Two-photon Microscopy Imaging Research Articles

Structural changes in the crystalline lens as a function of the postmortem interval assessed with two-photon imaging microscopy

Structural changes in the crystalline lens as a function of the postmortem interval assessed with two-photon imaging microscopy

Multicolor Two-Photon Microscopy Imaging of Lipid Droplets and Liver Capsule Macrophages In Vivo.

Lipid droplets (LDs) store energy and supply fatty acids and cholesterol. LDs are a hallmark of chronic nonalcoholic fatty liver disease (NAFLD). Recently, studies have focused on the role of hepatic macrophages in NAFLD. Green fluorescent protein (GFP) is used for labeling the characteristic targets in bioimaging analysis. Cx3cr1-GFP mice are widely used in studying the liver macrophages such as the NAFLD model. Here, we have developed a tool for two-photon microscopic observation to study the interactions between LDs labeled with LD2 and liver capsule macrophages labeled with GFP in vivo. LD2, a small-molecule two-photon excitation fluorescent probe for LDs, exhibits deep-red (700 nm) fluorescence upon excitation at 880 nm, high cell staining ability and photostability, and low cytotoxicity. This probe can clearly observe LDs through two-photon microscopy (TPM) and enables the simultaneous imaging of GFP+ liver capsule macrophages (LCMs) in vivo in the liver capsule of Cx3cr1-GFP mice. In the NAFLD mouse model, Cx3cr1+ LCMs and LDs increased with the progress of fatty liver disease, and spatiotemporal changes in LCMs were observed through intravital 3D TPM images. LD2 will aid in studying the interactions and immunological roles of hepatic macrophages and LDs to better understand NAFLD.

Integrated Microprism and Microelectrode Array for Simultaneous Electrophysiology and Two-Photon Imaging across All Cortical Layers.

Cerebral neural electronics play a crucial role in neuroscience research with increasing translational applications such as brain-computer interfaces for sensory input and motor output restoration. While widely utilized for decades, the understanding of the cellular mechanisms underlying this technology remains limited. Although two-photon microscopy (TPM) has shown great promise in imaging superficial neural electrodes, its application to deep-penetrating electrodes is technically difficult. Here, a novel device integrating transparent microelectrode arrays with glass microprisms, enabling electrophysiology recording and stimulation alongside TPM imaging across all cortical layers in a vertical plane, is introduced. Tested in Thy1-GCaMP6 mice for over 4 months, the integrated device demonstrates the capability for multisite electrophysiological recording/stimulationand simultaneous TPM calcium imaging. As a proof of concept, the impact of microstimulation amplitude, frequency, and depth on neural activation patterns is investigated using the setup. With future improvements in material stability and single unit yield, this multimodal tool greatly expands integrated electrophysiology and optical imaging from the superficial brain to the entire cortical column, opening new avenues for neuroscience research and neurotechnology development.

506 Deep Learning-Based Image-to-Image Translation to Identify Macrophage Infiltration in High-Grade Glioma Using Label-Free Stimulated Raman Histology

INTRODUCTION: 5-Aminolevulinic acid (5-ALA) is the most widely used fluorophore in image-guided glioma surgery, and previous work at our institution demonstrated that 5-ALA highlights tumor-associated macrophages (TAMs) in two-photon microscopy images of brain tumor tissue. Using a unique, paired dataset of stimulated Raman histology (SRH) and two-photon images that share one-to-one spatial resolution, we propose a deep-learning approach to identify macrophages from intraoperative SRH images without requiring fluorescent labels. METHODS: We compiled a dataset of 9,554 non-overlapping, 300-by-300 pixel fields of view from paired SRH and two-photon images representing 40 cases of high-grade glioma. A deep generative adversarial network (pix2pix), consisting of a U-Net generator and PatchGAN discriminator, was trained to generate synthetic two-photon images from each SRH patch. The model was trained for 200 epochs, and similarity between the synthetic and real distributions was assessed qualitatively while training and quantitatively using FrÉchet inception distance (FID). RESULTS: Our model was successfully trained to generate synthetic two-photon images nearly indistinguishable from ground truth examples. The FID between a held-out test set of real and synthetic images was 8.58, compared to a mean FID of 9.26 ± 0.24 between randomly sampled sets of real images. Our model consistently inferred the location of brightly fluorescing TAMs in held-out, previously unseen test images. CONCLUSIONS: We used deep learning to visualize TAMs in label-free SRH images. Analysis of TAM infiltration has the potential to identify patients most likely to benefit from immunotherapy clinical trials. Ongoing work will leverage additional deep-learning approaches to automatically identify and quantify TAM infiltration with SRH, enabling rapid patient-specific analysis of the glioma immune microenvironment.

Strategic Construction of meso-Aryl-Substituted N,N-Carbonyl-Bridged Dipyrrinones as Small, Bright, and Tunable Fluorophores.

A series of novel N,N-carbonyl-bridged dipyrrinone fluorophores have been directly constructed from α-halogenated dipyrrinones, which are conveniently obtained from the acid-catalyzed hydrolysis of readily available α,α'-dihalodipyrrins. This novel methodology affords efficient modulation of the functional groups at both the meso- and α-positions of this fluorophore. These resultant dyes show tunable absorption and emission wavelengths, good molar absorption coefficients, relatively large Stokes shifts, and excellent fluorescence quantum yields up to 0.99, and have been successfully applied in both one- and two-photon fluorescence microscopy imaging in living cells.

A 20 MHz Repetition Rate, Sub-Picosecond Ti-Sapphire Laser for Fiber Delivery in Nonlinear Microscopy of the Skin.

Nonlinear microscopy (NM) enables us to investigate the morphology or monitor the physiological processes of the skin through the use of ultrafast lasers. Fiber (or fiber-coupled) lasers are of great interest because they can easily be combined with a handheld, scanning nonlinear microscope. This latter feature greatly increases the utility of NM for pre-clinical applications and in vivo tissue imaging. Here, we present a fiber-coupled, sub-ps Ti-sapphire laser system being optimized for in vivo, stain-free, 3D imaging of skin alterations with a low thermal load of the skin. The laser is pumped by a low-cost, 2.1 W, 532 nm pump laser and delivers 0.5-1 ps, high-peak-power pulses at a ~20 MHz repetition rate. The spectral bandwidth of the laser is below 2 nm, which results in a low sensitivity for dispersion during fiber delivery. The reduction in the peak intensity due to the increased pulse duration is compensated by the lower repetition rate of our laser. In our proof-of-concept imaging experiments, a ~1.8 m long, commercial hollow-core photonic bandgap fiber was used for fiber delivery. Fresh and frozen skin biopsies of different skin alterations (e.g., adult hemangioma, basal cell cancer) and an unaffected control were used for high-quality, two-photon excitation fluorescence microscopy (2PEF) and second-harmonic generation (SHG) z-stack (3D) imaging.

Pyrene-fused dioxaborine-based merocyanines with high brightness, photostability, and fluorogenic function for deep-skin tissue imaging of a living mouse

Abstract Bright polymethine dyes, typified by carbocyanines, are employed in various fluorescence techniques such as the 3D visualization of living cell morphology and the tracking of extracellular vesicles in the blood vessels of a zebrafish. However, they often exhibit low photostability, particularly for dyes with red-shifted absorption/fluorescence wavelengths due to extended polymethine length, and limited photofunctionality. This limitation restricts their utility in specific applications requiring high-power excitation and/or a wash-free approaches. This study introduces novel merocyanine dyes, MCPY3 and MCPY5, comprising a newly developed pyrene-fused dioxaborine and polymethine chain. Despite their minimal polymethine lengths, their absorption/fluorescence wavelengths reside in the red to near infra-red regions due to the substantial π-conjugation system of pyrene. Moreover, they exhibit a considerably superior photostability to carbocyanine dyes and fluorogenic behavior between low (ON) and high (OFF) polar solvents, while maintaining brightness comparable to carbocyanine. Leveraging these advantages, the hydrophilic analogs of MCPY3, MCPY3S, were applied to two-photon microscopy imaging of the skin tissues on the finger of a living mouse. The dye clearly visualized the individual cell morphology in the epidermis and the elastin within the dermis, highlighting the potential of the new dye as a valuable tool for fundamental dermatological and histological studies.

Label-free multimodal imaging with simultaneous two-photon and three-photon microscopy and kernel-based nonlinear scaling denoising.

We report on a compact multimodal imaging system that can acquire two-photon microscopy (2PM) and three-photon microscopy (3PM) images simultaneously. With dual excitation wavelengths, multiple contrasts including two-photon-excitation-fluorescence (2PEF), second harmonic generation (SHG), and third harmonic generation (THG) are acquired simultaneously from cells, collagen fibers, and interfaces, all label-free. Challenges related to the excitation by two wavelengths and the effective separation of 2PM and 3PM signals are discussed and addressed. The data processing challenge where multiple contrasts can have significantly varying signal levels is also addressed. A kernel-based nonlinear scaling (KNS) denoising method is introduced to reduce noise from ultra-low signal images and generate high-quality multimodal images. Simultaneous 2PM and 3PM imaging is demonstrated on various tissue samples. The simultaneous acquisition speeds up the imaging process and minimizes the commonly encountered problem of motion artifacts and mechanical drift in sequential acquisition. Multimodal imaging with simultaneous 2PM and 3PM will have great potential for label-free in-vivo imaging of biological tissues.

TMIC-84. A DEEP LEARNING-BASED METHOD FOR RAPID, PATIENT-SPECIFIC ASSAY OF MACROPHAGE INFILTRATION IN HIGH-GRADE GLIOMA USING LABEL-FREE STIMULATED RAMAN HISTOLOGY

Abstract BACKGROUND 5-Aminolevulinic acid (5-ALA), the most widely used fluorophore in image-guided glioma surgery, identifies tumor-associated macrophages (TAM) in high-grade glioma (HGG) tissue. Infiltrating macrophages, the predominant immune cells in glioma, are highly implicated in tumor progression, recurrence, and treatment response – particularly in immunotherapy. Using a unique, paired dataset of label-free stimulated Raman histology (SRH) and two-photon microscopy (TPEF) images sharing one-to-one spatial resolution, we developed a deep-learning approach to automatically quantify TAM infiltration from intraoperatively acquired SRH images. METHODS We compiled a dataset of 906 paired whole-slide SRH/TPEF images from 79 patients with HGG. We first trained a pix2pix generative adversarial network to convert raw, label-free SRH into synthetic TPEF with identifiable macrophages. Next, we trained a MaskR-CNN model to locate and segment TAMs. The pix2pix network was trained using 5,531 hand-picked 300-by-300-pixel fields-of-view (FOV) best exemplifying TAMs. We used a human-in-the-loop approach to train the segmentation network on 1,000 hand-labeled FOVs. RESULTS Macrophage segmentation from purely histologic data was near-identical to the fluorescence-based ground truth, with a mean dice score of 90.3%. Predicted TAM density was highly correlated with r=0.735. Subgroup analyses of TAM density in 346,836 non-overlapping FOVs revealed significantly lower TAM density in IDH-mutant (p< 0.01) and MGMT-hypermethylated (p=0.03) tumors. Our end-to-end algorithm uses fresh, unlabeled tissue specimens in the operating room and takes just three minutes to analyze whole-slide SRH images. CONCLUSION We demonstrate how deep neural networks can be used to rapidly and quantitatively evaluate macrophage infiltration in HGG in the operating room. Our software enables analysis of glioma patients’ tumor immune environment without immunohistochemistry or fluorescent labels, and may be leveraged to study the effects of immune-modulating therapies in the tumor microenvironment. Future work will focus on evaluating macrophage density and distribution as biomarkers for response to immunotherapies showing efficacy in glioma patients.

Megakaryocytes Have Immune Characteristics in Murine Immune Bone Marrow Failure

Introduction Megakaryocytes (MKs) have familiar roles in the bone marrow (BM) microenvironment, including platelet production, maintenance of hematopoietic stem and progenitor cells (HSPCs) function, and more recent evidence suggests MK engagement in immune responses. Immune aplastic anemia (AA) is characterized by oligoclonal expansion of T-cells and death of HSPCs leading to bone marrow failure (BMF). As MKs demonstrate niche maintenance and immune effector function, we explored the potential roles of MKs in the development of AA in a murine model of immune BMF. Methods BMF was induced in CByB6F1 mice by intravenous infusion of lymph node (LN) cells from major histocompatibility complex mismatched C57BL/B6 (B6) donor mice at a dose of 30-40x10 6 cells/recipient. No irradiation was given to recipients to avoid extra damage to MKs and CByB6F1 recipient mice were sacrificed at D7, D10, D14, and D17 following LN cell infusion (Chen J, et al. Blood. 2004). Peripheral blood (PB) samples, tibias, femurs, and sterna were harvested at each timepoint. Immunophenotyping of MKs and T cells was performed by flow cytometry on flushed BM cells and two-photon excitation microscopy (2P) imaging was performed on fixed sternal samples, using fluorescence-labelled CD41 antibody to visualize the physical location of MKs in BM. To enrich for MKs, BM cells were filtered through a 10 µm pluriStrainer™ through which most lymphocytes, red blood cells, and other small cells were eluted while MKs and other large size cells remained on the strainer. Large cells and residual small cells were subsequently assessed by single cell RNA sequencing (sc-RNAseq) on the Parse Bio Evercode™ platform based on combinatorial barcoding technology, which was able to accommodate the large size of MKs, as no partitioning step is needed in the analysis workflow. Results BMF was successfully induced as measured by decreasing PB counts from point of lymphocyte infusion accompanied by increasing expansion of CD4 and CD8 T-cells and marked reduction of total BM cells. MKs persisted throughout the course of BMF as measured by both flow cytometry and 2P (Figure 1A) despite PB thrombocytopenia and progressive marrow hypocellularity. Concomitantly, MKs identified by large size on forward scatter versus side scatter and CD41+ expression had progressive upregulation of immune effector markers IA-IE and CD53 over the course of BMF. Following sc-RNAseq and bioinformatics analysis of enriched BM cells at D14, cell clustering identified a distinct cell cluster representing MKs (Figure 1B). MK identity was supported by co-expression of transcripts encoding platelet factor 4, TUBB1, Glycoprotein 5, and Itga2b (CD41). This MK cell population was distinct from macrophages which highly expressed Fcgr4, Cd5l, Ccr5, Cd74 and Gbp5 genes. Gene expression profiling demonstrated that MKs in BMF mice showed upregulation of immune pathways such as interferon gamma and interferon alpha response and downregulation of pathways related to platelet activation and platelet homeostasis, as compared with normal mice. Conclusions In a murine model of BMF, using multiple modalities, we demonstrate that MKs cease platelet production, manifested by reduction in peripheral blood platelet count and downregulation of gene expression pathways related to platelet production, while concurrently acquiring characteristics of immune effector cells. This suggests that MKs are active participants in immune BMF.

SNR enhanced high-speed two-photon microscopy using a pulse picker and time gating detection

Two-photon microscopy (TPM) is an attractive biomedical imaging method due to its large penetration depth and optical sectioning capability. In particular, label-free autofluorescence imaging offers various advantages for imaging biological samples. However, relatively low intensity of autofluorescence leads to low signal-to-noise ratio (SNR), causing practical challenges for imaging biological samples. In this study, we present TPM using a pulse picker to utilize low pulse repetition rate of femtosecond pulsed laser to increase the pulse peak power of the excitation source leading to higher emission of two-photon fluorescence with the same average illumination power. Stronger autofluorescence emission allowed us to obtain higher SNR images of arterial and liver tissues. In addition, by applying the time gating detection method to the pulse signals obtained by TPM, we were able to significantly reduce the background noise of two-photon images. As a result, our TPM system using the pulsed light source with a 19 times lower repetition rate allowed us to obtain the same SNR image more than 19 times faster with the same average power. Although high pulse energy can increase the photobleaching, we also observed that high-speed imaging with low total illumination energy can mitigate the photobleaching effect to a level similar to that of conventional illumination with a high repetition rate. We anticipate that this simple approach will provide guidance for SNR enhancement with high-speed imaging in TPM as well as other nonlinear microscopy.

Multi-color two-photon scanning structured illumination microscopy imaging of live cells.

Multi-color two-photon microscopy imaging of live cells is essential in biology. However, the limited diffraction resolution of conventional two-photon microscopy restricts its application to subcellular organelle imaging. Recently, we developed a laser scanning two-photon non-linear structured illumination microscope (2P-NLSIM), whose resolution improved three-fold. However, its ability to image polychromatic live cells under low excitation power has not been verified. Here, to improve the reconstruction super-resolution image quality under low excitation power, we increased the image modulation depth by multiplying the raw images with the reference fringe patterns in the reconstruction process. Simultaneously, we optimized the 2P-NLSIM system to image live cells, including the excitation power, imaging speed, and field of view. The proposed system could provide a new imaging tool for live cells.

Modified mouse model of repeated mild traumatic brain injury through a thinned-skull window and fluid percussion.

Mild traumatic brain injury (mTBI) is a clinically highly heterogeneous neurological disorder, none of the existing animal models can replicate the entire sequelae. This study aimed to develop a modified closed head injury (CHI) model of repeated mTBI (rmTBI) for investigating Ca2+ fluctuations of the affected neural network, the alternations of electrophysiology, and behavioral dysfunctions. The transcranial Ca2+ study protocol includes AAV-GCaMP6s infection in the right motor cortex, thinned-skull preparation, and two-photon laser scanning microscopy (TPLSM) imaging. The CHI rmTBI model is fabricated using the thinned-skull site and applying 2.0 atm fluid percussion with 48-h interval. The neurological dysfunction, minor motor performance, evident mood, spatial working, and reference deficits we found in this study mimic the clinically relevant syndromes after mTBI. Besides, our study revealed that there was a trend of transition from Ca2+ singlepeak to multipeak and plateau, and the total Ca2+ activities of multipeaks and plateaus (p < .001 vs. pre-rmTBI value) were significantly increased in ipsilateral layer 2/3 motor neurons after rm TBI. In parallel, there is a low-frequency power shift from delta to theta band (p < .01 vs. control) in the ipsilateral layer 2/3 of motor cortex of the rmTBI mice, and the overall firing rates significantly increased (p < .01 vs. control). Moreover, rmTBI causes slight cortical and hippocampal neuron damage and possibly induces neurogenesis in the dentate gyrus (DG). The alternations of Ca2+ and electrophysiological characteristics in layer 2/3 neuronal network, histopathological changes, and possible neurogenesis may play concertedly and partially contribute to the functional outcome post-rmTBI.

Synthesis and Application of Two-Photon Active Fluorescent Rhodol Dyes for Antibody Conjugation and In Vitro Cell Imaging.

A novel family of julolidine-containing fluorescent rhodols equipped with a wide variety of substituents was synthesized in a versatile two-step process. The prepared compounds were fully characterized and exhibited excellent fluorescence properties for microscopy imaging. The best candidate was conjugated to the therapeutic antibody trastuzumab through a copper-free strain-promoted azide-alkyne click reaction. The rhodol-labeled antibody was successfully applied for in vitro confocal and two-photon microscopy imaging of Her2+ cells.

Hypoxia-Preconditioned Bone Marrow Mesenchymal Stem Cells Improved Cerebral Collateral Circulation and Stroke Outcome in Mice.

Adequate collateral circulation can remarkably improve patient prognoses for patients experiencing ischemic stroke. Hypoxic preconditioning enhances the regenerative properties of bone marrow mesenchymal stem cells (BMSCs). Rabep2 (RAB GTPase binding effector protein 2) is a key protein in collateral remodeling. We investigated whether BMSCs and hypoxia-preconditioned BMSCs (H-BMSCs) augment collateral circulation poststroke, particularly through Rabep2 regulation. BMSCs or H-BMSCs (1×106) were delivered intranasally in ischemic mice with distal middle cerebral artery occlusion at 6 hours poststroke. Two-photon microscopic imaging and vessel painting methods were used to analyze collateral remodeling. Blood flow, vascular density, infarct volume, and gait analysis were assessed to evaluate poststroke outcomes. Expressions of proangiogenic marker VEGF (vascular endothelial growth factor) and Rabep2 were determined by Western blotting. Western blot, EdU (5-ethynyl-2'-deoxyuridine) incorporation, and tube formation assays were conducted on cultured endothelial cells treated with BMSCs. BMSCs were more effectively transplanted in the ischemic brain after hypoxic preconditioning. The ipsilateral collateral diameter was increased by BMSCs and strengthened by H-BMSCs (P<0.05). BMSCs increased peri-infarct blood flow and vascular density and reduced infarct volume, gait deficits (P<0.05), and furthermore by H-BMSCs (P<0.05). VEGF and Rabep2 protein expression was increased by BMSCs (P<0.05), which was enhanced by preconditioning (P<0.01). Additionally, BMSCs increased Rabep2 expression, proliferation, and tube formation of endothelial cells in vitro (P<0.05). H-BMSCs enhanced these effects (P<0.05), which were annulled by Rabep2 knockdown. BMSCs increased collateral circulation and improved poststroke outcomes, through the upregulation of Rabep2. These effects were enhanced by hypoxic preconditioning.

Ten-kilohertz two-photon microscopy imaging of single-cell dendritic activity and hemodynamics in vivo.

The studying of rapid neuronal signaling across large spatial scales in intact, living brains requires both high temporal resolution and versatility of the measurement device. We introduce a high-speed two-photon microscope based on a custom-built acousto-optic deflector (AOD). This microscope has a maximum line scan frequency of 400kHz and a maximum frame rate of 10,000 frames per second (fps) at . For stepwise magnification from population view to subcellular view with high spatial and temporal resolution, we combined the AOD with resonance-galvo (RS) scanning. With this combinatorial device that supports both large-view navigation and small-view high-speed imaging, we measured dendritic calcium propagation velocity and the velocity of single red blood cells (RBCs). We measured dendritic calcium propagation velocity ( ) in OGB-1-labeled single cortical neurons in mice in vivo. To benchmark the spatial precision and detection sensitivity of measurement in vivo, we also visualized the trajectories of single RBCs and found that their movement speed follows Poiseuille's law of laminar flow. This proof-of-concept methodological development shows that the combination of AOD and RS scanning two-photon microscopy provides both versatility and precision for quantitative analysis of single neuronal activities and hemodynamics in vivo.

Theoretical Insights on the Sensing Performance for Newly-synthesized Two-photon Fluorescent N2H4 Probes Based on Spirobifluorence.

The development of fluorescent probe for hydrazine (N2H4) detection has attracted much attention due to the important role of N2H4 plays in the fields of medicine, agriculture, biology and environments. In this paper, the optical properties and water solubility of two novel two-photon fluorescent molecular probes (Probe1 and Probe2) before and after the reaction with N2H4 are studied by using the density function theory. The results show that electronic distribution and transition dipole moment of the probes are obviously changed after the reaction with N2H4, thus the optical properties of the molecules are influenced and the detection of N2H4 are realized. In addition, photoinduced electron transfer processes for Probe1 and Probe2 in the presence of N2H4 are theoretically characterized, which explains the experimental observations from the microscopic mechanism. Special attention has been paid on the analysis of the two-photon absorption for the probes with the absence and presence of N2H4 by the response theory method. Both probes with good water solubility show large variation on the two-photon absorption cross section when reacts with N2H4. In particular, the two-photon absorption response of Probe2 is more obvious, so it possesses preferable two-photon fluorescence microscopic imaging ability. More importantly, the receptor effect on the sensing performances of the probes are demonstrated, providing a theoretical reference for the design and synthesis on more efficient two-photon fluorescence N2H4 probes. Our study provides necessary information on the response mechanism of the studied chemosensors and helps to establish the relationship between the structure and optical properties of two-photon fluorescence N2H4 probes.

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.

Novel Design and Application of High-NA Fiber Imaging Bundles for In Vivo Brain Imaging with Two-Photon Scanning Fluorescence Microscopy.

Here, we provide experimental verification supporting the use of short-section imaging bundles for two-photon microscopy imaging of the mouse brain. The 8 mm long bundle is made of a pair of heavy-metal oxide glasses with a refractive index contrast of 0.38 to ensure a high numerical aperture NA = 1.15. The bundle is composed of 825 multimode cores, ordered in a hexagonal lattice with a pixel size of 14 μm and a total diameter of 914 μm. We demonstrate successful imaging through custom-made bundles with 14 μm resolution. As the input, we used a 910 nm Ti-sapphire laser with 140 fs pulse and a peak power of 9 × 104 W. The excitation beam and fluorescent image were transferred through the fiber imaging bundle. As test samples, we used 1 μm green fluorescent latex beads, ex vivo hippocampal neurons expressing green fluorescent protein and cortical neurons in vivo expressing the fluorescent reporter GCaMP6s or immediate early gene Fos fluorescent reporter. This system can be used for minimal-invasive in vivo imaging of the cerebral cortex, hippocampus, or deep brain areas as a part of a tabletop system or an implantable setup. It is a low-cost solution, easy to integrate and operate for high-throughput experiments.

Two-photon ratiometric imaging of endogenous reactive inorganic sulfur species in the cellular cushion toward formaldehyde

To address the potential biological relationship between reactive inorganic sulfur species (RISSs) and formaldehyde, the novel two-photon ratiometric RISSs probes (PH-RISSs) were synthesized practically with high selectivity and sensitivity toward RISSs and the related two-photon microscopic imaging assay was established which could be employed in the interpretation of the potential interaction between RISSs and formaldehyde. It was disclosed that the level of RISSs would raise toward formaldehyde and this behavior could be prohibited by CSE (cystathionine gamma lyase) inhibitor and reactive oxygen species (ROS) inhibitor, which indicated that formaldehyde could facilitate the metabolism of biothiols by CSE and this motivation of RISSs should be related with ROS intimately. Thus, RISSs might provide the critical biological compensation toward formaldehyde in a short term for living system and this two-photon ratiometric technique could be a practical tool to understand further the detailed interaction between aldehydes and RISSs in living systems.