Two-photon Fluorescence Microscopy Research Articles

Temperature-Dependent Intensity Modulated Two-Photon Excited Fluorescence Microscopy for High Resolution Mapping of Charge Carrier Dynamics.

We present a temperature-dependent intensity modulated two-photon excited fluorescence microscopy technique that enables high-resolution quantitative mapping of charge carrier dynamics in perovskite microcrystal film. By disentangling the emission into harmonics of the excitation modulation frequency, we analyze the first and second order charge carrier recombination processes, including potential accumulation effects. Our approach allows for a quantitative comparison of different emission channels at a micrometer resolution. To demonstrate the effectiveness of the method, we applied it to a methylammonium lead bromide perovskite microcrystal film. We investigated the temperature-dependent modulated imaging, encompassing the exciton dissociation-association and charge carrier trapping-detrapping equilibrium. Additionally, we explored the potential freezing out of traps and the phase transition occurring at low temperatures.

Strongly Emitting Folic Acid-Derived Carbon Nanodots for One- and Two-Photon Imaging of Lyotropic Myelin Figures.

Non-invasive imaging of morphological changes in biologically relevant lipidic mesophases is essential for the understanding of membrane-mediated processes. However, its methodological aspects need to be further explored, with particular attention paid to the design of new excellent fluorescent probes. Here, we have demonstrated that bright and biocompatible folic acid-derived carbon nanodots (FA CNDs) may be successfully applied as fluorescent markers in one- and two-photon imaging of bioinspired myelin figures (MFs). Structural and optical properties of these new FA CNDs were first extensively characterized; they revealed remarkable fluorescence performance in linear and non-linear excitation regimes, justifying further applications. Then, confocal fluorescence microscopy and two-photon excited fluorescence microscopy were used to investigate a three-dimensional distribution of FA CNDs within the phospholipid-based MFs. Our results showed that FA CNDs are effective markers for imaging various forms and parts of multilamellar microstructures.

Adaptive Optical Two-Photon Fluorescence Microscopy Probes Cellular Organization of Ocular Lenses In Vivo.

The mammalian ocular lens is an avascular multicellular organ that grows continuously throughout life. Traditionally, its cellular organization is investigated using dissected lenses, which eliminates in vivo environmental and structural support. Therefore, in vivo optical imaging methods for studying lenses in their native context in live animals are urgently needed. Here, we demonstrated that two-photon fluorescence microscopy can visualize lens cells in vivo. To maintain subcellular resolution at depth, we used adaptive optics to correct aberrations owing to ocular and lens tissues, which led to substantial signal and resolution improvements. Imaging lens cells up to 980 µm deep, we observed novel cellular organizations including suture-associated voids, enlarged vacuoles, and large cavities, contrary to the conventional view of a highly ordered organization. We tracked these features longitudinally over weeks and observed the incorporation of new cells during growth. Taken together, noninvasive longitudinal in vivo imaging of lens morphology using adaptive optics two-photon fluorescence microscopy will allow us to observe the development or alterations of lens cellular organization in living animals directly.

A novel pyrimidine-based two-photon fluorogenic probe for rapidly visualizing nitroreductase activity in hypoxic cancer cells and in vivo

Nitroreductase (NTR) is an overexpressed enzyme in hypoxic tumor microenvironment that can be a specific recognition site for tumor diagnosis in clinical trials. Therefore, the development of probs for NTR activity with precision and instantaneity is significant. In this work, a NTR probe (ZJ) was constructed on the structure of pyrimidine derivatives with 4-animobenzyl carbamate groups. ZJ exhibited a responsive “turn-on” two-photon excited fluorescence by NTR recognition in cells and in vivo. The 4-nitrobenzyl carbamate groups in ZJ could be reduced by NTR to 4-animobenzyl carbamate groups in the presence of reduced nicotinamide adenine dinucleotide (NADH). Attributed to its self-immolation property, 4-animobenzyl carbamate would release to generate primary amine on pyrimidine, accompanied by fluorescence signal recovery of ZJ. Such responsive fluorescence turn-on process accomplished as fast as within 20 mins. The favorable stability and specificity of ZJ on NTR activity sensing was further illustrated according to the negligible interference by biological factors and the distinguishing on different NTR types. ZJ successfully visualized the distribution of over-expressed NTR in hypoxic cancer cells and in zebrafish using two-photon fluorescence microscopy, which demonstrated the potential application in studying the relationship between tumor hypoxic microenvironment and cancer.

Enhancing Total Optical Throughput of Microscopy with Deep Learning for Intravital Observation.

The significance of performing large-depth dynamic microscopic imaging in vivo for life science research cannot be overstated. However, the optical throughput of the microscope limits the available information per unit of time, i.e., it is difficult to obtain both high spatial and temporal resolution at once. Here, a method is proposed to construct a kind of intravital microscopy with high optical throughput, by making near-infrared-II (NIR-II, 900-1880nm) wide-field fluorescence microscopy learn from two-photon fluorescence microscopy based on a scale-recurrent network. Using this upgraded NIR-II fluorescence microscope, vessels in the opaque brain of a rodent are reconstructed three-dimensionally. Five-fold axial and thirteen-fold lateral resolution improvements are achieved without sacrificing temporal resolution and light utilization. Also, tiny cerebral vessel dilatations in early acute respiratory failure mice are observed, with this high optical throughput NIR-II microscope at an imaging speed of 30fps.

Retinal microvascular and neuronal pathologies probed in vivo by adaptive optical two-photon fluorescence microscopy.

The retina, behind the transparent optics of the eye, is the only neural tissue whose physiology and pathology can be non-invasively probed by optical microscopy. The aberrations intrinsic to the mouse eye, however, prevent high-resolution investigation of retinal structure and function in vivo. Optimizing the design of a two-photon fluorescence microscope (2PFM) and sample preparation procedure, we found that adaptive optics (AO), by measuring and correcting ocular aberrations, is essential for resolving putative synaptic structures and achieving three-dimensional cellular resolution in the mouse retina in vivo. Applying AO-2PFM to longitudinal retinal imaging in transgenic models of retinal pathology, we characterized microvascular lesions with sub-capillary details in a proliferative vascular retinopathy model, and found Lidocaine to effectively suppress retinal ganglion cell hyperactivity in a retinal degeneration model. Tracking structural and functional changes at high-resolution longitudinally, AO-2PFM enables microscopic investigations of retinal pathology and pharmacology for disease diagnosis and treatment in vivo.

A dataset of rodent cerebrovasculature from in vivo multiphoton fluorescence microscopy imaging

We present MiniVess, the first annotated dataset of rodent cerebrovasculature, acquired using two-photon fluorescence microscopy. MiniVess consists of 70 3D image volumes with segmented ground truths. Segmentations were created using traditional image processing operations, a U-Net, and manual proofreading. Code for image preprocessing steps and the U-Net are provided. Supervised machine learning methods have been widely used for automated image processing of biomedical images. While much emphasis has been placed on the development of new network architectures and loss functions, there has been an increased emphasis on the need for publicly available annotated, or segmented, datasets. Annotated datasets are necessary during model training and validation. In particular, datasets that are collected from different labs are necessary to test the generalizability of models. We hope this dataset will be helpful in testing the reliability of machine learning tools for analyzing biomedical images.

Fiber enhancement and 3D orientation analysis in label-free two-photon fluorescence microscopy

Fluorescence microscopy can be exploited for evaluating the brain’s fiber architecture with unsurpassed spatial resolution in combination with different tissue preparation and staining protocols. Differently from state-of-the-art polarimetry-based neuroimaging modalities, the quantification of fiber tract orientations from fluorescence microscopy volume images entails the application of specific image processing techniques, such as Fourier or structure tensor analysis. These, however, may lead to unreliable outcomes as they do not isolate myelinated fibers from the surrounding tissue. In this work, we describe a novel image processing pipeline that enables the computation of accurate 3D fiber orientation maps from both grey and white matter regions, exploiting the selective multiscale enhancement of tubular structures of varying diameters provided by a 3D implementation of the Frangi filter. The developed software tool can efficiently generate orientation distribution function maps at arbitrary spatial scales which may support the histological validation of modern diffusion-weighted magnetic resonance imaging tractography. Despite being tested here on two-photon scanning fluorescence microscopy images, acquired from tissue samples treated with a label-free technique enhancing the autofluorescence of myelinated fibers, the presented pipeline was developed to be employed on all types of 3D fluorescence images and fiber staining.

Imaging mitochondria through bone in live mice using two-photon fluorescence microscopy with adaptive optics.

Mitochondria are extremely important organelles in the regulation of bone marrow and brain activity. However, live imaging of these subcellular features with high resolution in scattering tissues like brain or bone has proven challenging. In this study, we developed a two-photon fluorescence microscope with adaptive optics (TPFM-AO) for high-resolution imaging, which uses a home-built Shack-Hartmann wavefront sensor (SHWFS) to correct system aberrations and a sensorless approach for correcting low order tissue aberrations. Using AO increases the fluorescence intensity of the point spread function (PSF) and achieves fast imaging of subcellular organelles with 400 nm resolution through 85 μm of highly scattering tissue. We achieved ~1.55×, ~3.58×, and ~1.77× intensity increases using AO, and a reduction of the PSF width by ~0.83×, ~0.74×, and ~0.9× at the depths of 0, 50 μm and 85 μm in living mouse bone marrow respectively, allowing us to characterize mitochondrial health and the survival of functioning cells with a field of view of 67.5× 67.5 μm. We also investigate the role of initial signal and background levels in sample correction quality by varying the laser power and camera exposure time and develop an intensity-based criteria for sample correction. This study demonstrates a promising tool for imaging of mitochondria and other organelles in optically distorting biological environments, which could facilitate the study of a variety of diseases connected to mitochondrial morphology and activity in a range of biological tissues.

Otolin-1, an otolith- and otoconia-related protein, controls calcium carbonate bioinspired mineralization

BackgroundOtoliths and otoconia are calcium carbonate biomineral structures that form in the inner ear of fish and humans, respectively. The formation of these structures is tightly linked to the formation of an organic matrix framework with otolin-1, a short collagen-like protein from the C1q family as one of its major constituents. MethodsIn this study, we examined the activity of recombinant otolin-1 originating from Danio rerio and Homo sapiens on calcium carbonate bioinspired mineralization with slow-diffusion method and performed crystals characterization with scanning electron microscopy, two-photon excited fluorescence microscopy, confocal laser scanning microscopy and micro-Raman spectroscopy. ResultsWe show that both proteins are embedded in the core of CaCO3 crystals that form through the slow-diffusion mineralization method. Both of them influence the morphology but do not change the polymorphic mineral phase. D.rerio otolin-1 also closely adheres to the crystal surface. General significanceThe results suggest, that otolin-1 is not a passive scaffold, but is directly involved in regulating the morphology of the resulting calcium carbonate biocrystals.

Time-resolved two-photon fluorescence dynamics of a novel mEGFP-linker-mScarlet-I crowding biosensor.

Laser-induced two-photon (2P) fluorescence microscopy is routinely used as a noninvasive, quantitative methodology for a wide range of cell and tissue studies due to its enhanced penetration depth, reduced laser scattering, and minimum extended photobleaching. Macromolecular crowding affects many cellular processes such as diffusion, biochemical reaction kinetics, protein-protein interactions, and protein folding. Here, we carried out time-resolved 2P fluorescence measurements of a novel mEGFP-linker-mScarlet-I macromolecular crowding construct (GE2.3) to characterize its environmental sensitivity in biomimetic crowded solutions (Ficoll-70, 0-300 g/L) using Förster resonance energy transfer (FRET) analysis. For these studies, the excited-state 2P-fluorescence dynamics of the donor (mEGFP) were investigated in the presence (intact) and absence (enzymatically cleaved) of the acceptor (mScarlet-I). In addition, time-resolved 2P-fluorescence measurements of intact GE2.3 were used to determine the corresponding equilibrium constant and the Gibbs free energy associated with the structural conformation equilibrium in response to environmental crowding. We further carried out time-resolved 2P-fluorescence depolarization anisotropy to examine both the macromolecular crowding and the linker flexibility effects on GE2.3 rotational dynamics within the context of Stokes-Einstein model as compared with theoretical predictions based on its molecular weight. Our results further the development of a rational engineering design of bioenvironmental sensors. Additionally, these results in well-defined environments will inform our future in vivo studies of genetically encoded GE2.3 towards the mapping of crowded intracellular environment under different physiological conditions. Importantly, this GE2.3 sensor allows for minimal interference of the intrinsic cellular autofluorescence with the FRET analysis and crowding studies.

Research advances in nondegenerate two-photonabsorption and its applications

Nondegenerate two-photon absorption is a nonlinear optical effect in which two photons with different energy are absorbed by a medium simultaneously, resulting in a single electron transition from ground state to excited state through an intermediate virtual state. Compared with the degenerate two-photon absorption coefficient, the absorption coefficient of nondegenerate two-photon absorption is increased by tens or even hundreds of times due to the intermediate resonance effect, so it has great potentials in many nonlinear optical applications. Firstly, the basic principle of two-photon absorption is introduced and the enhancement mechanism of non-degenerate two-photon absorption is explained in this paper. Secondly, the basic method of measuring two-photon absorption is introduced in detail. Thirdly, the reports on nondegenerate two-photon absorption of three-dimensional semiconductor materials and two-dimensional materials are reviewed. Finally, the application progress of infrared detection and imaging, two-photon fluorescence microscope, all-optical switch and optical modulation is summarized, and the future research in this field is summarized and prospected.

An astrocytic signaling loop for frequency-dependent control of dendritic integration and spatial learning

Dendrites of hippocampal CA1 pyramidal cells amplify clustered glutamatergic input by activation of voltage-gated sodium channels and N-methyl-D-aspartate receptors (NMDARs). NMDAR activity depends on the presence of NMDAR co-agonists such as D-serine, but how co-agonists influence dendritic integration is not well understood. Using combinations of whole-cell patch clamp, iontophoretic glutamate application, two-photon excitation fluorescence microscopy and glutamate uncaging in acute rat and mouse brain slices we found that exogenous D-serine reduced the threshold of dendritic spikes and increased their amplitude. Triggering an astrocytic mechanism controlling endogenous D-serine supply via endocannabinoid receptors (CBRs) also increased dendritic spiking. Unexpectedly, this pathway was activated by pyramidal cell activity primarily in the theta range, which required HCN channels and astrocytic CB1Rs. Therefore, astrocytes close a positive and frequency-dependent feedback loop between pyramidal cell activity and their integration of dendritic input. Its disruption in mice led to an impairment of spatial memory, which demonstrated its behavioral relevance.

Biomimetic design of bioartificial scaffolds for the in vitro modelling of human cardiac fibrosis.

In vitro models of pathological cardiac tissue have attracted interest as predictive platforms for preclinical validation of therapies. However, models reproducing specific pathological features, such as cardiac fibrosis size (i.e., thickness and width) and stage of development are missing. This research was aimed at engineering 2D and 3D models of early-stage post-infarct fibrotic tissue (i.e., characterized by non-aligned tissue organization) on bioartificial scaffolds with biomimetic composition, design, and surface stiffness. 2D scaffolds with random nanofibrous structure and 3D scaffolds with 150µm square-meshed architecture were fabricated from polycaprolactone, surface-grafted with gelatin by mussel-inspired approach and coated with cardiac extracellular matrix (ECM) by 3weeks culture of human cardiac fibroblasts. Scaffold physicochemical properties were thoroughly investigated. AFM analysis of scaffolds in wet state, before cell culture, confirmed their close surface stiffness to human cardiac fibrotic tissue. Following 3weeks culture, biomimetic biophysical and biochemical scaffold properties triggered the activation of myofibroblast phenotype. Upon decellularization, immunostaining, SEM and two-photon excitation fluorescence microscopy showed homogeneous decoration of both 2D and 3D scaffolds with cardiac ECM. The versatility of the approach was demonstrated by culturing ventricular or atrial cardiac fibroblasts on scaffolds, thus suggesting the possibility to use the same scaffold platforms to model both ventricular and atrial cardiac fibrosis. In the future, herein developed in vitro models of cardiac fibrotic tissue, reproducing specific pathological features, will be exploited for a fine preclinical tuning of therapies.

2-hydroxyisobutyric acid (2-HIBA) modulates ageing and fat deposition in Caenorhabditis elegans.

High levels of 2-hydroxyisobutyric acid (2-HIBA) were found in urines of patients with obesity and hepatic steatosis, suggesting a potential involvement of this metabolite in clinical conditions. The gut microbial origin of 2-HIBA was hypothesized, however its actual origin and role in biological processes are still not clear. We investigated how treatment with 2-HIBA affected the physiology of the model organism Caenorhabditis elegans, in both standard and high-glucose diet (HGD) growth conditions, by targeted transcriptomic and metabolomic analyses, Coherent Anti-Stokes Raman Scattering (CARS) and two-photon fluorescence microscopy. In standard conditions, 2-HIBA resulted particularly effective to extend the lifespan, delay ageing processes and stimulate the oxidative stress resistance in wild type nematodes through the activation of insulin/IGF-1 signaling (IIS) and p38 MAPK pathways and, consequently, through a reduction of ROS levels. Moreover, variations of lipid accumulation observed in treated worms correlated with transcriptional levels of fatty acid synthesis genes and with the involvement of peptide transporter PEP-2. In HGD conditions, the effect of 2-HIBA on C. elegans resulted in a reduction of the lipid droplets deposition, accordingly with an increase of acs-2 gene transcription, involved in β-oxidation processes. In addition, the pro-longevity effect appeared to be correlated to higher levels of tryptophan, which may play a role in restoring the decreased viability observed in the HGD untreated nematodes.

208 High-quality fluorescent solvatochromic three-dimensional imaging for dermatopathology with a two-photon excitation laser microscopy

Histopathological examination with a sliced tissue stained with hematoxylin-eosin dye (HE) is a fundamental procedure. However, a sliced tissue prepared as the vertical-cross section has only a few micrometers thick; hence several cross-section views need a composite image to see a horizontal cross-section view of a three-dimensional image. A confocal scanning laser microscope or two-photon excitation fluorescent microscopy is suitable for drawing fine pictures of three-dimensional and deep images to obtain a three-dimensional view of the skin tissue.

Long-Term Repeatable In Vivo Monitoring of Amyloid-β Plaques and Vessels in Alzheimer's Disease Mouse Model with Combined TPEF/CARS Microscopy.

Long-term, repeatable monitoring of the appearance and progress of Alzheimer's disease (AD) in real time can be extremely beneficial to acquire highly reliable diagnostic insights, which is crucial for devising apt strategies towards effective AD treatment. Herein, we present an optimized innovative cranial window imaging method for the long-term repeatable imaging of amyloid-β (Aβ) plaques and vessels in an AD mouse model. Basically, two-photon excitation fluorescence (TPEF) microscopy was used to monitor the fluorescently labeled Aβ plaques, whereas the label-free blood vessels were studied using coherent anti-Stokes Raman scattering (CARS) microscopy in the live in vivo AD mouse model. It was possible to clearly observe the Aβ deposition and vascular structure in the target cortex localization for 31 weeks in the AD mouse model using this method. The combined TPEF/CARS imaging studies were also instrumental in realizing the relationship between the tendency of Aβ deposition and ageing. Essentially, the progression of cerebral amyloid angiopathy (CAA) in the AD mouse model was quantitatively characterized, which revealed that the proportion Aβ deposition in the unit vessel can increase from 13.63% to 28.80% upon increasing the age of mice from 8 months old to 14 months old. The proposed imaging method provided an efficient, safe, repeatable platform with simple target localization aptitude towards monitoring the brain tissues, which is an integral part of studying any brain-related physiological or disease conditions to extract crucial structural and functional information.

SURG-24. QUANTITATIVE ANALYSIS OF TUMOR CELL DENSITY AND PROTOPORPHYRIN IX FLOURESCENCE IN GLIOMA PATIENTS USING PAIRED STIMULATED RAMAN HISTOLOGY AND TWO-PHOTON EXCITATION FLUORESCENCE MICROSCOPY

Abstract Fluorescence guidance is widely utilized to improve the precision of cancer surgery. 5-aminolevulinic acid, the most widely used fluorophore in glioma surgery, is thought to cause selective accumulation of fluorescent protoporphyrin IX (PpIX) in tumor cells by exploiting pathologic alterations in heme biosynthetic pathways. PpIX-induced fluorescence is highly specific for densely tumor-infiltrated tissue but less effective for visualizing the tumor periphery. To improve clinical detection of PpIX, we developed a microscope to perform paired stimulated Raman histology and two-photon excitation fluorescence microscopy (TPEF) and validated it in 175 fresh, unprocessed core tumor specimens from 75 high-grade glioma patients and three central nervous system lymphoma patients across three institutions. Surprisingly, intracellular PpIX accumulation was observed primarily in cells with histiocytic, rather than neoplastic morphology, and the number of cells concentrating PpIX within the cytoplasm was associated with the abundance of CD163 positive cells (p< 0.02). There was no correlation between the degree of tumor cellularity and the concentration of PpIX across all imaged specimens (R=-0.21). Our findings encourage reconsideration of the existing theory of 5-ALA-induced tumor cell fluorescence in gliomas and demonstrate how 5-ALA and TPEF imaging can provide a window into the immune microenvironment of human gliomas.

Glass-ionomer and calcium silicate-based cements interactions with human dentine in health and disease: Two-photon fluorescence microscopy and Raman spectroscopy analysis.

To investigate the potential mineralising effects of calcium silicate-based dentine replacement material (Biodentine™) in comparison with glass-ionomer cement (GIC) (Fuji IX™) on different human dentine substrates using a multimodal non-invasive optical assessment. Cements were applied on artificially demineralised or naturally carious dentine and stored for 4 weeks in phosphate-rich media +/- tetracycline used for mineralisation labelling. Interfacial dentine was examined from the same sample and location before and after aging using two-photon fluorescence microscopy, fluorescence lifetime imaging (FLIM) and second harmonic generation (SHG) imaging. Additionally, Raman spectroscopy was used to detect changes in the mineral content of dentine. Significant changes in the fluorescence intensity and lifetime were detected in partially demineralised dentine and caries-affected dentine underneath both tested cements, after storage (p<0.001). This was associated with a significant increase in the mineral content as indicated by the increased intensity of the phosphate Raman peak located at 959cm-1 (p<0.0001). Caries-infected dentine showed significant fluorescence changes under Biodentine™ after storage (p<0.001), but not under GIC (p=0.44). Tetracycline binding induced a reduction in the fluorescence lifetime with comparable increase in the fluorescence intensity in both cements' groups within the affected dentine (p<0.001). Significance Two-photon fluorescence microscopy can be used efficiently for non-destructive in-vitro dentine caries characterisation providing a technique for studying the same dentine-cement interface over time and detect changes. Biodentine™ demonstrated comparable remineralising potential to GIC, in addition to inducing remineralisation of caries-infected dentine. This may suggest using Biodentine™ as part of minimally invasive operative dentistry (MID) in caries management.

Compact multicolor two-photon fluorescence microscopy enabled by tailorable continuum generation from self-phase modulation and dispersive wave generation.

By precisely managing fiber-optic nonlinearity with anomalous dispersion, we have demonstrated the control of generating plural few-optical-cycle pulses based on a 24-MHz Chromium:forsterite laser, allowing multicolor two-photon tissue imaging by wavelength mixing. The formation of high-order soliton and its efficient coupling to dispersive wave generation leads to phase-matched spectral broadening, and we have obtained a broadband continuum ranging from 830 nm to 1200 nm, delivering 5-nJ pulses with a pulse width of 10.5 fs using a piece of large-mode-area fiber. We locate the spectral enhancement at around 920 nm for the two-photon excitation of green fluorophores, and we can easily compress the resulting pulse close to its limited duration without the need for active pulse shaping. To optimize the wavelength mixing for sum-frequency excitation, we have realized the management of the power ratio and group delay between the soliton and dispersive wave by varying the initial pulse energy without additional delay control. We have thus demonstrated simultaneous three-color two-photon tissue imaging with contrast management between different signals. Our source optimization leads to efficient two-photon excitation reaching a 500-µm imaging depth under a low 14-mW illumination power. We believe our source development leads to an efficient and compact approach for driving multicolor two-photon fluorescence microscopy and other ultrafast investigations, such as strong-field-driven applications.