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Gaining insight into how the nervous system functions is a challenge for scientists, particularly because the static morphology of the brain and the cells within tell little about how they actually work. Fixed specimens can provide critical structural information, but the jump to functional neurobiology in living cells is obviated with these preparations. In order to grasp the complexity of neuronal activity, it is necessary to observe the brain in action, from the level of subcellular signaling to the whole organism. Recent advances in nonlinear microscopy have given rise to a new era for biological research. In particular, the introduction of multiphoton excitation has drastically improved the depth and speed to which we can probe brain function. In order to better appreciate recent contributions of multiphoton microscopy to our current and future understanding of biological systems, an historical awareness of past microscopy applications is useful.

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To improve the luminescent signal in nonlinear optical imaging of biological specimens, specific coated standard microscope cover glasses were utilized to test a variety of specimens. Results demonstrated image quality can be exactly improved.

High-resolution multiphoton imaging of live cells has become an invaluable method to study protein dynamics in highly compartmentalized subcellular environments. Here we describe procedures that we recently developed to quantify rhodopsin mobility within and between retinal rod photoreceptor light signaling microcompartments, the disc membrane lobules, using multiphoton fluorescence relaxation after photoconversion.

The advantages offered by multiphoton microscopy enabled this technique to be applied for in vivo understanding of physiological processes. However, multiphoton intravital microscopy also requires associated technologies to be developed. In this work, we detailed the design of a dorsal skin fold chamber made of titanium alloy that allow high resolution multiphoton fluorescence and second harmonic generation (SHG) microscopy to be achieved. We demonstrate that our apparatus is capable of obtaining high resolution, images of blood vessels and collagen matrix in nude mice. Such an imaging chamber will allow physiological processes to be investigated at high resolution

A novel method for the fluorescence detection of proteins in cells is described in the present study. Proteins are labelled by the selective biosynthetic incorporation of 5-hydroxytryptophan and the label is detected via selective two-photon excitation of the hydroxyindole and detection of its fluorescence emission at 340 nm. The method is demonstrated in this paper with images of a labelled protein in yeast cells.

Since the initial images were taken using a multiphoton imaging technique the method has rapidly established itself as the preferred method for imaging deeply into biological samples with micron resolution in three dimensions. Multiphoton imaging has thus enabled researchers in the life sciences to undertake studies that had previously been believed to be impossible without significantly perturbing the sample. Many of these experiments have only been realized due to close cooperation between optical physicists, from a range of disciplines, and the biomedical researchers. This paper will provide a general review of the current state of the field demonstrating how the various aspects of the physics development have brought the multiphoton technique to its current position at the forefront of biological microscopy.

Ischemia-reperfusion injury, a common complication during liver surgery where steatotic livers are more prone to the injury, may become more prevalent in the growing obese population. This study characterizes liver morphology toward understanding changes in subcellular function in steatotic livers exposed to ischemia-reperfusion injury through quantitative description of fluoresce in distribution obtained by minimally invasive in vivo multiphoton microscopy using a physiologic pharmacokinetic model. Rats were fed a high-fat diet for 7 days to induce liver steatosis. Partial ischemia was induced after reperfusion for 4 hours, when fluoresce in (10 mg/kg) was injected intravenously. Liver images, bile, and blood were collected up to 180 minutes after injection. Ischemia-reperfusion injury was associated with an increase in alanine transaminase levels and apoptosis. In addition, steatosis featured lipid droplets and an increase in fluoresce in-associated fluorescence observed in hepatocytes via multiphoton imaging. Analysis of the hepatic concentration-time profiles has suggested that the steatosis-induced increase in fluoresce in-associated fluorescence mainly arises by inducing hepatic fluoresce in metabolism. The combination of ischemia-reperfusion with steatosis exacerbates these effects further. This was confirmed by fluorescence lifetime imaging microscopy showing a decreased average fluorescence lifetime of the liver, which is indicative of an increased production of the metabolite. Our results show the potential of noninvasive dye imaging for improving our understanding of liver disease induced by subcellular changes in vivo, providing further quantitative measures of metabolic and biliary liver function, and hence extending the qualitative liver function tests now available.

Photoactivated localization microscopy (PALM) and related single-molecule imaging techniques enable biological image acquisition at ~20 nm lateral and ~50–100 nm axial resolution. Although such techniques were originally demonstrated on single imaging planes close to the coverslip surface, recent technical developments now enable the 3D imaging of whole fixed cells. We describe methods for converting a 2D PALM into a system capable of acquiring such 3D images, with a particular emphasis on instrumentation that is compatible with choosing relatively dim, genetically expressed photoactivatable fluorescent proteins (PA-FPs) as PALM probes. After reviewing the basics of 2D PALM, we detail astigmatic and multiphoton imaging approaches well suited to working with PA-FPs. We also discuss the use of open-source localization software appropriate for 3D PALM.

International audience; The assembly of proteins into fibrillar structures is an important process that concerns different biological contexts, including molecular medicine and functional biomaterials. Engineering of hybrid biomaterials can advantageously provide synergetic interactions of the biopolymers with an inorganic component to ensure specific supramolecular organization and dynamics. To this aim, we designed hybrid systems associating collagen and surface-functionalized silica particles and we built a new strategy to investigate fibrillogenesis processes in such multicomponents systems, working at the crossroads of chemistry, physics and mathematics. The self-assembly process was investigated by bimodal multiphoton imaging coupling second harmonic generation (SHG) and 2 photon excited fluorescence (2PEF). The in-depth spatial characterization of the system was further achieved using the three-dimensional analysis of the SHG/2PEF data via mathematical morphology processing. Quantitation of collagen distribution around particles offers strong evidence that the chemically induced confinement of the protein on the silica nanosurfaces has a key influence on the spatial extension of fibrillogenesis. This new approach is unique in the information it can provide on 3D dynamic hybrid systems and may be extended to other associations of fibrillar molecules with optically responsive nano-objects.

We applied multiphoton microscopic imaging to observe freezing and heating effects in plant- and animal cell samples. The experimental setups consisted of a multiphoton imaging system and a heating and cooling stage which allows for precise temperature control from liquid nitrogen temperature (-196°C; 77 K) up to +600°C (873 K) with heating/freezing rates between 0.01 K/min and 150 K/min. Two multiphoton imaging systems were used: a system based on a modified optical microscope and a flexible mobile system. To illustrate the imaging capabilities, plant leafs as well as animal cells were microscopically imaged in vivo during freezing based on autofluorescence lifetime and intensity of intrinsic molecules. The measurements illustrate the usefulness of multiphoton imaging to investigate freezing effects on animal and plant cells.

Multiphoton microscopy has become staple tool for tracking cells within tissues and organs due to superior depth of penetration, low excitation volumes, and reduced phototoxicity. Many factors, ranging from laser pulse width to relay optics to detectors and electronics, contribute to the overall ability of these microscopes to excite and detect fluorescence deep within tissues. However, we have found that there are few standard ways already described in the literature to distinguish between microscopes or to benchmark existing microscopes to measure the overall quality and efficiency of these instruments. Here, we discuss some simple parameters and methods that can either be used within a multiphoton facility or by a prospective purchaser to benchmark performance. This can both assist in identifying decay in microscope performance and in choosing features of a scope that are suited to experimental needs.

The clinical application of Mesenchymal Stem Cells (MSCs) for the treatment of a variety of diseases is the focus of intense research. Despite large research efforts many questions regarding MSC biology in vivo remain unanswered. For instance, we do not know for certain whether MSCs exert their therapeutic effects directly within the target tissue or indirectly by influencing the polarization of other cell types, such as macrophages, which can then home to the target tissue microenvironment. To help address this issue, the application of intravital multiphoton microscopy allows for the determination of the dynamic action of intact MSCs versus endogenous host cells at the target tissue site in real time.

As the main input nucleus to the basal ganglia, the striatum is responsible for receiving and integrating highly convergent afferents to ultimately guide action selection and movement initiation. Although the majority of this synaptic integration occurs in the dendrites of striatal projection neurons (SPNs), their thin diameter makes them inaccessible with traditional recording electrodes. Recent advances in optical imaging technologies have allowed us and others to start lifting the veil on the mechanisms governing synaptic integration in the striatum by enabling direct dendritic measurements and manipulations. Here we describe how our lab has approached combining 2-photon imaging and photolysis with electrophysiological recordings to study dendritic excitability and synaptic integration in the striatum.

We discuss the development of compact, affordable multiphoton microscopes using robust femtosecond fiber lasers as the excitation source. Application in brain imaging and Barrett’s cancer imaging will be presented.

Through the development of simultaneous spatial and temporal focusing, platforms that can ablate tissue with excellent axial precision, and perform high-speed multiphoton imaging are converging. Recent developments in these platforms are presented.

Over the last two decades, multiphoton microscopy has created a renaissance in the brain imaging community. It has changed how we visualize neurons by providing high-resolution, non-invasive imaging capability deep within intact brain tissue. Multiphoton imaging will likely play an essential role in understanding how the brain works at the level of neural circuits, which will provide a bridge between microscopic interactions at the neuronal level and the macroscopic structures that perform complex computations.

The endomembrane system of a cell is a highly dynamic, ephemeral structure that is difficult to visualize. Reconstructions from sections of fixed material can provide high-resolution information on intercellular membrane architecture, but such techniques are fraught with artifacts and are of little help in understanding the dynamics of intracellular membrane traffic. Recently, the availability of fluorescent membrane probes and the development of techniques for optically sectioning intact specimens have allowed glimpses of membrane dynamics to be visualized in living tissue. In this review we discuss the potential of a new optical sectioning technique, multiphoton imaging, for visualizing membrane dynamics in living cells. Multiphoton microscopy offers an unparalleled ability to obtain images from deep within specimens while minimizing the effects of phototoxicity.

Advances in understanding the molecular regulation of longitudinal growth have led to development of novel drug therapies for growth plate disorders. Despite progress, a major unmet challenge is delivering therapeutic agents to avascular-cartilage plates. Dense extracellular matrix and lack of penetrating blood vessels create a semipermeable “barrier,” which hinders molecular transport at the vascular-cartilage interface. To overcome this obstacle, we used a hindlimb heating model to manipulate bone circulation in 5-wk-old female mice ( n = 22). Temperatures represented a physiological range of normal human knee joints. We used in vivo multiphoton microscopy to quantify temperature-enhanced delivery of large molecules into tibial growth plates. We tested the hypothesis that increasing hindlimb temperature from 22°C to 34°C increases vascular access of large systemic molecules, modeled using 10, 40, and 70 kDa dextrans that approximate sizes of physiological regulators. Vascular access was quantified by vessel diameter, velocity, and dextran leakage from subperichondrial plexus vessels and accumulation in growth plate cartilage. Growth plate entry of 10 kDa dextrans increased >150% at 34°C. Entry of 40 and 70 kDa dextrans increased

Acidity, hypoxia, and glucose levels characterize the tumor microenvironment rendering pH, pO2, and pGlucose, respectively, important indicators of tumor health. To this end, understanding how these parameters change can be a powerful tool for the development of novel and effective therapeutics. We have designed optical chemosensors that feature a quantum dot and an analyte-responsive dye. These noninvasive chemosensors permit pH, oxygen, and glucose to be monitored dynamically within the tumor microenvironment by using multiphoton imaging., National Cancer Institute (U.S.) (Grant R01-CA126642)

Three-dimensional culture systems bridge the experimental gap between in vivo and in vitro physiology. However, nonstandardized formation and limited downstream adaptability of 3D cultures have hindered mainstream adoption of these systems for biological applications, especially for low- and moderate-throughput assays commonly used in biomedical research. Here we build on our recent development of a 384-well hanging drop plate for spheroid culture to design a complementary spheroid transfer and imaging (TRIM) plate. The low-aspect ratio wells of the TRIM plate facilitated high-fidelity, user-independent, contact-based collection of hanging drop spheroids. Using the TRIM plate, we demonstrated several downstream analyses, including bulk tissue collection for flow cytometry, high-resolution low working-distance immersion imaging, and timely reagent delivery for enzymatic studies. Low working-distance multiphoton imaging revealed a cell type-dependent, macroscopic spheroid structure. Unlike ovarian cancer spheroids, which formed loose, disk-shaped spheroids, human mammary fibroblasts formed tight, spherical, and nutrient-limited spheroids. Beyond the applications we describe here, we expect the hanging drop spheroid plate and complementary TRIM plate to facilitate analyses of spheroids across the spectrum of throughput, particularly for bulk collection of spheroids and high-content imaging.

In recent years, there has been an increasing interest in non-invasive imaging techniques in clinical and investigational dermatology. Besides the growing number of biophysical methods for the assessment of skin properties, novel imaging tools have emerged and classical imaging methods are substantially improved.This review focuses on well established and some of the most promising imaging techniques for application in dermatology and cutaneous research. These comprise dermoscopy, sonography, confocal microscopy, multiphoton tomography and optical coherence tomography. Non-invasive imaging techniques are presented concerning their functional principles, the history of their development and their current clinical and research application. Advantages and limitations of each of the methods are discussed.Available non-invasive imaging techniques in dermatology show substantial differences concerning their limitations and opportunities, potential clinical applicability and practicability. Also, the current data available differ largely between these methods due to different levels of experience in research and clinical application. Future research will not only aim at improving current technical limitations, but also investigate the potential synergistic effects of combining two or more techniques in order to enhance their diagnostic impact.

Objective To identify and evaluate the ideal temperature for long-term storage of hearts from donation after cardiocirculatory death, in the novel organ preservation solution Somah. Methods DCD hearts from Sprague-Dawley rats were harvested after 30 minutes of euthanasia, preserved in Somah at 4°C, 10°C, 21°C, or 37°C for 24 hours and then reperfused with blood:Somah (3:1) perfusate at 37°C for 30 minutes. Myocardial biopsies were taken during storage and before and after reperfusion to assess the structural and functional viability of tissue using multiphoton imaging, biochemistry, and immunofluorescence. Results Myocyte viability, determined by Live-Dead and esterase assays, was similar at 4°C, 10°C, and 21°C (193, 198 and 217 normalized fluorescence counts [NFC]) with a significant decrease at 37°C (131 NFC). Upon reperfusion, esterase activity was enhanced in DCD hearts stored in Somah at 21°C but noticeably decreased at all other temperatures. High-energy adenosine triphosphate/creatine phosphate (ATP/CP) syntheses and the expression of structural/contractile proteins was well preserved at 21°C, both after 24-hour storage and upon reperfusion. In contrast, hearts stored at all other temperatures demonstrated variable degenerative changes, loss of protein expression, and/or deranged ATP/CP synthesis after 24 hours of storage and/or upon reperfusion. Conclusion The robust maintenance of structural/functional integrity of cardiac tissue and the preservation of protein expression and cellular energy metabolism in DCD hearts after long-term preservation at subnormothermic temperature suggests that 21°C is ideal for long-term storage of DCD hearts in Somah solution.

We have demonstrated a straightforward and noninvasive method to identify the distribution of grana and starch within an intact leaf. Grana and starch are the major functional structures for photosynthesis and energy storage of plant, respectively. Both exhibit highly ordered molecular structures and appear as micrometer-sized granules inside chloroplasts. In order to distinguish grana and starch, we used multiphoton microscopy, with simultaneous acquisition of two photon fluorescence (2PF) and second harmonic generation (SHG) signals. Consequently, SHG is found on both grana and starch while 2PF from chlorophyll indicates the identity of grana.

New labeling, imaging, or analysis tools could provide new retrospective insights when applied to archived, paraffin-embedded samples. Deep-tissue multiphoton microscopy of paraffin-embedded specimens is achieved using optical clearing with mineral oil. We tested a variety of murine tissue specimens including skin, lung, spleen, kidney, and heart, acquiring multiphoton autofluorescence and second-harmonic generation, and pump-probe images This technique introduces the capability for non-destructive 3-dimensional microscopic imaging of existing archived pathology specimens, enabling retrospective studies.

We present a multiphoton microscope system which combines the advantages of multiphoton imaging with precise control of the sample temperature. The microscope provides online insight in temperature-induced changes and effects in plant tissue and animal cells with subcellular resolution during cooling and thawing processes. Image contrast is based on multiphoton fluorescence intensity or fluorescence lifetime in the range from liquid nitrogen temperature up to +600°C. In addition, micro spectra from the imaged regions can be recorded. We present measurement results from plant leaf samples as well as Chinese hamster ovary cells.

We show the recent progress on superresolution multiphoton imaging and tweezing of micro/nano fluorescent particles in a fibre-optical nonlinear optical endoscope.

Intravital imaging by multiphoton microscopy is a powerful tool to gain invaluable insight into tissue biology and function. Here, we provide a step-by-step tissue preparation protocol for imaging the mouse tibialis anterior skeletal muscle. Additionally, we include steps for jugular vein catheterization that allow for well-controlled intravenous reagent delivery. Preparation of the tibialis anterior muscle is minimally invasive, reducing the chances of inducing damage and inflammation prior to imaging. The tibialis anterior muscle is useful for imaging leukocyte interaction with vascular endothelium, and to understand muscle contraction biology. Importantly, this model can be easily adapted to study neuromuscular diseases and myopathies.

Standard histopathology techniques (including paraffin embedding) are incompatible with thick tissue multiphoton imaging, and standard clearing techniques on those specimens destroy some molecular information. We demonstrate multiphoton imaging in specimens prepared according to standard histopathology techniques. This permits unlabeled 3-dimensional histology on archival tissue banks, which is of great value in evaluating prognostic indicators.

Intercellular glial calcium waves (GCW) constitute a signaling pathway which can be visualized by fluorescence imaging of cytosolic Ca(2+) changes. Reliable detection of calcium waves in multiphoton imaging data is challenging because of low signal-to-noise ratio. We modified the multiscale vision model (MVM), originally employed to detect faint objects in astronomy data to process stacks of fluorescent images. We demonstrate that the MVM identified and characterized GCWs with much higher sensitivity and detail than pixel thresholding. Origins of GCWs were often associated with prolonged secondary Ca(2+) elevations. The GCWs had variable shapes, and secondary GCWs were observed to bud from the primary, larger GCW. GCWs evaded areas shortly before occupied by a preceding GCW instead circulating around the refractory area. Blood vessels uniquely reshaped GCWs and were associated with secondary GCW events. We conclude that the MVM provides unique possibilities to study spatiotemporally correlated Ca(2+) signaling in brain tissue.

Alzheimer’s disease (AD) is a protein conformational disorder characterized by two major neuropathological features: extracellular accumulations of amyloid-β peptides in the form of plaques and intracellular tangles, consisting of hyperphosphorylated tau proteins. Several morphological and functional changes are associated with these lesions in the diseased brain, such as dendritic and synaptic alterations, as well as microglial and astroglial recruitment and their activation. The availability of transgenic mouse models that mimic key aspects of the disease in conjunction with recent advances in two-photon imaging facilitate the study of fundamental aspects of AD pathogenesis and allow for longitudinally monitoring the efficacy of therapeutic interventions. Here, we review the ambitious efforts to understand the relationship between the main neuropathological hallmarks of AD and their associated structural and functional abnormalities by means of in vivo two-photon imaging.

In this work we present how to entirely remove the scattering ambiguity present in existing multiphoton multifocal systems. This is achieved through the development and implementation of single-element detection systems that incorporate high-speed photon-counting electronics. These systems can be used to image entire volumes in the time it takes to perform a single transverse scan (four depths simultaneously at a rate of 30 Hz). In addition, this capability is further exploited to accomplish single-element detection of multiple modalities (two photon excited fluorescence and second harmonic generation) and to perform efficient image deconvolution. Finally, we demonstrate a new system that promises to significantly simplify this promising technology.

Manipulations of mouse vessels are difficult due to their small sizes, limiting the possibilities for developing clinically relevant thrombosis models. Currently used models of arterial and venous thrombosis have short-comings not only in terms of clinical relevance but also with respect to collection of data usable and commensurate with thrombodynamic processes. Confocal and multiphoton imaging systems offer continuous evaluation of more direct measures, such as fluorophore-labeled, thrombus-targeting factors, but have been predominantly confined to studies in microvessels (arterioles and venules) with questionable transference of findings to large-vessel thrombotic activities. Going forward, we should select more clinically relevant models, using evaluative measures that yield optimal quantitative data, to refine our understanding of critical aspects of thrombotic processes.

Multiphoton microscopy is a powerful tool in neuroscience, promising to deliver important data on the spatiotemporal activity within individual neurons as well as in networks of neurons. A major limitation of current technologies is the relatively slow scan rates along the z direction compared to the kHz rates obtainable in the x and y directions. Here, we describe a custom-built microscope system based on an architecture that allows kHz scan rates over hundreds of microns in all three dimensions without introducing aberration. We further demonstrate how this high-speed 3D multiphoton imaging system can be used to study neuronal activity at millisecond resolution at the subcellular as well as the population level.

Multiphoton imaging methods are excellent for non-invasive imaging of living tissue without any need of additional contrast agents. The increasing demand for endoscopic techniques has forced the development of multiphoton endoscopes for imaging of areas with reduced accessibility like chronic wounds. Gradient index (GRIN) lenses can miniaturize the bulky distal focusing optics of conventional tomographs to a diameter of less than 1.4 mm and a numerical aperture (NA) of 0.8. We combined a high NA clinical multiphoton endoscope with existing multiphoton tomographs like the DermaInspect® and the MPTflex® to enable the examination of wound healing processes.

Multiphoton imaging systems are capable of high-resolution 3-D image acquisition of deep tissue. A first commercially available CE-certified biomedical system for subcelluar resolution of human skin has been launched by JenLab company with the DermaInspectR in 2002. The demand for more flexibility caused the development of the MPTflexR, which provides an increased flexibility and accessibility especially for clinical and cosmetic examinations. However the high resolution of clinical multiphoton tomographs are adherent with a small field-of-view (FOV) of about 360×360μm2. Especially time-consuming is the relocation of areas of interest (AOI) like lesions, sweat glands or hair shafts during a multiphoton examination. This limitation can be be overcome by macroscopic large-area (wide-field-ofview) multiphoton tomography, which is tested first within this work.

An Yb fiber laser oscillator with sub-30 fs pulses compressed by MIIPS is tested for multiphoton microscopy. It leads to greatly improved third harmonic generation images. Multiphoton fluorescence, second and third harmonic generation modalities are compared on stained microspheres and unstained biological tissues.

Multiphoton (MP) microscopy enables the direct in vivo visualization, with high spatial and temporal resolution, of fluorescently tagged immune cells, extracellular matrix and vasculature in tissues. This approach, therefore, represents a powerful alternative to traditional methods of assessing immune cell function in the skin, which are mainly based on flow cytometry and histology. Here we provide a step-by-step protocol describing experimental procedures for intravital MP imaging of the mouse ear skin, which can be easily adapted to address many specific skin-related biological questions. We demonstrate the use of this procedure by characterizing the response of neutrophils during cutaneous inflammation, which can be used to perform in-depth analysis of neutrophil behavior in the context of the skin microanatomy, including the epidermis, dermis and blood vessels. Such experiments are typically completed within 1 d, but as the procedures are minimally invasive, it is possible to perform longitudinal studies through repeated imaging.