Multi-Photon Microscopy.

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.

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

Multiphoton microscopy is an established technique for deep in vivo neuroimaging. However, the volume of the adult brain in most model organisms (e.g. mouse) is beyond the reach of high resolution optical imaging techniques, limiting the study of neurobiological questions in adulthood. <i>Danionella</i>, a genus closely related to zebrafish, which have smaller brains and lack ossification above the brain in adulthood have been introduced for in vivo studying of brain circuits in adult stages. Here, we present a quantitative comparison of optical accessibility within the <i>Danionella dracula</i> brain with two- and three-photon microscopy. We demonstrate that the entire brain of this animal is optically accessible in adulthood by imaging fluorescently-labeled vasculature throughout the deepest part of the brain. While both two- and three-photon microscopy can penetrate through the entire brain, images obtained by three-photon microscopy maintain higher contrast and optical sectioning in deeper regions. To determine the source of low signal to background ratio in the deep images we characterized the distribution of blood vessels to find the staining density in the brain and compared our findings to theoretical expectations of signal to background ratio as a function of depth in multiphoton microscopy. We found that low quality of images in not just due to background generation. Optical aberrations due to tissue inhomogeneity in the cone of light likely play a role in low image quality of the deeper regions. We also demonstrate longitudinal imaging enabled by the non-invasive nature of multiphoton microscopy.

In vivo microscopy

Chapter 19 - Applications of Multiphoton Microscopy in Dermatology

581 Background: Multiphoton endomicroscopy is the recentely updated technique for endoscopy and virtual image and optical sectioning. However optimized probe has not been established for multiphoton endomicroscopic image. Therefore we developed novel probe for mitochondria and applied for colon neoplasm tissues. In cancer cell, abnormally increased mitochondrial replication is related mitochondrial dysfunction and Warburg effect. Methods: We used newly developed multiphoton probe for micochondria imaging which are made using benzofuran derivative (BFP, maximal multiphoton fluorescence at 570 nm). Fresh mucosal tissues of colonic adenoma and adenocarcinoma were obtained from endoscopic biopsy. Multiphoton probe BFP for mitochondria was stained for tissues and imaging performed using multiphoton microscopy. Results: BFP shows high enhancement factor upon binding mitochondria, good selectivity, cell permeability, and can readily detect mitochondria in human tissues by multiphoton microscopy. Mitochondria were detected in human colon mucosa tissues. Calculated mitochondria area were increased in adenocarcinoma tissues compared to normal mucosal tissues. Conclusions: Newly developed multiphoton probe for mitochondria are usable to image human live colon tissues.

The ultrafast laser for material processing concentrates the pulse energy into the scale of picosecond or femtosecond temporal duration. The high peak power would induce nonlinear multiphoton fluorescence, material modification and photoablation with higher pulse energy [1]–[3]. With the increase of processing resolution and complexity, we would propose the noninvasive inspection method by integrating with multiphoton excited fluorescence microscopy (MPEFM) to directly monitor the processed structure in specimen for verification and analysis [1]. In this paper, we would show the setup of FPGA-based MPEFM and adopt the single photon counting (SPC) technique for high signal-to-noise (SNR) images. The image spatial resolution is submicron level. To the need of the high bandwidth electrical components and applications in industry, companies are developing different kinds of composite materials and insulators together with the technology of different kinds of laser processing methods and protocols including direct writing, drilling, and modification with etching-assistance. Glass and polyimide (PI) are important materials for the insulation layer in PCB (printed circuit board) design and the narrow electrical routing structures are especially required to be confirmed after processing [4], [5]. We have shown the MPEFM can detect the axial-resolved images of the laser modified surface on the silicon glass and the laser-cut structure on the PI film without damaging the specimen. The mechanism shows the potential for the rapid 3D inspection of the laser processed specimen.

It is highly desirable to be able to optically probe biological activities deep inside live organisms. By employing a spatially confined excitation via a nonlinear transition, multiphoton fluorescence microscopy has become indispensable for imaging scattering samples. However, as the incident laser power drops exponentially with imaging depth due to scattering loss, the out-of-focus fluorescence eventually overwhelms the in-focal signal. The resulting loss of imaging contrast defines a fundamental imaging-depth limit, which cannot be overcome by increasing excitation intensity. Herein we propose to significantly extend this depth limit by multiphoton activation and imaging (MPAI) of photo-activatable fluorophores. The imaging contrast is drastically improved due to the created disparity of bright-dark quantum states in space. We demonstrate this new principle by both analytical theory and experiments on tissue phantoms labeled with synthetic caged fluorescein dye or genetically encodable photoactivatable GFP.

Creating a model for intravital visualization of femoral bone marrow, a major site of hematopoiesis in adult mammalian organisms, poses a serious challenge, in that it needs to overcome bone opacity and the inaccessibility of marrow. Furthermore, meaningful analysis of bone marrow developmental and differentiation processes requires the repetitive observation of the same site over long periods of time, which we refer to as chronic imaging. To surmount these issues, we developed a chronic intravital imaging model that allows the observation of split femurs, ectopically transplanted into a dorsal skinfold chamber of a host mouse. Repeated, long term observations are facilitated by multiphoton microscopy, an imaging technique that combines superior imaging capacity at greater tissue depth with low phototoxicity. The transplanted, ectopic femur was stabilized by its sterile environment and rapidly connected to the host vasculature, allowing further development and observation of extended processes. After optimizing transplant age and grafting procedure, we observed the development of new woven bone and maturation of secondary ossification centers in the transplanted femurs, preceded by the sprouting of a sinusoidal-like vascular network, which was almost entirely composed of femoral endothelial cells. After two weeks, the transplant was still populated with stromal and haematopoietic cells belonging both to donor and host. Over this time frame, the transplant partially retained myeloid progenitor cells with single and multi-lineage differentiation capacity. In summary, our model allowed repeated intravital imaging of bone marrow angiogenesis and hematopoiesis. It represents a promising starting point for the development of improved chronic optical imaging models for femoral bone marrow.

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 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.

Introduction: In vivo multiphoton autofluorescence microscopy provides live, label free, single cell imaging of metabolic changes. These metabolic changes are quantified via the metabolic coenzymes NAD(P)H and FAD which are autofluorescent molecules endogenous to all cells. Metabolic reprogramming of tumor and immune cells is closely associated with cancer progression and cell phenotype. We aim to study metabolic changes during administration of an effective, triple-combination immunotherapy within murine melanoma tumors. This therapy includes external beam radiation, intratumoral hu14.18-IL2 immunocytokine (anti-GD2 mAb fused to IL2, provided by Anyxis Immuno-Oncology GmbH of Vienna, Austria), and intraperitoneal anti-CTLA-4 leading to in situ vaccination and cure of GD2+ murine tumors. Previous work has shown that a T cell response is critical to the efficacy of this therapy, so we created an mCherry-labeled T cell mouse model to study this response. Methods: We implanted syngeneic B78 (GD2+) melanoma cells into the flanks of mCherry-labeled CD8 T cell reporter mice (C57Bl/6 background) to induce tumors. Under anesthesia, skin flap surgery was performed and tumors were imaged at several time points during therapy. Multiphoton imaging was performed to collect NAD(P)H, FAD, mCherry, and collagen signal with a 40X objective. Fluorescence lifetime data was collected using time correlated single photon counting electronics. Tissues were harvested and analyzed via flow cytometry and multiplex immunofluorescence to corroborate intravital imaging findings and characterize the immune infiltrate. Results: Here we demonstrate that our in vivo imaging is sensitive to metabolic changes in both B78 melanoma and CD8 T cells from immunotherapy treated versus control tumors. These metabolic differences include changes in protein binding, redox balance, and fluorescence lifetime. We also show remodeling of collagen, a major component of the extracellular matrix, during immunotherapy that may be explained by an observed decrease in macrophages. Flow cytometry and multiplex immunofluorescence illustrate changes in the immune infiltrate composition, activation, cytotoxicity, and spatial distribution during therapy. We are also currently investigating metabolic changes in an MC38 mouse model - a hot tumor model - that we anticipate will provide excellent metabolic contrast to our B78 immunologically cold tumor model. Conclusions: These results show that in vivo metabolic imaging enables single cell quantification of metabolic changes in tumor and immune cells during therapy. Combined with other traditional assays, we can elucidate key immune cell populations and the crucial timepoints during therapy where changes are occurring. With continued efforts, this imaging platform may be leveraged to develop new combinations of immunotherapies. Citation Format: Alexa R. Heaton, Anna Hoefges, Peter R. Rehani, Nathaniel J. Burkard, Arika S. Feils, Dan V. Spiegelman, Noah W. Tsarovsky, Alina A. Hampton, Amy K. Erbe Gurel, Alexander L. Rakhmilevich, Paul M. Sondel, Melissa C. Skala. In vivo multiphoton autofluorescence imaging is sensitive to CD8 T cell and tumor cell metabolic changes during immunotherapy in a murine melanoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2389.

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.