Multiphoton Excitation Fluorescence Microscopy Research Articles

Laser-based techniques for the non-invasive characterisation of grisaille paints on stained-glass windows

Grisaille was the first paint applied on stained-glass panels, used in Europe since the twelfth century. Historical written sources described the use of iron and copper together with a high lead-silica base glass in the grisailles production. This project aims to study the evolution of the grisaille paint composition throughout time and the changes in the raw materials used in their production using non-destructive and non-invasive techniques. To achieve this objective, 23 grisaille samples dated from the 13th to the twentieth centuries from nine different countries (Portugal, Poland, United Kingdom, Sweden, Norway, Belgium, Low Countries, Germany, and France) were studied by means of micro particle-induced X-ray emission (μ-PIXE), micro energy dispersive X-ray fluorescence (μ-EDXRF), laser-induced breakdown spectroscopy (LIBS), laser-induced fluorescence (LIF), non-linear optical microscopy (NLOM) in the modality of multiphoton excitation fluorescence (MPEF) and optical microscopy (OM). The results showed that it was possible to identify compositional differences and patterns throughout the samples when compared with literature results. The preference for using copper in central and south-central European countries and the addition of new compounds (CoO, Cr2O3, MnO) as colouring agents since the nineteenth century was verified. The LIBS analyses allow the identification of boron on two samples, confirming the change of base glass components since the seventeenth century. The NLOM-MPEF showed the capability of this technique to measure the grisaille paint layers’ thickness. This non-invasive multi-analytical and complementary approach proves itself efficient in identifying changes in the grisaille’s composition throughout time, which can be interpreted as changes in the raw materials and manufacture used in the production of these paint materials.

Spectrally Resolved Nonlinear Optical Properties of Doped Versus Undoped Quasi-2D Semiconductor Nanocrystals: Copper and Silver Doping Provokes Strong Nonlinearity in Colloidal CdSe Nanoplatelets

Nonlinear optical processes are crucial for emerging applications including multiphoton-excited fluorescence microscopy and optical power limiting. Therefore, searching for materials of high multiphoton absorption cross sections is essential for the development of these techniques. We present synthesis of 4.5 monolayer CdSe nanoplatelets (NPLs) doped with silver and copper ions along with the evaluation of their two-photon absorption (TPA) and three-photon absorption (3PA) cross sections. Doping significantly increases the TPA cross section of each NPL sample, which reaches up to 1.33 × 107 GM for the most absorbing copper-doped ones. We also detected 1–2 orders of magnitude-enhanced 3PA cross sections for the doped NPLs in comparison with their undoped counterparts. As TPA and 3PA peaks appear, in the first and the second biological transmission windows, respectively, doped NPLs are promising candidates for multiphoton fluorescence microscopy as bioimaging agents. Moreover, the strong nonlinear response suggests application as active optoelectronic materials in optical sensors.

Characterization of medieval-like glass alteration layers by laser spectroscopy and nonlinear optical microscopy

Historical glass-based objects undergo, since the time of their manufacture, different degradation phenomena that are related to their composition and to the environment to which they were exposed. Three-dimensional (3D) structural and chemical characterization of the degradation layers is important to select the most adequate conservation strategies for glass objects. Optical microscopy (OM) is the most frequently used non-destructive method to examine the surface of historical glasses; however, the 3D structural assessment of alteration layers requires applying the destructive modality of this technique to conduct a cross-sectional study. In this work, a different approach for structural and compositional characterization of alteration layers on model medieval-like glasses is presented, based on the combination of the laser spectroscopies of laser-induced breakdown spectroscopy (LIBS), laser-induced fluorescence (LIF) and FT-Raman, and the emerging, cutting edge technique of nonlinear optical microscopy (NLOM) in the modality of multiphoton excitation fluorescence (MPEF). The results obtained through this multi-analytical photonic approach were compared with those retrieved by examination of the surface and cross sections of the samples by OM and scanning electron microscopy–energy-dispersive X-ray spectroscopy (SEM–EDS). While the combination of LIBS, LIF and FT-Raman served to assess the composition of the various alteration layers, the use of MPEF microscopy allowed the non-destructive determination of the thicknesses of these layers, showing for both thickness and composition a good agreement with the OM and SEM–EDS results. Thus, the proposed approach, which avoids sample preparation, illustrates the capability of non-destructive, or micro-destructive in the case of LIBS, laser spectroscopies and microscopies for the in situ study of glass objects of historic or/and artistic value.Graphic

Canine mammary cancer tumour behaviour and patient survival time are associated with collagen fibre characteristics

Precise diagnosis and prognosis are key in prevention and reduction of morbidity and mortality in all types of cancers. Here we show that changes in the collagen fibres in the main histological subtypes of canine mammary gland carcinomas are directly associated with the tumour behaviour and the animal survival time and could become a useful tool in helping with diagnosis. Imaging by second harmonic generation and multiphoton excited fluorescence microscopy were performed to evaluate the collagen and cellular segment parameters in cancer biopsies. We present a retrospective study of 45 cases of canine mammary cancer analysing 836 biopsies regions including normal mammary gland tissue, benign mixed tumours, carcinoma in mixed tumour, carcinosarcoma, micropapillary carcinoma and solid carcinoma. The image analyses and the comparison between the tumour types allowed to assess the collagen fibre changes during tumour progression. We demonstrate that the collagen parameters correlate with the clinical and pathological data, the results show that in neoplastic tissues, the collagen fibres are more aligned and shorter as compared to the normal tissues. There is a clear association of the mean fibre length with the dogs survival times, the carcinomas presenting shorter collagen fibres indicate a worse survival rate.

Innate Immune Mechanism of Hemarthrosis in Hemophilia-a Mice

Hemarthrosis is a chronic arthropathy responsible for irreversible joint damage, disability, and acute joint pain in hemophilia patients. Hemarthrosis is caused by spontaneous or traumatic bleeding into joints, which when recurrent, leads to synovial inflammation and cartilage degeneration. Although release of erythrocyte-derived damage associated molecular pattern molecules (eDAMPs) is believed to promote sterile inflammation in the synovium, the innate immune mechanism of hemarthrosis remains poorly understood and the current therapy is limited to factor replacement and pain management. Here, we use factor 8 total knock-out (F8TKO) hemophilia A mice (C57BL/6J background) that manifest a complete deletion of the F8 coding region, expressing no detectable F8 mRNA and exhibiting a severe hemophilia phenotype. Right knee joint capsules of F8TKO mice were punctured with a 30-g needle below the patella between the anterior portions of the femur and tibia, followed by assessment of bleeding severity score and histological analysis one-week post injury. Intravital multiphoton excitation fluorescence microscopy of injured synovium was performed to assess the role of thrombo-inflammatory events in promoting hemarthrosis. Neutrophil, platelets, and blood vessels were visualized by intravenous administration of fluorescent anti-Ly6G mAb, anti-CD49b mAb, and dextran, respectively. Protein and mRNA levels of proinflammatory cytokines were measured in plasma, joint, synovium, and cartilage tissue using Real-Time PCR and ELISA, respectively. F8TKO but not wild-type (WT) control (C57BL/6J) mice manifested unresolved joint bleeding, cartilage degeneration and synovitis leading to impaired mobility and high bleeding severity scores. Significantly more recruitment of neutrophils and neutrophil-platelet aggregates as well as elevated IL-1b levels were observed in the synovium of F8TKO compared to WT mice. These results are the first to suggest that sterile inflammation contributing to hemarthrosis in hemophilia involves enhanced neutrophil-platelet recruitment to the synovium. Currently, experiments are underway to identify the role of eDAMPs (heme and hemoglobin) mediated activation of TLR4 and inflammasome pathway in promoting IL-1b generation, neutrophil-platelet aggregation, and progression of joint injury in F8TKO mice Disclosures Ragni: OPKO: Research Funding; Sangamo: Research Funding; Shire/Takeda: Other: Advisory Board, Research Funding; Spark Therapeutics: Other: Advisory Board, Research Funding; Alnylam/Sanofi: Other: Advisory Board, Research Funding; BioMarin: Other: Advisory Board, Research Funding; Bioverativ/Sanofi: Other: Advisory Board, Research Funding. Gladwin:Globin Solutions, Inc: Patents & Royalties: Provisional patents for the use of recombinant neuroglobin and heme-based molecules as antidotes for CO poisoning; Bayer Pharmaceuticals: Other: Co-investigator; United Therapeutics: Patents & Royalties: Co-inventor on an NIH government patent for the use of nitrite salts in cardiovascular diseases .

Characterization of Pancreatic Cancer Tissue Using Multiphoton Excitation Fluorescence and Polarization-Sensitive Harmonic Generation Microscopy.

Thin tissue sections of normal and tumorous pancreatic tissues stained with hematoxylin and eosin were investigated using multiphoton excitation fluorescence (MPF), second harmonic generation (SHG), and third harmonic generation (THG) microscopies. The cytoplasm, connective tissue, collagen and extracellular structures are visualized with MPF due to the eosin stain, whereas collagen is imaged with endogenous SHG contrast that does not require staining. Cellular structures, including membranous interfaces and nuclear components, are seen with THG due to the aggregation of hematoxylin dye. Changes in the collagen ultrastructure in pancreatic cancer were investigated by a polarization-sensitive SHG microscopy technique, polarization-in, polarization-out (PIPO) SHG. This involves measuring the orientation of the linear polarization of the SHG signal as a function of the linear polarization orientation of the incident laser radiation. From the PIPO SHG data, the second-order non-linear optical susceptibility ratio, χ(2)zzz'/χ(2)zxx', was obtained that serves as a structural parameter for characterizing the tissue. Furthermore, by assuming C6 symmetry, an additional second-order non-linear optical susceptibility ratio, χ(2)xyz'/χ(2)zxx', was obtained, which is a measure of the chirality of the collagen fibers. Statistically-significant differences in the χ(2)zzz'/χ(2)zxx' values were found between tumor and normal pancreatic tissues in periductal, lobular, and parenchymal regions, whereas statistically-significant differences in the full width at half maximum (FWHM) of χ(2)xyz'/χ(2)zxx' occurrence histograms were found between tumor and normal pancreatic tissues in periductal and parenchymal regions. Additionally, the PIPO SHG data were used to determine the degree of linear polarization (DOLP) of the SHG signal, which indicates the relative linear depolarization of the signal. Statistically-significant differences in DOLP values were found between tumor and normal pancreatic tissues in periductal and parenchymal regions. Hence, the differences observed in the χ(2)zzz'/χ(2)zxx' values, the FWHM of χ(2)xyz'/χ(2)zxx' values and the DOLP values could potentially be used to aid pathologists in diagnosing pancreatic cancer.

SRNA-FISH: versatile fluorescent insitu detection of small RNAs in plants.

Localization of mRNA and small RNAs (sRNAs) is important for understanding their function. Fluorescent insitu hybridization (FISH) has been used extensively in animal systems to study the localization and expression of sRNAs. However, current methods for fluorescent insitu detection of sRNA in plant tissues are less developed. Here we report a protocol (sRNA-FISH) for efficient fluorescent detection of sRNAs in plants. This protocol is suitable for application in diverse plant species and tissue types. The use of locked nucleic acid probes and antibodies conjugated with different fluorophores allows the detection of two sRNAs in the same sample. Using this method, we have successfully detected the co-localization of miR2275 and a 24-nucleotide phased small interfering RNA in maize anther tapetal and archesporial cells. We describe how to overcome the common problem of the wide range of autofluorescence in embedded plant tissue using linear spectral unmixing on a laser scanning confocal microscope. For highly autofluorescent samples, we show that multi-photon fluorescence excitation microscopy can be used to separate the target sRNA-FISH signal from background autofluorescence. In contrast to colorimetric insitu hybridization, sRNA-FISH signals can be imaged using super-resolution microscopy to examine the subcellular localization of sRNAs. We detected maize miR2275 by super-resolution structured illumination microscopy and direct stochastic optical reconstruction microscopy. In this study, we describe how we overcame the challenges of adapting FISH for imaging in plant tissue and provide a step-by-step sRNA-FISH protocol for studying sRNAs at the cellular and even subcellular level.

Effects of artepillin C on model membranes displaying liquid immiscibility.

It has been hypothesized that the therapeutic effects of artepillin C, a natural compound derived from Brazilian green propolis, are likely related to its partition in the lipid bilayer component of biological membranes. To test this hypothesis, we investigated the effects of the major compound of green propolis, artepillin C, on model membranes (small and giant unilamelar vesicles) composed of ternary lipid mixtures containing cholesterol, which display liquid-ordered (lo) and liquid-disordered (ld) phase coexistence. Specifically, we explored potential changes in relevant membrane parameters upon addition of artepillin C presenting both neutral and deprotonated states by means of small angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), and confocal and multiphoton excitation fluorescence microscopy. Thermotropic analysis obtained from DSC experiments indicated a loss in the lipid cooperativity of lo phase at equilibrium conditions, while at similar conditions spontaneous formation of unilamellar vesicles from SAXS experiments showed that deprotonated artepillin C preferentially located at the surface of the membrane. Time-resolved experiments using fluorescence microscopy showed that at doses above 100 µM, artepillin C in its neutral state interacted with both liquid-ordered and liquid-disordered phases, inducing curvature stress and promoting dehydration at the membrane interface.

Cycad Coralloid Roots Contain Bacterial Communities Including Cyanobacteria and Caulobacter spp. That Encode Niche-Specific Biosynthetic Gene Clusters.

Cycads are the only early seed plants that have evolved a specialized root to host endophytic bacteria that fix nitrogen. To provide evolutionary and functional insights into this million-year old symbiosis, we investigate endophytic bacterial sub-communities isolated from coralloid roots of species from Dioon (Zamiaceae) sampled from their natural habitats. We employed a sub-community co-culture experimental strategy to reveal both predominant and rare bacteria, which were characterized using phylogenomics and detailed metabolic annotation. Diazotrophic plant endophytes, including Bradyrhizobium, Burkholderia, Mesorhizobium, Rhizobium, and Nostoc species, dominated the epiphyte-free sub-communities. Draft genomes of six cyanobacteria species were obtained after shotgun metagenomics of selected sub-communities. These data were used for whole-genome inferences that suggest two Dioon-specific monophyletic groups, and a level of specialization characteristic of co-evolved symbiotic relationships. Furthermore, the genomes of these cyanobacteria were found to encode unique biosynthetic gene clusters, predicted to direct the synthesis of specialized metabolites, mainly involving peptides. After combining genome mining with detection of pigment emissions using multiphoton excitation fluorescence microscopy, we also show that Caulobacter species co-exist with cyanobacteria, and may interact with them by means of a novel indigoidine-like specialized metabolite. We provide an unprecedented view of the composition of the cycad coralloid root, including phylogenetic and functional patterns mediated by specialized metabolites that may be important for the evolution of ancient symbiotic adaptations.

Quantitative Assessment of Type-1 Collagen in Charcot Neuroarthropathy with Use of Harmonic Generation and Multiphoton Microscopy

Category: Diabetes Introduction/Purpose: Diabetic Charcot neuroarthropathy is characterized by rapid loss of bone with accompanied disruption to the surrounding joints and is often in the absence of trauma or infection. Previous research has demonstrated neuropeptide release and osteoclast activation in addition to increased collagen turnover. Currently, there is limited understanding on the impact of the disease on type-1 collagen within the joint synovium. The purpose of this study was to analyze qualitative and quantitative differences in type-1 collagen between diabetic, neuropathic patient controls and those with Charcot neuroarthropathy. Methods: A prospective case-control study using routinely discarded surgical waste synovial tissue from diabetic, neuropathic patients with and without Charcot. The tissue was prepared in the laboratory for analysis. Microscopy was performed using multiphoton excitation fluorescence (MPEF) and second harmonic generation (SHG) microscopy techniques, utilizing a mode locked femto-second i: Sapphire Tsunami (Spectra-Physics, Mountain View, CA) synchronously pumped by a Millenia Xs J (Spectra-Physics) diode-pumped solid state laser. Qualitative and quantitative microscopic analysis was performed on Charcot specimens and controls. A statistical analysis was performed to detect a quantitative difference in type-1 collagen. Results: An analysis was performed on 8 samples; 4 neuropathic controls and 4 Charcot neuroarthropathy samples. There was a statistically significant difference between the amount, the quality and organization of type-1 collagen (p<0.0001). The Charcot group had significantly less type-1 collagen when compared to the control (p<0.0001). Qualitative analysis was performed using a mean intensity value. The control group’s mean intensity was 2561.4 nm while the Charcot group’s was 884.1 nm. The Charcot group had a significantly smaller mean intensity indicating poorer quality of collagen (p<0.0001). Orientation index was used to assess the organization of type-1 collagen in the synovium. A smaller orientation index signifies an increase in disorganization. The Charcot group had a significantly smaller orientation index of 16.5 compared to the control group’s 27.8 (p<0.0001). Conclusion: Charcot neuroarthropathy leads to significant abnormalities in type-1 collagen within the joint synovium when compared to the diabetic controls. Further research will determine if these differences are attributable to neuropeptide release and osteoclastic activation.

Scanless multitarget-matching multiphoton excitation fluorescence microscopy

Using the combination of a reflective blazed grating and a reflective phase-only diffractive spatial light modulator (SLM), scanless multitarget-matching multiphoton excitation fluorescence microscopy (SMTM-MPM) was achieved. The SLM shaped an incoming mode-locked, near-infrared Ti:sapphire laser beam into an excitation pattern with addressable shapes and sizes that matched the samples of interest in the field of view. Temporal and spatial focusing were simultaneously realized by combining an objective lens and a blazed grating. The fluorescence signal from illuminated areas was recorded by a two-dimensional sCMOS camera. Compared with a conventional temporal focusing multiphoton microscope, our microscope achieved effective use of the laser power and decreased photodamage with higher axial resolution.

Generation of multi-wavelength femtosecond laser pulse based on nonlinear propagation of high peak power ultrashort laser pulse in single-mode fiber and spectral selectivity technology

Highly-integrated high-reliability widely-tunable femtosecond laser sources have important application values in various research and application fields, such as ultrafast spectroscopy, quantum optics, remote sensing and bio-imaging. In multi-photon excited fluorescence microscopy, femtosecond laser sources with moderate pulse energy and wide wavelength tunable range can not only meet the application requirements of the different tissue structures for the peak power and excitation wavelength, but also improve the nonlinear fluorescence efficiency and imaging resolution of the sample, and thus enhancing the penetration depth. Considering the extensive application prospect and important scientific research significance of the widely tunable femtosecond laser, in this paper we conduct an experimental research of the high repetition rate multi-wavelength femtosecond laser generation in compact sized and low-cost configuration based on the nonlinear propagation scheme of the high peak power femtosecond laser pulses in single-mode fiber. In experiment, we first construct a highly-integrated reliable all-polarization-maintaining fiber femtosecond laser amplifier, which mainly consists of an environmentally stable all-polarization-maintaining fiber mode-locked laser oscillator, single-mode fiber stretcher, a single-mode power pre-amplifier, a dual-cladding Yb-fiber amplifier, and transmission grating-pair compressor. Self-starting mode-locked operation is assured with a semiconductor saturable absorber mirror, and intra-cavity dispersion compensation is realized by a chirped fiber Bragg grating in the mode-locked oscillator. The mode-locked oscillator, which delivers laser pulses with center wavelength peaked at 1035 nm, is robust operation as temperature changes from 10℃ to 40℃ and the measured power fluctuation is less than 1% RMS over 168 hours at 23℃. The amplified high repetition rate laser pulses are compressed in a double-pass 1000 lines/mm transmission grating-pair compressor. After compression, laser pulses with 5.83 W average power and 264 fs pulse duration at 34 MHz repetition rate can be obtained. Simultaneously, we also study the dependence of the compressed pulse duration on the amplified output power. Employing a home-made high reliable compact sized all-polarization-maintaining fiber femtosecond laser as a pump source and low-cost single-mode fiber as a nonlinear medium, the generation technology of the widely tunable femtosecond laser in only fiber format is also studied based on the self-phase modulation nonlinear spectral broadening mechanism. Simultaneously, in order to reduce the effect of the dispersion on the spectral broadening as much as possible, an 80-mm-long fiber is used in experiment. The used single-mode spectral broadening fiber has a 6-m-diameter core and 20 fs2/mm dispersion coefficient. By coupling the femtosecond pump laser pulses into the 6-m-diameter fiber core, the output spectrum presents a significant nonlinear broadening. The coupled pump power can be continuously adjusted by a combination of a half-wave plate and a Glan laser polarizer. After bandpass filtering the leftmost and rightmost spectral lobes in self-phase modulation and self-steeping induced broadened spectrum with bandpass filters centered at 980, 1000, 1050, 1070 and 1100 nm, the laser pulses with 203, 195, 196, 187, and 194 fs pulse duration can be obtained at the corresponding center wavelengths. The experimental scheme presented in this paper, which is based on the nonlinear spectral broadening of the highreliability femtosecond laser pulse in single-mode fiber and the spectral selectivity technology, provides a new research approach to the realization of the highly-compacted reliable widely-tunable femtosecond laser sources and has important research significance.

High-Resolution Morphological Approach to Analyse Elastic Laminae Injuries of the Ascending Aorta in a Murine Model of Marfan Syndrome

In Marfan syndrome, the tunica media is disrupted, which leads to the formation of ascending aortic aneurysms. Marfan aortic samples are histologically characterized by the fragmentation of elastic laminae. However, conventional histological techniques using transverse sections provide limited information about the precise location, progression and 3D extension of the microstructural changes that occur in each lamina. We implemented a method using multiphoton excitation fluorescence microscopy and computational image processing, which provides high-resolution en-face images of segmented individual laminae from unstained whole aortic samples. We showed that internal elastic laminae and successive 2nd laminae are injured to a different extent in murine Marfan aortae; in particular, the density and size of fenestrae changed. Moreover, microstructural injuries were concentrated in the aortic proximal and convex anatomical regions. Other parameters such as the waviness and thickness of each lamina remained unaltered. In conclusion, the method reported here is a useful, unique tool for en-face laminae microstructure assessment that can obtain quantitative three-dimensional information about vascular tissue. The application of this method to murine Marfan aortae clearly shows that the microstructural damage in elastic laminae is not equal throughout the thickness of the tunica media and in the different anatomical regions of the ascending aorta.

Using quantitative intravital multiphoton microscopy to dissect hepatic transport in rats.

Hepatic solute transport is a complex process whose disruption is associated with liver disease and drug-induced liver injury. Intravital multiphoton fluorescence excitation microscopy provides the spatial and temporal resolution necessary to characterize hepatic transport at the level of individual hepatocytes in vivo and thus to identify the mechanisms and cellular consequences of cholestasis. Here we present an overview of the use of fluorescence microscopy for studies of hepatic transport in living animals, and describe how we have combined methods of intravital microscopy and digital image analysis to dissect the effects of drugs and pathological conditions on the function of hepatic transporters in vivo.

Evidence of proteolipid domain formation in an inner mitochondrial membrane mimicking model

BackgroundMitochondrial creatine kinase (mtCK) is highly abundant in mitochondria; its quantity is equimolecular to the Adenylic Nucleotide Translocator and represents 1% of the mitochondrial proteins. It is a multitask protein localized in the mitochondria intermembrane space where it binds to the specific cardiolipin (CL) phospholipid. If mtCK was initially thought to be exclusively implicated in energy transfer between mitochondria and cytosol through a mechanism referred to as the phosphocreatine shuttle, several recent studies suggested an additional role in maintaining mitochondria membrane structure. MethodsTo further characterized mtCK binding process we used multiphoton excitation fluorescence microscopy coupled with Giant Unilamellar Vesicles (GUV) and laurdan as fluorescence probe. ResultsWe gathered structural and dynamical information on the molecular events occurring during the binding of mtCK to the mitochondria inner membrane.We present the first visualization of mtCK-induced CL segregation on a bilayer model forming micrometer-size proteolipid domains at the surface of the GUV. Those microdomains, which only occurred when CL is included in the lipid mixture, were accompanied by the formation of protein multimolecular assembly, vesicle clamping, and changes in both vesicle curvature and membrane fluidity ConclusionThose results highlighted the importance of the highly abundant mtCK in the lateral organization of the mitochondrial inner membrane. General significanceMicrodomains were induced in mitochondria-mimicking membranes composed of natural phospholipids without cholesterol and/or sphingolipids differing from the proposed cytoplasmic membrane rafts. Those findings as well as membrane curvature modification were discussed in relation with protein-membrane interaction and protein cluster involvement in membrane morphology.

Micro-endoscopic system for functional assessment of neural circuits in deep brain regions: Simultaneous optical and electrical recordings of auditory responses in mouse’s inferior colliculus

In vivo Ca2+ imaging is a powerful method for the functional assessment of neural circuits. Although multi-photon excitation fluorescence microscopy has been widely used, observation of circuits in deep brain regions remains challenging. Recently, observing these deep regions has become possible via an endoscope consisting of an optical fiber bundle or gradient-index lens. We have designed a micro-endoscope system that enables simultaneous optical recording of fluorescence and electrical recording of neural activity. Using this system, we recorded auditory responses by simultaneously detecting changes in the fluorescence intensity of a Ca2+ indicator dye, multi-unit activities (MUA), and local field potentials (LFP) in the mouse’s inferior colliculus (IC). Such simultaneous optical and electrical recordings enabled detailed comparison of electrically recorded phenomena (MUA and LFP) and optically recorded Ca2+ response. By systematically changing sound frequency and intensity, we determined the frequency tuning of the recording site. The best frequency shifted higher as the probe advanced more deeply, demonstrating that the system is capable of optically measuring the dorso-ventral organization of IC (i.e., tonotopicity). Thus, our new micro-endoscope system will be useful in the neurophysiological studies of a wide range of brain circuits, including those within the auditory system.

Second Harmonic Generation and Multiphoton Excited Fluorescence Microscopy as a Phenotypic Tool in an Animal Model.

Second Harmonic Generation and Multiphoton Excited Fluorescence Microscopy as a Phenotypic Tool in an Animal Model.

In vivo microscopy

This article summarizes the past, present, and future promise of multiphoton excitation fluorescence microscopy for intravital kidney imaging. During the past 15years, several high-power visual research approaches have been developed using multiphoton imaging to study the normal functions of the healthy, intact, living kidney, and the various molecular and cellular mechanisms of the development of kidney diseases. In this review, the main focus will be on intravital multiphoton imaging of the glomerulus, the structure and function of the glomerular filtration barrier, especially the podocyte. Examples will be given for the combination of two powerful research tools, in vivo multiphoton imaging and mouse genetics using commercially available whole animal models for the detailed characterization of glomerular cell types, their function and fate, and for the better understanding of the molecular mechanisms of glomerular pathologies. One of the new modalities of multiphoton imaging, serial imaging of the same glomerulus in the same animal over several days will be emphasized for its potential for further advancing the field of nephrology research.

Multiphoton imaging of myogenic differentiation in gelatin-based hydrogels as tissue engineering scaffolds.

BackgroundHydrogels can serve as three-dimensional (3D) scaffolds for cell culture and be readily injected into the body. Recent advances in the image technology for 3D scaffolds like hydrogels have attracted considerable attention to overcome the drawbacks of ordinary imaging technologies such as optical and fluorescence microscopy. Multiphoton microscopy (MPM) is an effective method based on the excitation of two-photons. In the present study, C2C12 myoblasts differentiated in 3D gelatin hydroxyphenylpropionic acid (GHPA) hydrogels were imaged by using a custom-built multiphoton excitation fluorescence microscopy to compare the difference in the imaging capacity between conventional microscopy and MPM.ResultsThe physicochemical properties of GHPA hydrogels were characterized by using scanning electron microscopy and Fourier-transform infrared spectroscopy. In addition, the cell viability and proliferation of C2C12 myoblasts cultured in the GHPA hydrogels were analyzed by using Live/Dead Cell and CCK-8 assays, respectively. It was found that C2C12 cells were well grown and normally proliferated in the hydrogels. Furthermore, the hydrogels were shown to be suitable to facilitate the myogenic differentiation of C2C12 cells incubated in differentiation media, which had been corroborated by MPM. It was very hard to get clear images from a fluorescence microscope.ConclusionsOur findings suggest that the gelatin-based hydrogels can be beneficially utilized as 3D scaffolds for skeletal muscle engineering and that MPM can be effectively applied to imaging technology for tissue regeneration.

Platelet-Neutrophil Aggregates Promote Pulmonary Arteriole Microembolism in Sickle Cell Disease

Introduction: Sickle cell disease (SCD) is an autosomal recessive genetic disorder that affects ~100,000 Americans and millions of people worldwide. The acute chest syndrome (ACS), a form of acute lung injury, is a major cause of morbidity among SCD patients. The current treatment for ACS is primarily supportive and the molecular mechanism remains largely unknown. SCD patients hospitalized with vaso-occlusive pain crisis (VOC) often develop ACS within the ensuing days, suggesting a role for pulmonary vaso-occlusion in the onset of ACS. However, the cellular and molecular mechanism and the anatomical site of pulmonary vaso-occlusion are still elusive.Materials and Methods: Intravenous (IV) bacterial lipopolysaccharide (LPS) was used to induce VOC in SCD mice. Intravital multiphoton excitation (MPE) fluorescence microscopy was used to study the blood cell trafficking within the pulmonary microcirculation of live SCD or control mice. Fluorochrome-conjugated anti-mouse Ly-6G, Ter-119, and CD49b antibodies were administered IV for in vivo staining of circulating neutrophils, red blood cells and platelets, respectively. Cellular trafficking was recorded at baseline and 2 hours after IV challenge with LPS. Image sequences were analyzed to identify vaso-occlusion, which was defined as cellular aggregation and stasis of blood flow within the pulmonary blood vessels.Results and Discussion: Preliminary data using MPE imaging in transgenic SCD mice revealed that vaso-occlusion was absent at baseline in unchallenged SCD mice and the cellular trafficking within the pulmonary microcirculation was comparable in SCD and control mice. Doses of IV LPS (0.01-5 mg/kg of body weight), which were innocuous to control mice were found to be lethal to SCD mice. Remarkably, MPE imaging of the lung microcirculation revealed that IV LPS led to microembolism of the pre-capillary pulmonary arterioles by platelet-neutrophil aggregates in SCD but not control mice. The microembolism involved either entrapment of circulating platelet-neutrophil aggregates or in situ aggregation through sequential steps of neutrophil arrest on the arteriolar endothelium, followed by platelet nucleation on arrested neutrophils and microthrombus formation.Conclusions: Initial findings demonstrate that pulmonary vaso-occlusion in SCD mice involves microembolism of the pre-capillary pulmonary arterioles by platelet-neutrophil aggregates. Future studies will determine the molecular interactions responsible for pulmonary arteriolar microembolism.Acknowledgments: This study is supported by the 11SDG7340005 from the American Heart Association (P.S.) and the VMI startup account (P.S.). M.F.B is supported by the NIH NHLBI VMI T32 training grant T32HL110849. DisclosuresNo relevant conflicts of interest to declare.