Measuring Exocytosis in Endocrine Tissue Slices

A recent challenge in the field of bioimaging is to image vital, thick, and complex tissues in real time and in non-invasive mode. Among the different tools available for diagnostics, nonlinear optical (NLO) multi-photon microscopy allows label-free non-destructive investigation of physio-pathological processes in live samples at sub-cellular spatial resolution, enabling to study the mechanisms underlying several cellular functions. In this review, we discuss the fundamentals of NLO microscopy and the techniques suitable for biological applications, such as two-photon excited fluorescence (TPEF), second and third harmonic generation (SHG-THG), and coherent Raman scattering (CRS). In addition, we present a few of the most recent examples of NLO imaging employed as a label-free diagnostic instrument to functionally monitor in vitro and in vivo vital biological specimens in their unperturbed state, highlighting the technological advantages of multi-modal, multi-photon NLO microscopy and the outstanding challenges in biomedical engineering applications.

Chapter thirty-one - Multiphoton Excitation Microscopy and Spectroscopy of Cells, Tissues and Human Skin In Vivo

Metabolism of xenobiotics is often seen as an exclusive function of the liver, but some current findings support the notion that the lungs, kidneys and intestine may contribute considerably. After the establishment of the use of liver slices as a useful in vitro model to study metabolism and toxicity of xenobiotics, the same concept is currently being used for slices from lung, kidney and intestine. It is the aim of this review to discuss the use of organ slices in biotransformation research. The basic idea behind the use of tissue slices in biomedical research is the assumption that the cells under study will function optimally in vitro if they are cultivated in an environment that is most alike to their natural in vivo embedding, which is the case in tissue slices. Advantages in the use of organ slices are the relatively easy preparation as well as the potential standardization of both the preparation and use. Moreover, a direct interspecies comparison can be made between liver, lungs, kidneys and intestines, for example with respect to their metabolic capacity and their sensitivity for toxicants. Of major importance is that organ slices can be made with a similar procedure from organs/tissues originating from different species, including man. This latter aspect is useful in drug development in general but also for a better insight in the metabolic fate of compounds in man. Importantly the use of slices may largely contribute to a reduction in the use of experimental animals.

Since the first report more than 10 years ago of two-photon excited fluorescence microscopy (Denk et al., 1990), multiphoton microscopy (MPM) has found uses in many research areas. Multiphoton microscopy has had a particularly strong impact on life sciences because several of its properties provide special advantages for studying living cells in intact tissue (for reviews see Denk and Svoboda, 1997; Piston, 1999; König, 2000; So et al., 2000; Helmchen and Denk, 2002). In this chapterwe focus on applications in neuroscience. The cerebral cortex in human contains about 1010 neurons with about 1014 synaptic connections among them (Braendgaard et al., 1990; Tang et al., 2001). To understand the integrated function of these structures, it is necessary to study them in their native configuration. Multiphoton microscopy has helped to reveal neural signaling in near-intact living tissue at levels ranging from individual synapses to entire neural networks (fig. 3.1 [Garaschuk et al., 2000; Wang et al., 2000b]). It has been used to investigate development, structural plasticity, modulation of excitability, and synaptic integration. Multiphoton microscopy has proved particularly powerful when used in conjunction with advances in fluorescent tissue labeling techniques, electrophysiology, and molecular biology.

Multiphoton fluorescence microscopy for in vivo imaging

Pilot Study of the Correlation of Multiphoton Tomography of Ex Vivo Human Testis with Histology

Multiphoton fluorescence microscopy is a powerful, important tool in biomedical research that offers low photon toxicity and higher spatial and temporal resolution than other in vivo imaging modalities. The capability to collect images hundreds of micrometers into biological tissues provides an invaluable tool for studying cellular and subcellular processes in the context of tissues and organs in living animals. Multiphoton microscopy is based upon two-photon excitation of fluorescence that occurs only in a sub-femtoliter volume at the focus; by scanning the focus through a sample, 2- and 3-dimensional images can be collected. The complex 3-dimensional organization of the kidney makes it especially appropriate for multiphoton microscopic analysis, which has been used to characterize numerous aspects of renal physiology and pathophysiology in living rats and mice. However, the ability to collect fluorescence images deep into biological tissues raises unique problems not encountered in other forms of optical microscopy, including issues of probe access, and tissue optics. Future improvements in multiphoton fluorescence microscopy will involve optimizing objectives for the unique characteristics of multiphoton fluorescence imaging, improving the speed at which images may be collected and extending the depth to which imaging may be conducted.

In this series of papers on light microscopy imaging, we have covered the fundamentals of microscopy, super-resolution microscopy, and lightsheet microscopy. This last review covers multi-photon microscopy with a brief reference to intravital imaging and Brainbow labeling. Multi-photon microscopy is often referred to as two-photon microscopy. Indeed, using two-photon microscopy is by far the most common way of imaging thick tissues; however, it is theoretically possible to use a higher number of photons, and three-photon microscopy is possible. Therefore, this review is titled "multi-photon microscopy." Another term for describing multi-photon microscopy is "non-linear" microscopy because fluorescence intensity at the focal spot depends upon the average squared intensity rather than the squared average intensity; hence, non-linear optics (NLO) is an alternative name for multi-photon microscopy. It is this non-linear relationship (or third exponential power in the case of three-photon excitation) that determines the axial optical sectioning capability of multi-photon imaging. In this paper, the necessity for two-photon or multi-photon imaging is explained, and the method of optical sectioning by multi-photon microscopy is described. Advice is also given on what fluorescent markers to use and other practical aspects of imaging thick tissues. The technique of Brainbow imaging is discussed. The review concludes with a description of intravital imaging of the mouse. © 2023 Wiley Periodicals LLC.

Two-photon excitation microscopy enables in vivo deep-tissue imaging within organisms. This technique is based on two-photon excitation, a nonlinear optical process that uses near-infrared light for excitation, resulting in high tissue permeability. Notably, two-photon excitation occurs only near the focal plane; therefore, minimally invasive tomographic images can be obtained. Owing to these features, two-photon excitation microscopy is currently widely used in medical and life-science research, particularly in the domain of neuroscience for in vivo visualization of deep tissues. However, the use of long-wavelength excitation light in two-photon excitation microscopy has resulted in a larger focused spot size and relatively low spatial resolution, which is a limitation of this technique for further applications. Recent studies have described super-resolution microscopy techniques applied to two-photon excitation microscopy in an attempt to observe living organisms "as they are in their natural state" with high spatial resolution. We have also addressed this topic using an optical approach (two-photon stimulated emission depletion microscopy) and an image analysis approach (two-photon super-resolution radial fluctuation). Here, we describe these approaches together with a discussion of our recent accomplishments.

Multi-photon excitation (MPE) microscopy plays a growing role among microscopical techniques utilized for studying biological matter. In conjunction with confocal microscopy it can be considered the imaging workhorse of life science laboratories. Its roots can be found in a fundamental work written by Maria Goeppert Mayer more than 70 years ago. Nowadays, 2PE and MPE microscopes are expected to increase their impact in areas such biotechnology, neurobiology, embryology, tissue engineering, materials science where imaging can be coupled to the possibility of using the microscopes in an active way, too. As well, 2PE implementations in noninvasive optical bioscopy or laser-based treatments point out to the relevance in clinical applications. Here we report about some basic aspects related to the phenomenon, implications in three-dimensional imaging microscopy, practical aspects related to design and realization of MPE microscopes, and we only give a list of potential applications and variations on the theme in order to offer a starting point for advancing new applications and developments.

Immune Regulatory Mechanisms Influence Early Pathology in Spinal Cord Injury and in Spontaneous Autoimmune Encephalomyelitis

Methods for calculating the stereotaxic coordinates of rat brain structures by pixel coordinates of the image obtained by confocal and two-photon laser scanning microscopy

Time- and dose-response effects of the mycotoxin, fumonisin B 1 on sphingoid base elevations in precision-cut rat liver and kidney slices

Calcium (Ca2+) is an intracellular secondary messenger with multiple important physiological roles; disturbances in intracellular Ca2+ homeostasis have been implicated in the pathogenesis of various kidney diseases. We have recently demonstrated that multiphoton microscopy can be used to image a range of intracellular signals in ex vivo rat kidney preparations such as tissue slices [1, 2] and isolated perfused organs [3].Here, we demonstrate that the tissue slice model can be used to load established Ca2+ indicators into both tubular and endothelial cells in the kidney. Kidney slices (200 μm thick) were obtained from adult, male sprague dawley rat kidneys (following cervical dislocation) and maintained in a physiological saline solution, bubbled with 95% O2/5% CO2. Loading of exogenous Ca2+ sensitive dyes (Fluo4, Rhod‐2, Fura Red) was achieved ex vivo by using a re‐circulating perfusion system. Confocal microscopy was used to image Ca2+ loading in the renal medulla, and to record changes in signal in response to physiological stimuli, i.e. ATP, Angiotensin II.We believe that the application of these imaging techniques have much potential to enhance understanding of the roles of intracellular Ca2+ signalling in renal physiology and pathophysiology.Research supported by the Medical Research Council

Multiphoton fluorescence excitation microscopy is almost invariably conducted with samples whose refractive index differ from that of the objective immersion medium, conditions that cause spherical aberration. Due to the quadratic nature of multiphoton fluorescence excitation, spherical aberration is expected to profoundly affect the depth dependence of fluorescence excitation. In order to determine the effect of refractive index mismatch in multiphoton fluorescence excitation microscopy, we measured signal attenuation, photobleaching rates and resolution degradation with depth in homogeneous samples with minimal light scattering and absorption over a range of refractive indices. These studies demonstrate that signal levels and resolution both rapidly decline with depth into refractive index mismatched samples. Analyses of photobleaching rates indicate that the preponderance of signal attenuation with depth results from decreased rates of fluorescence excitation, even in a system with a descanned emission collection pathway. Similar results were obtained in analyses of fluorescence microspheres embedded in rat kidney tissue, demonstrating that spherical aberration is an important limiting factor in multiphoton fluorescence excitation microscopy of biological samples.