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Significance: Two-photon fluorescence microscopy (TPFM) excited by Gaussian beams requires axial tomographic scanning for three-dimensional (3D) volumetric imaging, which is a time-consuming process, and the slow imaging speed hinders its application for in vivo brain imaging. The Bessel focus, characterized by an extended depth of focus and constant resolution, facilitates the projection of a 3D volume onto a two-dimensional image, which significantly enhances the speed of volumetric imaging. Aim: We aimed to demonstrate the ability of a TPFM with a sidelobe-free Bessel beam to provide a promising tool for research in live biological specimens. Approach: Comparative in vivo imaging was conducted in live mouse brains and transgenic zebrafish to evaluate the performance of TPFM and Bessel-beam-based TPFM. Additionally, an image-difference method utilizing zeroth-order and third-order Bessel beams was introduced to effectively suppress background interference introduced by sidelobes. Results: In comparison with traditional TPFM, the Bessel-beams-based TPFM demonstrated a 30-fold increase in imaging throughput and speed. Furthermore, the effectiveness of the image-difference method was validated in live biological specimens, resulting in a substantial enhancement of image contrast. Importantly, our TPFM with a sidelobe-free Bessel beam exhibited robustness against axial displacements, a feature of considerable value for in vivo experiments. Conclusions: We achieved rapid, high-contrast, and robust volumetric imaging of the vasculature in live mouse brains and transgenic zebrafish using our TPFM with a sidelobe-free Bessel beam.
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Encéfalo , Peixe-Zebra , Animais , Camundongos , Encéfalo/diagnóstico por imagem , Microscopia de Fluorescência , Distribuição Normal , FótonsRESUMO
Introduction: 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. Methods: 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. Results: 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. Discussion: 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.
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The analysis of histological alterations in all types of tissue is of primary importance in pathology for highly accurate and robust diagnosis. Recent advances in tissue clearing and fluorescence microscopy made the study of the anatomy of biological tissue possible in three dimensions. The combination of these techniques with classical hematoxylin and eosin (H&E) staining has led to the birth of three-dimensional (3D) histology. Here, we present an overview of the state-of-the-art methods, highlighting the optimal combinations of different clearing methods and advanced fluorescence microscopy techniques for the investigation of all types of biological tissues. We employed fluorescence nuclear and eosin Y staining that enabled us to obtain hematoxylin and eosin pseudo-coloring comparable with the gold standard H&E analysis. The computational reconstructions obtained with 3D optical imaging can be analyzed by a pathologist without any specific training in volumetric microscopy, paving the way for new biomedical applications in clinical pathology.
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Imageamento Tridimensional , Hematoxilina , Amarelo de Eosina-(YS) , Microscopia de Fluorescência/métodos , Coloração e Rotulagem , Imageamento Tridimensional/métodos , Microscopia ConfocalRESUMO
For in vivo two-photon fluorescence microscopy (2PM) imaging, the development of techniques that can improve the observable depth and temporal resolution is an important challenge to address biological and biomedical concerns such as vascular dynamics in the deep brain (typically the hippocampal region) of living animals. Improvements have been achieved through two approaches: an optical approach using a highly tissue-penetrating excitation laser oscillating in the second near-infrared wavelength region (NIR-II, 1100-1350 nm) and a chemical approach employing fluorescent probes with high two-photon brightness (characterized by the product of the two-photon absorption cross section, σ2, and the fluorescence quantum yield, Φ). To integrate these two approaches, we developed a fluorescent dye exhibiting a sufficiently high σ2Φ value of 68 Goeppert-Mayer units at 1100 nm. When a nanoemulsion encapsulating >1000 dye molecules per particle and a 1100 nm laser were employed for 2PM imaging, capillary blood vessels in almost the entire hippocampal CA1 region of the mouse brain (approximately 1.1-1.5 mm below the surface) were clearly visualized at a frame rate of 30 frames s-1 (averaged over eight frames, practically 3.75 frames s-1). This observable depth and frame rate are much higher than those in previous reports on 2PM imaging. Furthermore, this nanoemulsion allowed for the visualization of blood vessels at a depth of 1.8 mm, corresponding to the hippocampal dentate gyrus. These results highlight the advantage of combining bright probes with NIR-II lasers. Our probe is a promising tool for studying the vascular dynamics of living animals and related diseases.
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Região CA1 Hipocampal , Tomografia Computadorizada por Raios X , Animais , Corantes Fluorescentes/química , Camundongos , Microscopia de Fluorescência/métodos , Imagem Óptica , FótonsRESUMO
Genetically encoded voltage indicators are emerging tools for monitoring voltage dynamics with cell-type specificity. However, current indicators enable a narrow range of applications due to poor performance under two-photon microscopy, a method of choice for deep-tissue recording. To improve indicators, we developed a multiparameter high-throughput platform to optimize voltage indicators for two-photon microscopy. Using this system, we identified JEDI-2P, an indicator that is faster, brighter, and more sensitive and photostable than its predecessors. We demonstrate that JEDI-2P can report light-evoked responses in axonal termini of Drosophila interneurons and the dendrites and somata of amacrine cells of isolated mouse retina. JEDI-2P can also optically record the voltage dynamics of individual cortical neurons in awake behaving mice for more than 30 min using both resonant-scanning and ULoVE random-access microscopy. Finally, ULoVE recording of JEDI-2P can robustly detect spikes at depths exceeding 400 µm and report voltage correlations in pairs of neurons.
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Microscopia , Neurônios , Animais , Interneurônios , Camundongos , Microscopia/métodos , Neurônios/fisiologia , Fótons , VigíliaRESUMO
OBJECTIVES: For the development and validation of diagnostic procedures based on microscopic methods, knowledge about the imaging depth and achievable resolution in tissue is crucial. This poses the challenge to develop a microscopic artificial phantom focused on the microscopic instead of the macroscopic optical tissue characteristics. METHODS: As existing artificial tissue phantoms designed for image forming systems are primarily targeted at wide field applications, they are unsuited for reaching the formulated objective. Therefore, a microscopy- and microendoscopy-suited artificial tissue phantom was developed and characterized. It is based on a microstructured glass surface coated with fluorescent beads at known depths covered by a scattering agent with modifiable optical properties. The phantom was examined with different kinds of microscopy systems in order to characterize its quality and stability and to demonstrate its usefulness for instrument comparison, for example, regarding structural as well as fluorescence lifetime analysis. RESULTS: The analysis of the manufactured microstructured glass surfaces showed high regularity in their physical dimensions in accordance with the specifications. Measurements of the optical parameters of the scattering medium were consistent with simulations. The fluorescent beads coating proved to be stable for a respectable period of time (about a week). The developed artificial tissue phantom was successfully used to detect differences in image quality between a research microscope and an endoscopy based system. Plausible causes for the observed differences could be derived based on the well known microstructure of the phantom. CONCLUSIONS: The artificial tissue phantom is well suited for the intended use with microscopic and microendoscopic systems. Due to its configurable design, it can be adapted to a wide range of applications. It is especially targeted at the characterization and calibration of clinical imaging systems that often lack extensive positioning capabilities such as an intrinsic z-stage.
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Microscopia , Imagem Óptica , Imagens de FantasmasRESUMO
Rationale: Mild traumatic brain injury (mTBI), the most common type of brain trauma, frequently leads to chronic cognitive and neurobehavioral deficits. Intervening effectively is impeded by our poor understanding of its pathophysiological sequelae. Methods: To elucidate the long-term neurovascular sequelae of mTBI, we combined optogenetics, two-photon fluorescence microscopy, and intracortical electrophysiological recordings in mice to selectively stimulate peri-contusional neurons weeks following repeated closed-head injury and probe individual vessel's function and local neuronal reactivity. Results: Compared to sham-operated animals, mTBI mice showed doubled cortical venular speeds (115 ± 25%) and strongly elevated cortical venular reactivity (53 ± 17%). Concomitantly, the pericontusional neurons exhibited attenuated spontaneous activity (-57 ± 79%) and decreased reactivity (-47 ± 28%). Post-mortem immunofluorescence revealed signs of peri-contusional senescence and DNA damage, in the absence of neuronal loss or gliosis. Alteration of neuronal and vascular functioning was largely prevented by chronic, low dose, systemic administration of a GABA-A receptor inverse agonist (L-655,708), commencing 3 days following the third impact. Conclusions: Our findings indicate that repeated mTBI leads to dramatic changes in the neurovascular unit function and that attenuation of tonic inhibition can prevent these alterations. The sustained disruption of the neurovascular function may underlie the concussed brain's long-term susceptibility to injury, and calls for development of better functional assays as well as of neurovascularly targeted interventions.
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Concussão Encefálica/metabolismo , Concussão Encefálica/fisiopatologia , Acoplamento Neurovascular/fisiologia , Animais , Encéfalo/fisiopatologia , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Endogâmicos , Microscopia de Fluorescência/métodos , Neurônios/fisiologia , Optogenética/métodosRESUMO
Sensorineural hearing loss (SNHL) is the most common sensory deficit worldwide, and it typically originates from the cochlea. Methods to visualize intracochlear cells in living people are currently lacking, limiting not only diagnostics but also therapies for SNHL. Two-photon fluorescence microscopy (TPFM) is a high-resolution optical imaging technique. Here we demonstrate that TPFM enables visualization of sensory cells and auditory nerve fibers in an unstained, non-decalcified adult human cochlea.
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Kombucha pellicles are often used as inoculum to produce this beverage and have become a signature feature. This cellulosic biofilm produced by acetic acid bacteria (AAB) involves yeasts, which are also part of the kombucha consortia. The role of microbial interactions in the de novo formation and structure of kombucha pellicles was investigated during the 3 days following inoculation, using two-photon microscopy coupled with fluorescent staining. Aggregated yeast cells appear to serve as scaffolding to which bacterial cellulose accumulates. This initial foundation leads to a layered structure characterized by a top cellulose-rich layer and a biomass-rich sublayer. This sublayer is expected to be the microbiologically active site for cellulose production and spatial optimization of yeast-AAB metabolic interactions. The pellicles then grow in thickness while expanding their layered organization. A comparison with pellicles grown from pure AAB cultures shows differences in consistency and structure that highlight the impact of yeasts on the structure and properties of kombucha pellicles.
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Rationale: Delivery of therapeutic agents to the brain is limited by the presence of the blood-brain barrier (BBB). An emerging strategy to temporarily and locally increase the permeability of the BBB is the use of transcranial focused ultrasound (FUS) and systematically injected microbubbles (MBs). FUS+MB BBB treatments cause an acute inflammatory response, marked by a transient upregulation of pro-inflammatory genes; however, the cellular immune response remains unknown. Methods: FUS+MB BBB treatments were monitored in real-time using two-photon fluorescence microscopy and transgenic EGFP Wistar rats, which harbour several fluorescent cell types. Leukocyte identification and counts were confirmed using magnetic resonance imaging-guided FUS+MB BBB treatments. Participation of leukocytes in reducing ß-amyloid pathology following repeated FUS+MB BBB treatments was investigated in the TgCRND8 mouse model of Alzheimer's disease. Results: Intravascular leukocyte activity indicative of acute inflammation were identified, including transendothelial migration, formation of cell aggregates, and cell masses capable of perturbing blood flow. Leukocyte responses were only observed after the onset of sonication. Neutrophils were identified to be a key participating leukocyte. Significantly more neutrophils were detected in the sonicated hemisphere compared to the contralateral hemisphere, and to untreated controls. Three to five biweekly FUS+MB BBB treatments did not induce significantly more neutrophil recruitment, nor neutrophil phagocytosis of ß-amyloid plaques, in TgCRND8 mice compared to untreated controls. Conclusions: This study provides evidence that the cellular aspect of the peripheral immune response triggered by FUS+MB BBB treatments begins immediately after sonication, and emphasizes the importance for further investigations to be conducted to understand leukocyte dynamics and cerebral blood flow responses to FUS+MB BBB treatments.
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Doença de Alzheimer/imunologia , Barreira Hematoencefálica/metabolismo , Permeabilidade Capilar , Leucócitos/imunologia , Microbolhas , Infiltração de Neutrófilos/imunologia , Sonicação/métodos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/radioterapia , Precursor de Proteína beta-Amiloide/fisiologia , Animais , Transporte Biológico , Barreira Hematoencefálica/efeitos da radiação , Feminino , Proteínas de Fluorescência Verde , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Inflamação/radioterapia , Masculino , Camundongos , Camundongos Transgênicos , Placa Amiloide/patologia , Ratos , Ratos WistarRESUMO
SIGNIFICANCE: Recent advances in nonlinear optics in neuroscience have focused on using two ultrafast lasers for activity imaging and optogenetic stimulation. Broadband femtosecond light sources can obviate the need for multiple lasers by spectral separation for chromatically targeted excitation. AIM: We present a photonic crystal fiber (PCF)-based supercontinuum source for spectrally resolved two-photon (2P) imaging and excitation of GCaMP6s and C1V1-mCherry, respectively. APPROACH: A PCF is pumped using a 20-MHz repetition rate femtosecond laser to generate a supercontinuum of light, which is spectrally separated, compressed, and recombined to image GCaMP6s (930 nm excitation) and stimulate the optogenetic protein, C1V1-mCherry (1060 nm excitation). Galvanometric spiral scanning is employed on a single-cell level for multiphoton excitation and high-speed resonant scanning is employed for imaging of calcium activity. RESULTS: Continuous wave lasers were used to verify functionality of optogenetic activation followed by directed 2P excitation. Results from these experiments demonstrate the utility of a supercontinuum light source for simultaneous, single-cell excitation and calcium imaging. CONCLUSIONS: A PCF-based supercontinuum light source was employed for simultaneous imaging and excitation of calcium dynamics in brain tissue. Pumped PCFs can serve as powerful light sources for imaging and activation of neural activity, and overcome the limited spectra and space associated with multilaser approaches.
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Multiple peripheral nerves are known to degenerate after nerve compression injury but the correlation between the extent of nerve alteration and pain severity remains unclear. Here, we used intravital two-photon fluorescence microscopy to longitudinally observe changes in cutaneous fibers in the hind paw of Nav1.8-Cre-tdTomato mice after chronic constriction injury (CCI). Results showed that the CCI led to variable loss of the skin nerve plexus and intraepidermal nerve fibers. The timing of Nav1.8 nerve fiber loss correlated with the development of mechanical hypersensitivity. We compared a scoring approach that assessed whole-paw nerve degeneration with an index that quantified changes in the nerve plexus and terminals in multiple small regions of interest (ROI) from intravital images of the third and fifth toe tips. We found that the number of surviving nerve fibers was not linearly correlated with mechanical hypersensitivity. On the contrary, at 14 days after CCI, the moderately injured mice showed greater mechanical hypersensitivity than the mildly or severely injured mice. This indicates that both surviving and injured nerves are required for evoked neuropathic pain. In addition, these two methods may have the estimative effect as diagnostic and prognostic biomarkers for the assessment of neuropathic pain.
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Hiperalgesia/patologia , Fibras Nervosas/patologia , Neuralgia/patologia , Animais , Comportamento Animal , Doença Crônica , Constrição Patológica , Epiderme/inervação , Feminino , Hiperalgesia/complicações , Microscopia Intravital , Masculino , Camundongos Endogâmicos C57BL , Degeneração Neural/complicações , Degeneração Neural/patologia , Neuralgia/complicações , Limiar da DorRESUMO
OBJECTIVES: The investigation of cochlear hair cells and lateral wall is a time-consuming and labor-intensive process. However, it is a mandatory experiment in audiology research. Here we suggest a novel method for investigating the inner ear microstructures from intact cochleae using two-photon laser scanning microscopy (TPLSM). This technique guarantees fewer artifacts and technical simplicity. METHODS: Using TPLSM, we investigated the whole mount cochleae, decalcified cochleae, and cleared cochleae of wild type C57BL/6 mice. CX3CR1+/GFP mice were used to investigate the feasibility of visualizing cellular structures in the cochlear spiral ligament. All samples were investigated without staining. RESULTS: Endogenous fluorescence emission from the outer hair cells was strong enough to be distinguished from the other structures in all samples. From the single apical view, 50 and 90% of the whole hair cells of the decalcified cochleae and cleared cochleae, respectively, could be visualized without staining using TPLSM. Capillary structure of stria vascularis and spiral ligament could be visualized by endogenous fluorescence without staining. CONCLUSION: We successfully investigated the hair cells and lateral wall of mouse cochleae using TPLSM without using staining or any destructive procedures. This method is easier, faster, and more reliable than conventional methods.
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Cóclea , Estria Vascular , Animais , Células Ciliadas Auditivas , Camundongos , Camundongos Endogâmicos C57BL , Microscopia ConfocalRESUMO
Monoamine oxidases have two functionally distinct but structurally similar isoforms (MAO-A and MAO-B). The ability to differentiate them by using fluorescence detection/imaging technology is of significant biological relevance, but highly challenging with available chemical tools. Herein, we report the first MAO-A-specific two-photon fluorogenic probe (F1), capable of selective imaging of endogenous MAO-A enzymatic activities from a variety of biological samples, including MAO-A-expressing neuronal SY-SY5Y cells, the brain of tumor-bearing mice and human Glioma tissues by using two-photon fluorescence microscopy (TPFM) with minimal cytotoxicity.
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Neoplasias Encefálicas/enzimologia , Corantes Fluorescentes/síntese química , Glioma/enzimologia , Monoaminoxidase/química , Animais , Linhagem Celular , Desenho de Fármacos , Humanos , Camundongos , Microscopia de Fluorescência por Excitação Multifotônica , Neurônios/enzimologiaRESUMO
Deep tissue imaging using two-photon fluorescence (TPF) techniques have revolutionized the optical imaging community by providing in depth molecular information at the single-cell level. These techniques provide structural and functional aspects of mammalian brain at unprecedented depth and resolution. However, wavefront distortions introduced by the optical system as well as the biological sample (tissue) limit the achievable fluorescence signal-to-noise ratio and resolution with penetration depth. In this review, we discuss on the advances in TPF microscopy techniques for in vivo functional imaging and offer guidelines as to which technologies are best suited for different imaging applications with special reference to adaptive optics.
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Encéfalo/diagnóstico por imagem , Microscopia de Fluorescência , Neuroimagem , Óptica e Fotônica , Fótons , Animais , Imageamento TridimensionalRESUMO
BACKGROUND: Fatty liver (FL) is now a worldwide disease. For decades, researchers have been kept trying to elucidate the mechanism of FL at the molecular level, but rarely involve the study of morphology and medical physics. Traditionally, it was believed that hemodynamic changes occur only when fibrosis occurs, but it has been proved that these changes already show in steatosis stage, which may help to reveal the pathogenesis and its progress. Because the pseudolobules are not formed during the steatosis stage, this phenomenon may be caused by the compression of the liver microcirculation and changes in the hemodynamics. AIM: To understand the pathogenesis of hepatic steatosis and to study the hemodynamic changes associated with hepatic steatosis. METHODS: Eight-week-old male C57BL/6 mice were divided into three groups randomly (control group, 2-wk group, and 4-wk group), with 16 mice per group. A hepatic steatosis model was established by subcutaneous injection of carbon tetrachloride in mice. After establishing the model, liver tissue from mice was stained with hematoxylin and eosin (HE), and oil red O stains. Blood was collected from the angular vein, and hemorheological parameters were estimated. A two-photon fluorescence microscope was used to examine the flow properties of red blood cells in the hepatic sinusoids. RESULTS: Oil red O staining indicated lipid accumulation in the liver after CCl4 treatment. HE staining indicated narrowing of the hepatic sinusoidal vessels. No significant difference was observed between the 2-wk and 4-wk groups of mice on morphological examination. Hemorheological tests included whole blood viscosity (mPas, γ = 10 s-1/γ = 100 s-1) (8.83 ± 2.22/4.69 ± 1.16, 7.73 ± 2.46/4.22 ± 1.32, and 8.06 ± 2.88/4.22 ± 1.50), red blood cell volume (%) (51.00 ± 4.00, 42.00 ± 5.00, and 40.00 ± 3.00), the content of plasma fibrinase (g/L) (3.80 ± 0.50, 2.90 ± 0.80, and 2.30 ± 0.70), erythrocyte deformation index (%) (44.49 ± 5.81, 48.00 ± 15.29, and 44.36 ± 15.01), erythrocyte electrophoresis rate (mm/s per V/m) (0.55 ± 0.11, 0.50 ± 0.11, and 0.60 ± 0.20), revealing pathological changes in plasma components and red blood cells of hepatic steatosis. Assessment of blood flow velocity in the hepatic sinusoids with a laser Doppler flowmeter (mL/min per 100 g) (94.43 ± 14.64, 80.00 ± 12.12, and 67.26 ± 5.92) and two-photon laser scanning microscope (µm/s) (325.68 ± 112.66, 213.53 ± 65.33, and 173.26 ± 44.02) revealed that as the modeling time increased, the blood flow velocity in the hepatic sinusoids decreased gradually, and the diameter of the hepatic sinusoids became smaller (µm) (10.28 ± 1.40, 6.84 ± 0.93, and 5.82 ± 0.79). CONCLUSION: The inner diameter of the hepatic sinusoids decreases along with the decrease in the blood flow velocity within the sinusoids and the changes in the systemic hemorheology.
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Capilares/fisiopatologia , Fígado Gorduroso/fisiopatologia , Imageamento Tridimensional/métodos , Microcirculação/fisiologia , Animais , Velocidade do Fluxo Sanguíneo/fisiologia , Capilares/diagnóstico por imagem , Tetracloreto de Carbono/toxicidade , Modelos Animais de Doenças , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/diagnóstico por imagem , Humanos , Fluxometria por Laser-Doppler , Fígado/irrigação sanguínea , Fígado/diagnóstico por imagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia ConfocalRESUMO
The rapid growth in the use of optogenetics for neuroscience applications is largely driven by two important advantages: highly specific cellular targeting through genetic manipulations; and precise temporal control of neuronal activation via temporal modulation of the optical stimulation. The difference between the most commonly used stimulation modalities, namely diffused (i.e. synchronous) and focused (i.e. asynchronous) stimulation has not been described. Furthermore, full realization of optogenetics' potential is hindered by our incomplete understanding of the cellular and network level response to photoactivation. Here we address these gaps by examining the neuronal and cerebrovascular responses to focused and diffuse photostimulation of channelrhodopsin in the Thy1-ChR2 mouse. We presented the responses of photoactivation via 470-nm fiber optic illumination (diffuse) alongside 458-nm raster-scan (focused) stimulation of the barrel field. Local field potentials (LFP) assessment of intracerebral electrophysiology and two-photon fluorescence microscopy measurements of red blood cell (RBC) speed (vRBC) in cortical penetrating vessels revealed â¼40% larger LFP responses (pâ¯=â¯0.05) and twice as large cerebrovascular responses (pâ¯=â¯0.002) under focused vs. diffuse photostimulation (focused: 1.64⯱â¯0.84â¯mV LFP amplitude and 75⯱â¯48% increase in vRBC; diffuse: 1.14⯱â¯0.75â¯mV LFP amplitude and 35⯱â¯23% increase in vRBC). Compared to diffuse photostimulation, focused photostimulation resulted in a â¼65% increase in the yield of cerebrovascular responses (73⯱â¯10% for focused and 42⯱â¯29% for diffuse photostimulation) and a doubling of the signal-to-noise ratio of the cerebrovascular response (20.9⯱â¯14.7 for focused and 10.4⯱â¯1.4 for diffuse photostimulation). These data reveal important advantages of focused optogenetic photoactivation, which can be easily integrated into single- or two-photon fluorescence microscopy platforms, as a means of assessing neuronal excitability and cerebrovascular reactivity, thus paving the way for broader application of optogenetics in preclinical models of CNS diseases.
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Encéfalo/irrigação sanguínea , Circulação Cerebrovascular/fisiologia , Channelrhodopsins/metabolismo , Optogenética/métodos , Animais , Encéfalo/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
In recent years, two-photon fluorescence microscopy has gained significant interest in bioimaging. It allows the visualization of deeply buried inhomogeneities in tissues. The near-infrared (NIR) dyes are also used for deep tissue imaging. Indocyanine green (ICG) is the only U.S. Food and Drug Administration (FDA) approved exogenous contrast agent in the NIR region for clinical applications. However, despite its potential candidature, it had never been used as a two-photon contrast agent for biomedical imaging applications. This letter provides an insight into the scope and application of the two-photon excitation property of ICG to the second excited singlet (S2 ) state in aqueous solution. Furthermore, in this work, we demonstrate the two-photon cellular imaging application of ICG using direct fluorescence emission from S2 state for the first time. Our results show that two-photon excitation to S2 state of ICG could be achieved with approximately 790 nm wavelength of femtosecond laser, which lies in well-known "tissue-optical window." This property would enable light to penetrate much deeper in the turbid medium such as biological tissues. Thus, ICG could be used as the first FDA approved NIR exogenous contrast agent for two-photon imaging. These findings can make remarkable influence on preclinical and clinical cell imaging.
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Corantes/química , Aprovação de Drogas , Verde de Indocianina/química , Raios Infravermelhos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Fótons , United States Food and Drug Administration , Células HeLa , Humanos , Fenômenos Ópticos , Estados UnidosRESUMO
Optical imaging is a key modality for observing biological specimen with higher spatial resolution. However, scattering and absorption of light in tissues are inherent barriers in maximizing imaging depth in biological tissues. To achieve this goal, use of light at near-infrared spectrum can improve the present situation. Here, the capability of saturated two-photon saturated excitation (TP-SAX) fluorescence microscopy to image at depths of >2.0 mm, with submicron resolution in transparent mouse brain imaging, is demonstrated. At such depths with scattering-enlarged point spread function (PSF), we find that TP-SAX is capable to provide spatial resolution improvement compared to its corresponding TPFM, which is on the other hand already providing a much improved resolution compared with single-photon confocal fluorescence microscopy. With the capability to further improve spatial resolution at such deep depth with scattering-enlarged PSF, TP-SAX can be used for exquisite visualization of delicate cerebral neural structure in the scattering regime with a submicron spatial resolution inside intact mouse brain.
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Encéfalo/diagnóstico por imagem , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Rede Nervosa/diagnóstico por imagem , Animais , Encéfalo/metabolismo , Carbocianinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Rede Nervosa/metabolismo , Fenômenos ÓpticosRESUMO
The application of two-photon absorption (2PA) materials is a classical research field and has recently attracted increasing interest. It has generated a demand for new dyes with high 2PA cross-sections. In this short review, we briefly cover the structure-2PA property relationships of organic fluorophores, organic-inorganic nanohybrids and metal complexes explored by our group. (1) The two-photon absorption cross-section (δ) of organic fluorophores increases with the extent of charge transfer, which is important to optimize the core, donor-acceptor pair, and conjugation-bridge to obtain a large δ value. Among the various cores, triphenylamine appears to be an efficient core. Lengthening of the conjugation with styryl groups in the D-π-D quadrupoles and D-π-A dipoles increased δ over a long wavelength range than when vinylene groups were used. Large values of δ were observed for extended conjugation length and moderate donor-acceptors in the near-IR wavelengths. The δ value of the three-arm octupole is larger than that of the individual arm, if the core has electron accepting groups that allow significant electronic coupling between the arms; (2) Optical functional organic/inorganic hybrid materials usually show high thermal stability and excellent optical activity; therefore the design of functional organic molecules to build functional organic-inorganic hybrids and optimize the 2PA properties are significant. Advances have been made in the design of organic-inorganic nanohybrid materials of different sizes and shapes for 2PA property, which provide useful examples to illustrate the new features of the 2PA response in comparison to the more thoroughly investigated donor-acceptor based organic compounds and inorganic components; (3) Metal complexes are of particular interest for the design of new materials with large 2PA ability. They offer a wide range of metals with different ligands, which can give rise to tunable electronic and 2PA properties. The metal ions, including transition metals and lanthanides, can serve as an important part of the structure to control the intramolecular charge-transfer process that drives the 2PA process. As templates, transition metal ions can assemble simple to more sophisticated ligands in a variety of multipolar arrangements resulting in interesting and tailorable electronic and optical properties, depending on the nature of the metal center and the energetics of the metal-ligand interactions, such as intraligand charge-transfer (ILCT) and metal-ligand charge-transfer (MLCT) processes. Lanthanide complexes are attractive for a number of reasons: (i) their visible emissions are quite long-lived; (ii) their absorption and emission can be tuned with the aid of appropriate photoactive ligands; (iii) the accessible energy-transfer path between the photo-active ligands and the lanthanide ion can facilitate efficient lanthanide-based 2PA properties. Thus, the above materials with excellent 2PA properties should be applied in two-photon applications, especially two-photon fluorescence microscopy (TPFM) and related emission-based applications. Furthermore, the progress of research into the use of those new 2PA materials with moderate 2PA cross section in the near-infrared region, good Materials 2017, 10, 223 2 of 37 biocompatibility, and enhanced two-photon excited fluorescence for two-photon bio-imaging is summarized. In addition, several possible future directions in this field are also discussed (146 references).