challenging. Here, single-crystalline quasi-2D (HA)2(MA)n-1PbnI3n+1 perovskites with layer numbers of n from 1 to 3 have been synthesized in a controllable manner, exhibiting stable lattice structures and tunable optical emission. Structurally, hexylamine (HA) with longer alkyl chains was used as an organic spacer in Ruddlesden-Popper (RP) phase perovskites to improve the chemical stability of perovskites. Various ratios of precursor materials were adopted for separating pure and large-scale quasi-2D (HA)2(MA)n-1PbnI3n+1 perovskites, in which different layer-number perovskites with tunable band gaps were obtained with the modulated fluorescence lifetimes. Our lattice structural and fluorescence-lifetime characterizations of RP-phase perovskites provide critical information on the emerging quasi-2D perovskite-crystal systems and their fluorescence relaxation dynamics. The employed strategy to synthesize high-quality perovskites (HA)2(MA)n-1PbnI3n+1 is technically essential to develop the rising perovskite-based photovoltaic and light-emitting applications.

Pioglitazone Phases and Metabolic Effects in Nanoparticle-Treated Cells Analyzed via Rapid Visualization of FLIM Images.

Fluorescence lifetime imaging microscopy (FLIM) has proven to be a useful method for analyzing various aspects of material science and biology, like the supramolecular organization of (slightly) fluorescent compounds or the metabolic activity in non-labeled cells; in particular, FLIM phasor analysis (phasor-FLIM) has the potential for an intuitive representation of complex fluorescence decays and therefore of the analyzed properties. Here we present and make available tools to fully exploit this potential, in particular by coding via hue, saturation, and intensity the phasor positions and their weights both in the phasor plot and in the microscope image. We apply these tools to analyze FLIM data acquired via two-photon microscopy to visualize: (i) different phases of the drug pioglitazone (PGZ) in solutions and/or crystals, (ii) the position in the phasor plot of non-labelled poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs), and (iii) the effect of PGZ or PGZ-containing NPs on the metabolism of insulinoma (INS-1 E) model cells. PGZ is recognized for its efficacy in addressing insulin resistance and hyperglycemia in type 2 diabetes mellitus, and polymeric nanoparticles offer versatile platforms for drug delivery due to their biocompatibility and controlled release kinetics. This study lays the foundation for a better understanding via phasor-FLIM of the organization and effects of drugs, in particular, PGZ, within NPs, aiming at better control of encapsulation and pharmacokinetics, and potentially at novel anti-diabetics theragnostic nanotools.

Net O2 exchange rates under dark and light conditions across different stem compartments.

Woody plants display some photosynthetic activity in stems, but the biological role of stem photosynthesis and the specific contributions of bark and wood to carbon uptake and oxygen evolution remain poorly understood. We aimed to elucidate the functional characteristics of chloroplasts in stems of different ages in Fraxinus ornus. Our investigation employed diverse experimental approaches, including microsensor technology to assess oxygen production rates in whole stem, bark, and wood separately. Additionally, we utilized fluorescence lifetime imaging microscopy (FLIM) to characterize the relative abundance of photosystems I and II (PSI : PSII chlorophyll ratio) in bark and wood. Our findings revealed light-induced increases in O2 production in whole stem, bark, and wood. We present the radial profile of O2 production in F. ornus stems, demonstrating the capability of stem chloroplasts to perform light-dependent electron transport. Younger stems exhibited higher light-induced O2 production and dark respiration rates than older ones. While bark emerged as the primary contributor to net O2 production under light conditions, our data underscored that wood chloroplasts are also photosynthetically active. The FLIM analysis unveiled a lower PSI abundance in wood than in bark, suggesting stem chloroplasts are not only active but also acclimate to the spectral composition of light reaching inner compartments.

Multimodal non-invasive probing of stress-induced carotenogenesis in the cells of microalga Bracteacoccus aggregatus.

Microalgae are the richest source of natural carotenoids-accessory photosynthetic pigments used as natural antioxidants, safe colorants, and nutraceuticals. Microalga Bracteacoccus aggregatus IPPAS C-2045 responds to stresses, including high light, with carotenogenesis-gross accumulation of secondary carotenoids (the carotenoids structurally and energetically uncoupled from photosynthesis). Precise mechanisms of cytoplasmic transport and subcellular distribution of the secondary carotenoids under stress are still unknown. Using multimodal imaging combining micro-Raman imaging (MRI), fluorescent lifetime (τ) imaging (FLIM), and transmission electron microscopy (TEM), we monitored ultrastructural and biochemical rearrangements of B. aggregatus cells during the stress-induced carotenogenesis. MRI revealed a decline in the diversity of molecular surrounding of the carotenoids in the cells compatible with the relocation of the bulk of the carotenoids in the cell from functionally and structurally heterogeneous photosynthetic apparatus to the more homogenous lipid matrix of the oleosomes. Two-photon FLIM highlighted the pigment transformation in the cell during the stress-induced carotenogenesis. The structures co-localized with the carotenoids with shorter τ (mainly chloroplast) shrunk, whereas the structures harboring secondary carotenoids with longer τ (mainly oleosomes) expanded. These changes were in line with the ultrastructural data (TEM). Fluorescence of B. aggregatus carotenoids, either in situ or in acetone extracts, possessed a surprisingly long lifetime. We hypothesize that the extension of τ of the carotenoids is due to their aggregation and/or association with lipids and proteins. The propagation of the carotenoids with prolonged τ is considered to be a manifestation of the secondary carotenogenesis suitable for its non-invasive monitoring with multimodal imaging.

Fluorescence lifetime imaging microscopy of flexible and rigid dyes probes the biophysical properties of synthetic and biological membranes

Sensing of the biophysical properties of membranes using molecular reporters has recently regained widespread attention. This was elicited by the development of new probes of exquisite optical properties and increased performance, combined with developments in fluorescence detection. Here, we report on fluorescence lifetime imaging of various rigid and flexible fluorescent dyes to probe the biophysical properties of synthetic and biological membranes at steady state as well as upon the action of external membrane-modifying agents. We tested the solvatochromic dyes Nile red and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt) (NBD), the viscosity sensor Bodipy C12, the flipper dye FliptR, as well as the dyes 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO), Bodipy C16, lissamine-rhodamine, and Atto647, which are dyes with no previous reported environmental sensitivity. The performance of the fluorescent probes, many of which are commercially available, was benchmarked with well-known environmental reporters, with Nile red and Bodipy C12 being specific reporters of medium hydration and viscosity, respectively. We show that some widely used ordinary dyes with no previous report of sensing capabilities can exhibit competing performance compared to highly sensitive commercially available or custom-based solvatochromic dyes, molecular rotors, or flipper in a wide range of biophysics experiments. Compared to other methods, fluorescence lifetime imaging is a minimally invasive and nondestructive method with optical resolution. It enables biophysical mapping at steady state or assessment of the changes induced by membrane-active molecules at subcellular level in both synthetic and biological membranes when intensity measurements fail to do so. The results have important consequences for the specific choice of the sensor and take into consideration factors such as probe sensitivity, response to environmental changes, ease and speed of data analysis, and the probe’s intracellular distribution, as well as potential side effects induced by labeling and imaging.

In Vitro Effect of 9,9'-Norharmane Dimer against Herpes Simplex Viruses.

Herpes simplex virus (HSV) infections are highly widespread among humans, producing symptoms ranging from ulcerative lesions to severe diseases such as blindness and life-threatening encephalitis. At present, there are no vaccines available, and some existing antiviral treatments can be ineffective or lead to adverse effects. As a result, there is a need for new anti-HSV drugs. In this report, the in vitro anti-HSV effect of 9,9'-norharmane dimer (nHo-dimer), which belongs to the β-carboline (βC) alkaloid family, was evaluated. The dimer exhibited no virucidal properties and did not impede either the attachment or penetration steps of viral particles. The antiviral effect was only exerted under the constant presence of the dimer in the incubation media, and the mechanism of action was found to involve later events of virus infection. Analysis of fluorescence lifetime imaging data showed that the nHo-dimer internalized well into the cells when present in the extracellular incubation medium, with a preferential accumulation into perinuclear organelles including mitochondria. After washing the host cells with fresh medium free of nHo-dimer, the signal decreased, suggesting the partial release of the compound from the cells. This agrees with the observation that the antiviral effect is solely manifested when the alkaloid is consistently present in the incubation media.

Preparation and optical properties of rare earth fluoride and its related composite materials

Rare earth fluoride and its composite materials have shown broad application prospects in fields such as optoelectronics and photocatalysis. In this study, we prepared rare earth fluoride (β-NaYF4 nanocrystals) and its related composite materials (β-NaYF4/ZnO composite and GQDs@NaYF4 nanomaterials) using a solvothermal method and investigated their optical performance. The characterization and performance of the prepared materials were analyzed using a transmission electron microscope, X-ray diffraction, upconversion emission spectra, and fluorescence lifetime imaging microscopy. The results show that the crystal thickness of β-NaYF4 nanocrystals is approximately 45.0 nm when the F−: Ln3+ value is 6. Furthermore, X-ray diffraction analysis reveals that diffraction peak positions in β-NaYF4 nanocrystals correspond to the peak positions of standard card at different doping concentrations of Zn2+ Moreover, it was observed that the highest fluorescence lifetime of Tm3+ ion in 1G4 is 778 μs when the doping concentration of Zn2+ is 1 mol%. When the concentration of exonuclease III increases to 22.5U, the upconversion fluorescence emission intensity of GQDs@NaYF4 nanomaterial is about 34 ∗ 106 a.u. When the activation reaction time is 90 minutes, the upconversion emission intensity of GQDs@NaYF4 nanomaterial is about 37 ∗ 106 a.u. In conclusion, we successfully prepared rare earth fluoride and its composite materials with excellent optical properties through the solvothermal method.

MAdCAM-1 co-stimulation combined with retinoic acid and TGF-β induces blood CD8+ T cells to adopt a gut CD101+ TRM phenotype

Resident memory T cells (TRMs) help control local immune homeostasis and contribute to tissue protective immune responses. The local cues that guide their differentiation and localization are poorly defined. We demonstrate that MAdCAM-1, a ligand for the gut homing receptor α4β7 integrin, in the presence of retinoic acid and TGF-β provide a costimulatory signal that induces blood CD8+ T cells to adopt a TRM -like phenotype. These cells express CD103 (integrin αE) and CD69, the two major TRM cell surface markers, along with CD101. They also express CCR5, CCR9 and α4β7, three receptors associated with gut homing. A subset also express E-cadherin, a ligand for αEβ7. Fluorescent lifetime imaging indicated an αEβ7 and E-cadherin cis interaction on the plasma membrane. This report advances our understanding of the signals that drive the differentiation of CD8+ T cells into TRMs and provides a means to expand these cells in vitro, thereby affording an avenue to generate more effective tissue specific immunotherapies.

Spatial photoluminescence and lifetime mappings of quasi-2D perovskites coupled with a dielectric metasurface.

Light-matter interaction between quantum emitters and optical cavities plays a vital role in fundamental quantum photonics and the development of optoelectronics. Resonant metasurfaces are proven to be an efficient platform for tailoring the spontaneous emission (SE) of the emitters. In this work, we study the interplay between quasi-2D perovskites and dielectric TiO2 metasurfaces. The metasurface, functioning as an open cavity, enhances electric fields near its plane, thereby influencing the emissions of the perovskite. This is verified through angle-resolved photoluminescence (PL) studies. We also conducted reflectivity measurements and numerical simulations to validate the coupling between the quasi-2D perovskites and photonic modes. Notably, our work introduces a spatial mapping approach to study Purcell enhancement. Using fluorescence lifetime imaging microscopy (FLIM), we directly link the PL and lifetimes of the quasi-2D perovskites in spatial distribution when positioned on the metasurface. This correlation provides unprecedented insights into emitter distribution and emitter-resonator interactions. The methodology opens a new (to the best of our knowledge) approach for studies in quantum optics, optoelectronics, and medical imaging by enabling spatial mapping of both PL intensity and lifetime, differentiating between uncoupled quantum emitters and those coupled with different types of resonators.

Trident anchoring-lipid droplets strategy for fluorescence/super-resolution/lifetime imaging and diagnosis of fatty liver in vivo

Lipid droplets (LDs) are crucial dynamic organelles within cells that play an indispensable role in cellular energy supply, membrane formation, and signal transduction. Therefore, detecting and tracking LDs with high selectivity and resolution is particularly important. In this study, we describe a “trident” anchoring strategy (based on polarity, lipophilicity, and viscosity sensitivity) to achieve highly specific and dynamic imaging of LDs. A series of carbazole-substituted fluorescent protein (Car-FP) mimics conjugated with different electron-nature substituents (A–H) were synthesized and characterized. Spectroscopic properties indicate that the electron-donating triphenylamine (TPA)-conjugated H exhibits high sensitivity to polarity, viscosity response, and lipophilicity. Co-localization experiments in cells also demonstrate that H selectively localizes at LDs in living cells, displaying a bright yellow fluorescence signal (λex = 488 nm; λem = 577 nm) with a larger Stokes shift (89 nm) than the commercial LD dye bodipy 493/503 (10 nm). Super-resolution fluorescence imaging (SIM) and lifetime imaging (FLIM) are employed to visualize the distribution and aggregation in cell models of nonalcoholic fatty liver disease as well as zebrafish and mice models, demonstrating the potential applications of H in LD biology research and diagnosis of related diseases.

Implementation of FRET Spectrometry Using Temporally Resolved Fluorescence: A Feasibility Study.

Förster resonance energy transfer (FRET) spectrometry is a method for determining the quaternary structure of protein oligomers from distributions of FRET efficiencies that are drawn from pixels of fluorescence images of cells expressing the proteins of interest. FRET spectrometry protocols currently rely on obtaining spectrally resolved fluorescence data from intensity-based experiments. Another imaging method, fluorescence lifetime imaging microscopy (FLIM), is a widely used alternative to compute FRET efficiencies for each pixel in an image from the reduction of the fluorescence lifetime of the donors caused by FRET. In FLIM studies of oligomers with different proportions of donors and acceptors, the donor lifetimes may be obtained by fitting the temporally resolved fluorescence decay data with a predetermined number of exponential decay curves. However, this requires knowledge of the number and the relative arrangement of the fluorescent proteins in the sample, which is precisely the goal of FRET spectrometry, thus creating a conundrum that has prevented users of FLIM instruments from performing FRET spectrometry. Here, we describe an attempt to implement FRET spectrometry on temporally resolved fluorescence microscopes by using an integration-based method of computing the FRET efficiency from fluorescence decay curves. This method, which we dubbed time-integrated FRET (or tiFRET), was tested on oligomeric fluorescent protein constructs expressed in the cytoplasm of living cells. The present results show that tiFRET is a promising way of implementing FRET spectrometry and suggest potential instrument adjustments for increasing accuracy and resolution in this kind of study.

Benefits of Combined Fluorescence Lifetime Imaging Microscopy and Fluorescence Correlation Spectroscopy for Biomedical Studies Demonstrated by Using a Liposome Model System

Drug delivery systems play a pivotal role in targeted pharmaceutical transport and controlled release at specific sites. Liposomes, commonly used as drug carriers, constitute a fundamental part of these systems. Moreover, the drug–liposome model serves as a robust platform for investigating interaction processes at both cellular and molecular levels. To advance our understanding of drug carrier uptake mechanisms, we employed fluorescence lifetime imaging microscopy (FLIM) and fluorescence correlation spectroscopy (FCS), leveraging the unique benefits of two-photon (2P) excitation. Our approach utilized giant unilamellar vesicles (GUVs) as a simplified model system for cell membranes, labelled with the amphiphilic fluorescent dye 3,3′-dioctadecyloxa-carbocyanine (DiOC18(3)). Additionally, large unilamellar vesicles (LUVs) functioned as a drug carrier system, incorporating the spectrally distinct fluorescent sulforhodamine 101 (SRh101) as a surrogate drug. The investigation emphasized the diverse interactions between GUVs and LUVs based on the charged lipids employed. We examined the exchange kinetics and structural alterations of liposome carriers during the uptake process. Our study underscores the significance of employing 2P excitation in conjunction with FLIM and FCS. This powerful combination offers a valuable methodological approach for studying liposome interactions, positioning them as an exceptionally versatile model system with a distinct technical advantage.

Delignification of wood fibers using a eutectic carvacrol–methanesulfonic acid mixture analyses of the structure and fractional distribution of lignin, cellulose, and hemicellulose

AbstractDelignification and fractional pretreatments are essential for valorization of wood biomass in various bioproducts. Herein, lignocellulose wood fibers were exposed to a eutectic mixture (EM) of carvacrol and methanesulfonic acid for different times. The resulting structural and chemical alterations in biomass were explored in terms of the fiber morphology and fractional chemical composition through fiber image analysis, field-emission scanning electron microscopy, high-performance liquid chromatography, and a novel approach based on fluorescence-lifetime imaging microscopy (FLIM). The autofluorescence of the lignocellulose fibers, which was primarily due to lignin with contributions from cellulose and hemicellulose, enabled application of FLIM in lignocellulose compositional analysis in micro-scale. FLIM analysis revealed that EM treatment efficiently removed lignin from the outer fiber layers. Furthermore, the effective EM treatment time was 3 h (with a residual lignin content of ~ 7 wt%), after which defects were observed on the fibers and the cellulose chains started breaking. This degradation was also indicated by a shift of the lifetime spectra toward the fluorescence lifetime of cellulose with increasing treatment time. Overall, our findings provide valuable insights to the response of lignocellulose fibers to EM treatment, contributing to the important goal of wood biomass application in bioproducts.

Exploring Skin Interactions with 5G Millimeter-Wave through Fluorescence Lifetime Imaging Microscopy

The ongoing expansion of fifth-generation (5G) and future sixth-generation (6G) mobile communications is expected to result in widespread human exposure to millimeter-wave (mmWave) radiation globally. Given the short penetration depth of mmWaves and their high absorption by the skin, it is imperative to investigate the potential effects of 5G radiation not only in terms of temperature increase but also at the cellular level. To understand the biological mechanisms of mmWave effects, accurate methods for assessing mmWave absorption in the skin are crucial. In this study, we use fluorescence lifetime imaging microscopy (FLIM) to explore these effects. Employing a mmWave exposure system operating at 26 gigahertz (GHz), porcine skin is irradiated for varying durations (5, 10, 20, and 30 min). We investigate changes in tissue temperature and the autofluorescence of flavin adenine dinucleotide (FAD). Our findings suggest that operating our mmWave exposure systems at the configured power level of 26 GHz is unlikely to cause damage to FADs, even after a 30 min exposure duration.

Identification of different plastic types and natural materials from terrestrial environments using fluorescence lifetime imaging microscopy

Environmental pollution by plastics is a global issue of increasing concern. However, microplastic analysis in complex environmental matrices, such as soil samples, remains an analytical challenge. Destructive mass-based methods for microplastic analysis do not determine plastics’ shape and size, which are essential parameters for reliable ecological risk assessment. By contrast, nondestructive particle-based methods produce such data but require elaborate, time-consuming sample preparation. Thus, time-efficient and reliable methods for microplastic analysis are needed. The present study explored the potential of frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) for rapidly and reliably identifying as well as differentiating plastics and natural materials from terrestrial environments. We investigated the fluorescence spectra of ten natural materials from terrestrial environments, tire wear particles, and eleven different transparent plastic granulates <5 mm to determine the optimal excitation wavelength for identification and differentiation via FD-FLIM under laboratory conditions. Our comparison of different excitation wavelengths showed that 445 nm excitation exhibited the highest fluorescence intensities. 445 nm excitation was also superior for identifying plastic types and distinguishing them from natural materials from terrestrial environments with a high probability using FD-FLIM. We could demonstrate that FD-FLIM analysis has the potential to contribute to a streamlined and time-efficient direct analysis of microplastic contamination. However, further investigations on size-, shape-, color-, and material-type detection limitations are necessary to evaluate if the direct identification of terrestrial environmental samples of relatively low complexity, such as a surface inspection soil, is possible.Graphical

Dissolvable microneedles in the skin: Determination the impact of barrier disruption and dry skin on dissolution

Dissolvable microneedles (DMNs), fabricated from biocompatible materials that dissolve in both water and skin have gained popularity in dermatology. However, limited research exists on their application in compromised skin conditions. This study compares the hyaluronic acid-based DMNs penetration, formation of microchannels, dissolution, and diffusion kinetics in intact, barrier-disrupted (tape stripped), and dry (acetone-treated) porcine ear skin ex vivo. After DMNs application, comprehensive investigations including dermoscopy, stereomicroscope, skin hydration, transepidermal water loss (TEWL), optical coherence tomography (OCT), reflectance confocal laser scanning microscopy (RCLSM), confocal Raman micro-spectroscopy (CRM), two-photon tomography combined with fluorescence lifetime imaging (TPT-FLIM), histology, and scanning electron microscopy (SEM) were conducted. The 400 µm long DMNs successfully penetrated the skin to depths of ≈200 µm for dry skin and ≈200–290 µm for barrier-disrupted skin. Although DMNs fully inserted into all skin conditions, their dissolution rates were high in barrier-disrupted and low in dry skin, as observed through stereomicroscopy and TPT-FLIM. The dissolved polymer exhibited a more significant expansion in barrier-disrupted skin compared to intact skin, with the smallest increase observed in dry skin. Elevated TEWL and reduced skin hydration levels were evident in barrier-disrupted and dry skins compared to intact skin. OCT and RCLSM revealed noticeable skin indentation and pronounced microchannel areas, particularly in barrier-disrupted and dry skin. Additional confirmation of DMN effects on the skin and substance dissolution was obtained through histology, SEM, and CRM techniques. This study highlights the impact of skin condition on DMN effectiveness, emphasizing the importance of considering dissolvability and dissolution rates of needle materials, primarily composed of hyaluronic acid, for optimizing DMN-based drug delivery.

Combining sophisticated fast FLIM, confocal microscopy, and STED nanoscopy for live-cell imaging of tunneling nanotubes.

Cell-to-cell communication via tunneling nanotubes (TNTs) is a challenging topic with a growing interest. In this work, we proposed several innovative tools that use red/near-infrared dye labeling and employ lifetime-based imaging strategies to investigate the dynamics of TNTs in a living mesothelial H28 cell line that exhibits spontaneously TNT1 and TNT2 subtypes. Thanks to a fluorescence lifetime imaging microscopy module being integrated into confocal microscopy and stimulated emission depletion nanoscopy, we applied lifetime imaging, lifetime dye unmixing, and lifetime denoising techniques to perform multiplexing experiments and time-lapses of tens of minutes, revealing therefore structural and functional characteristics of living TNTs that were preserved from light exposure. In these conditions, vesicle-like structures, and tubular- and round-shaped mitochondria were identified within living TNT1. In addition, mitochondrial dynamic studies revealed linear and stepwise mitochondrial migrations, bidirectional movements, transient backtracking, and fission events in TNT1. Transfer of Nile Red-positive puncta via both TNT1 and TNT2 was also detected between living H28 cells.

Effect of time post warming to embryo transfer on human blastocyst metabolism and pregnancy outcome.

This study is aiming to test whether variation in post warming culture time impacts blastocyst metabolism or pregnancy outcome. In this single center retrospective cohort study, outcomes of 11,520 single frozen embryo transfer (FET) cycles were analyzed from January 2015 to December 2020. Patient treatments included both natural and programmed cycles. Time categories were determined using the time between blastocyst warming and embryo transfer: 0 (0- <1h), 1 (1-<2h), 2 (2-<3h), 3(3-<4h), 4 (4-<5), 5 (5-<6), 6 (6-<7) and 7 (7-8h). Non-invasive metabolic imaging of discarded human blastocysts for up to 10h was also performed using Fluorescence lifetime imaging microscopy (FLIM) to examine for metabolic perturbations during culture. The mean age of patients across all time categories were comparable (35.6 ± 3.9). Live birth rates (38-52%) and miscarriage rate (5-11%) were not statistically different across post-warming culture time. When assessing pregnancy outcomes based on the use of PGT-A, miscarriage and live birth rates were not statistically different across culture hours in both PGT-A and non-PGT cycles. Further metabolic analysis of blastocysts for the duration of 10h of culture post warming, revealed minimal metabolic changes of embryos in culture. Overall, our results show that differences in the time of post warming culture have no significant impact on miscarriage or live birth rate for frozen embryo transfers. This information can be beneficial for clinical practices with either minimal staffing or a high number of patient cases.

Two-photon autofluorescence lifetime assay of rabbit photoreceptors and retinal pigment epithelium during light-dark visual cycles in rabbit retina

Two-photon excited fluorescence (TPEF) is a powerful technique that enables the examination of intrinsic retinal fluorophores involved in cellular metabolism and the visual cycle. Although previous intensity-based TPEF studies in non-human primates have successfully imaged several classes of retinal cells and elucidated aspects of both rod and cone photoreceptor function, fluorescence lifetime imaging (FLIM) of the retinal cells under light-dark visual cycle has yet to be fully exploited. Here we demonstrate a FLIM assay of photoreceptors and retinal pigment epithelium (RPE) that reveals key insights into retinal physiology and adaptation. We found that photoreceptor fluorescence lifetimes increase and decrease in sync with light and dark exposure, respectively. This is likely due to changes in all-trans-retinol and all-trans-retinal levels in the outer segments, mediated by phototransduction and visual cycle activity. During light exposure, RPE fluorescence lifetime was observed to increase steadily over time, as a result of all-trans-retinol accumulation during the visual cycle and decreasing metabolism caused by the lack of normal perfusion of the sample. Our system can measure the fluorescence lifetime of intrinsic retinal fluorophores on a cellular scale, revealing differences in lifetime between retinal cell classes under different conditions of light and dark exposure.

Classification of Neural Stem Cell Activation State In Vitro using Autofluorescence.

Neural stem cells (NSCs) divide and produce newborn neurons in the adult brain through a process called adult neurogenesis. Adult NSCs are primarily quiescent, a reversible cell state where they have exited the cell cycle (G0) yet remain responsive to the environment. In the first step of adult neurogenesis, quiescent NSCs (qNSCs) receive a signal and activate, exiting quiescence and re-entering the cell cycle. Thus, understanding the regulators of NSC quiescence and quiescence exit is critical for future strategies targeting adult neurogenesis. However, our understanding of NSC quiescence is limited by technical constraints in identifying quiescent NSCs (qNSCs) and activated NSCs (aNSCs). This protocol describes a new approach to identify and enrich qNSCs and aNSCs generated in in vitro cultures by imaging NSC autofluorescence. First, this protocol describes how to use a confocal microscope to identify autofluorescent markers of qNSCs and aNSCs to classify NSC activation state using autofluorescence intensity. Second, this protocol describes how to use a fluorescent activated cell sorter (FACS) to classify NSC activation state and enrich samples for qNSCs or aNSCs using autofluorescence intensity. Third, this protocol describes how to use a multiphoton microscope to perform fluorescence lifetime imaging (FLIM) at single-cell resolution, classify NSC activation state, and track the dynamics of quiescent exit using both autofluorescence intensitiesand fluorescence lifetimes. Thus, this protocol provides a live-cell, label-free, single-cell resolution toolkit for studying NSC quiescence and quiescence exit.