Multiphoton Microscopy

Two-photon excitation fluorescence (2PEF) allows imaging of tissue up to about one millimeter in thickness. Typically, reducing fluorescence excitation exposure reduces the quality of the image. However, using deep learning super resolution techniques, these low-resolution images can be converted to high-resolution images. This work explores improving human tissue imaging by applying deep learning to maximize image quality while reducing fluorescence excitation exposure. We analyze two methods: a method based on U-Net, and a patch-based regression method. Both methods are evaluated on a skin dataset and an eye dataset. The eye dataset includes 1200 paired high power and low power images of retinal organoids. The skin dataset contains multiple frames of each sample of human skin. High-resolution images were formed by averaging 70 frames for each sample and low-resolution images were formed by averaging the first 7 and 15 frames for each sample. The skin dataset includes 550 images fo...

Background/purpose: Multiphoton microscopy (MPM) enables the assessment of unstained living biological tissue with submicron resolution, whereas fluorescence lifetime imaging microscopy (FLIM) generates image contrast between different states of tissue characterized by various fluorescence decay rates. The aim of this study was to compare the healthy skin of young individuals with that of older subjects, as well as to assess the skin at different body sites, by means of MPM and FLIM. Methods: Nineteen elderly patients were examined on the outer side of the forearm, whereas 30 young individuals were assessed on the dorsal and volar sides of the forearm and on the thigh. Results: Cell and nucleus diameters, cell density and FLIM vary according to the epidermal cell depth and the skin site. In elderly subjects, epidermal cells show morphologic al-terations in shape and size, with smaller cell and nucleus diameters; the number of basal cells is decreased, whereas the mean fluorescence lifetimes at both the upper and the lower layers increase. This study provides quantitative and qualitative data on normal epidermis at different skin sites at different ages and represents a reference for the clinician attempting to understand the effectiveness of MPM and FLIM in discriminating diseased states of the skin from normal ones.

a technique enabling real-time imaging of bacterial proliferation and tissue responses in a live animal. Spatial and temporal control of the infection process was achieved by microinjecting GFP + -expressing uropathogenic Escherichia coli (UPEC) into tubules of exteriorized kidneys in live rats. GFP + was introduced in the clinical UPEC strain CFT073 as a single-copy chromosomal gene fusion. Within hours, bacterial colonization was accompanied by marked ischaemic effects, perivascular leakage, loss of tubular integrity and localized recruitment of immune cells. The pathophysiology was altered in response to an isogenic bacterial strain lacking the exotoxin haemolysin, revealing the subtle and temporal roles of bacterial virulence factors in vivo. Microdissection and RNA extraction of the injected nephron allowed molecular analysis of prokaryotic and eukaryotic gene expression. The techniques described here can be applied to study the integrated cell communication evoked by a variety of bacterial pathogens, assisting in the design of strategies to combat bacterial infections.

There is a need for biochemical contrast mediators with high signal-to-noise ratios enabling noninvasive biomedical sensing, for example, for neural sensing and protein-protein interactions, in addition to cancer diagnostics. The translational challenge is to develop a biocompatible approach ensuring high biochemical contrast while avoiding a raise of the background signal. We here present a concept where gold nanoparticles (AuNPs) can be utilized as a stimuli responsive contrast medium by chemically triggering their ability to exhibit multiphoton-induced luminescence (MIL) when performing multiphoton laser scanning microscopy (MPM). Proof-of-principle is demonstrated using peptidefunctionalized AuNPs sensitive to zinc ions (Zn 2+ ). Dispersed particles are invisible in the MPM until addition of millimolar concentrations of Zn 2+ upon which MIL is enabled through particle aggregation caused by specific peptide interactions and folding. The process can be reversed by removal of the Zn 2+ using a chelator, thereby resuspending the AuNPs. In addition, the concept was demonstrated by exposing the particles to matrix metalloproteinase-7 (MMP-7) causing peptide digestion resulting in AuNP aggregation, significantly elevating the MIL signal from the background. The approach is based on the principle that aggregation shifts the plasmon resonance, elevating the absorption cross section in the near-infrared wavelength region enabling onset of MIL. This Letter demonstrates how biochemical sensing can be obtained in far-field MPM and should be further exploited as a future tool for noninvasive optical biosensing.

Background: Diabetic nephropathy (DN) is a leading cause of end-stage renal disease, characterized by early structural and functional changes in the glomerular filtration barrier, notably the glomerular basement membrane (GBM). Traditional histopathological tools often lack the resolution and dynamic imaging capacity needed for early detection. Objective: This study explores the application of multiphoton microscopy (MPM) to assess GBM integrity in early DN, integrating proteomic profiling to correlate structural abnormalities with molecular alterations. Methods: Renal biopsies from diabetic patients and matched controls underwent MPM imaging and mass spectrometry-based proteomic analysis. Key structural markers such as GBM thickness and continuity were quantified alongside protein expression patterns. Results: MPM revealed significant GBM thickening and focal discontinuities in early DN samples. Proteomic analysis identified upregulation of fibronectin, laminin α2, and downregulation of collagen IV α3 chains. Correlations were observed between MPM-derived thickness measurements and specific protein expression levels (R² > 0.75). Conclusion: This integrative approach demonstrates the utility of MPM in visualizing early GBM alterations and underscores the value of proteomic biomarkers for characterizing early DN. These findings support potential diagnostic and therapeutic applications for early intervention in diabetic nephropathy.

Multiphoton microscopy is a powerful tool for imaging sub-cellular distribution of luminescent compounds present in living cells. We have used this tool to study the distribution and pharmacology of photosensitizers in tissue and tissue culture. Murine hepatoma tumor cells dosed with a photosensitizer were briefly photoactivated, then imaged for periods up to several hours. Using the photosensitizer Rose Bengal with green light activation, nearly immediate photolytic release of lysosomal enzymes resulted in catastrophic cell destruction within 5-30 minutes. The magnitude and rapidity of this response is markedly different than that observed with other photosensitizer agents, and is consistent with in vivo studies illustrating that Rose Bengal is capable of causing extremely rapid destruction of treated tumors.

Structural dynamics of dendritic spines is one of the key correlative measures of synaptic plasticity for encoding short-term and long-term memory. Optical studies of structural changes in brain tissue using confocal microscopy face difficulties of scattering. This results in low signal-to-noise ratio and thus limiting the imaging depth to few tens of microns. Multiphoton microscopy (MpM) overcomes this limitation by using low-energy photons to cause localized excitation and achieve high resolution in all three dimensions. Multiple low-energy photons with longer wavelengths minimize scattering and allow access to deeper brain regions at several hundred microns. In this article, we provide a basic understanding of the physical phenomena that give MpM an edge over conventional microscopy. Further, we highlight a few of the key studies in the field of learning and memory which would not have been possible without the advent of MpM.

The microstructural deformation-mechanical loading relation of the blood vessel wall is essential for understanding the overall mechanical behavior of vascular tissue in health and disease. We employed simultaneous mechanical loading-imaging to quantify in situ deformation of individual collagen and elastin fibers on unstained fresh porcine coronary adventitia under a combination of vessel inflation and axial extension loading. Specifically, the specimens were imaged under biaxial loads to study microscopic deformation-loading behavior of fibers in conjunction with morphometric measurements at the zero-stress state. Collagen fibers largely orientate in the longitudinal direction, while elastin fibers have major orientation parallel to collagen, but with additional orientation angles in each sublayer of the adventitia. With an increase of biaxial load, collagen fibers were uniformly stretched to the loading direction, while elastin fibers gradually formed a network in sublayers, whic...

We have developed a new descanned parallel ͑32-fold͒ pinhole and photomultiplier detection array for multifocal multiphoton microscopy that effectively reduces the blurring effect originating from scattered fluorescence photons in strongly scattering biological media. With this method, we achieve a fourfold improvement in photon statistics for detecting ballistic photons and an increase in spatial resolution by 21% in the lateral and 35% in the axial direction compared to single-beam non-descanned multiphoton microscopy. The new detection concept has been applied to plant leaves and pollen grains to verify the improvements in imaging quality.

In the field of topical application without or with little systemic side-effects to reach antiinflammatory or anti-androgeneous effects, nanoparticles as carriers for drugs as betamethason-17-valerate, prednicarbate, prednisolone, RU 58841-myristate or cyproterone acetate have proven to enhance the transdermal delivery. This enhancement is closely connected to the interaction of the drug molecules with the lipid carrier systems, i.e. incorporation into the carriers or attachment to their surfaces. Whereas the techniques to measure the penetration profiles in the cutaneous region of the skin are well established in the case of fluorescence microscopy applied to thin slices of epidermis or being established in the case of multiphoton microscopy to monitor this fluorescence, the methods for the investigation of the type of interaction between drugs and carrier systems are relatively new: In the case of electron spin resonance the sample volumes have to be restricted to capillary sizes to avoid parelectric losses in the microwave cavities, in the case of the novel method of parelectric spectroscopy we are free from such restrictions. The application of the latter method will be presented here in detail concerning the underlying theory, the experimental aspects as well as the algorithms to extract the parameters of interest from the measured samples. As samples we restrict ourselves to solid lipid nanoparticles coated with different surfactants as carriers for drug-, dye-or spinlabel molecules.

Ultrasound (US) enhanced with microbubble contrast agents may transiently disrupt the blood–brain barrier (BBB) with minimal damage, providing a technique for noninvasive, localized drug-delivery deep within the brain. The mechanism and temporal profile of disruption are not understood, owing to the limitations of imaging modalities used previously. In this study, we monitored US-induced BBB disruption with multiphoton microscopy, providing high-resolution temporal and spatial information about the permeabilization mechanism and immediate effects of US exposure. Anesthetized C57 mice were prepared with a craniotomy and injected intravenously with fluorescent dyes to permit visualization of the vasculature and BBB integrity. The animals were imaged through a cranial window while exposed to low-intensity US ( f =1.029 MHz, power = 0.2 W) with a coincident intravenous injection of Optison (a microbubble contrast agent). We observed arteriolar vasoconstriction on US exposure that disrup...

The uptake and subcellular partitioning of benzo[a]pyrene (BaP) were examined in a rat-liver cell line (Clone 9) using confocal and multiphoton microscopy. Following a 16-h treatment, intracellular accumulation of BaP increased with increasing concentration, and cytoplasmic BaP fluorescence reached saturation at 10 M. Analysis of the kinetics of BaP uptake at this concentration indicated that BaP is rapidly partitioned into all cytoplasmic membranes within several min, although saturation was not reached until 4 h. Based upon the rapid uptake of BaP into membranes, the chronology of changes in gap junction-mediated intercellular communication (GJIC), plasma membrane potential (PMP), and steady state levels of intracellular Ca 2؉ in relation to the time-course for induction of microsomal ethoxyresorufin-0deethylase (EROD) activity were examined. EROD activity in Clone 9 cells treated for 16 h increased with increasing concentrations of BaP and reached the highest levels at 40 M BaP. In addition, kinetic analysis of EROD activity in Clone 9 cells treated with 10 M BaP indicated that significant induction of EROD activity was not detected before 3 h, and it reached maximal levels by 16 h of treatment at this concentration. Both GJIC and PMP were directly affected by the partitioning of BaP into cellular membranes. The most sensitive index of BaP-induced changes in membrane function was GJIC which revealed a 25% suppression in cells exposed to 0.4 M BaP for 16 h. Kinetic analysis revealed that suppression of GJIC occurred within 15 min of exposure of cells to 10 M BaP, whereas significant suppression of PMP was not detected prior to 30-min exposure at this concentration. Elevation of basal Ca 2؉ level was also detected simultaneously with PMP at this dose. These data suggest that early changes in cellular membrane functions occur prior to detectable induction of EROD activity, although basal metabolic activation of BaP may contribute to these changes.

In this study, a multiple parallel perfused microbioreactor platform, TissueFlex Ò , was developed which can be used to perform cell and tissue culture under almost uniform and precisely controlled environment in a mid-throughput and parallel manner. These microbioreactors were used to culture human bone marrow cells (hBMCs) in three-dimensional (3D) scaffolds and also in two-dimensional (2D) monolayer for comparison for upto 7 days. Several scaffolding materials were evaluated for this purpose in terms of easiness in handling, ability to support the hBMC growth, and feasibility for non-destructive optical assays. The feasibility and efficacy of using the developed 3D-hBMCs-based model tissue-constructs cultured in TissueFlex Ò microbioreactors for drug evaluation and toxicity testing was then studied. As a demonstration case study, the cultured cells were challenged with two chemicals, trimethoprim and pyrimethamine, both known to be harmful to cellular activities, with different protocols. Cytotoxicity in terms of cell viability and growth was determined using the AlamarBlue assay. The 3D spatial variations in cell morphology and cell survival were also monitored using 3D optical imaging using non-linear multiphoton microscopy. The results show that (i) the data obtained from 3D hBMCs culture and from (2D) monolayer cultures on the effect of the tested chemicals on cell growth are significantly different, and that (ii) the perfused microbioreactor technology could provide a highly controlled and prolonged cell culture environment for testing of various drugs and chemicals. The outcome of this study demonstrated the feasibility and potentials of the using 3D stem cell based model tissues in TissueFlex Ò microbioreactors for drug evaluation and toxicity testing of chemicals as an efficient and standardized alternative testing method.

Deterioration in mitochondrial function leads to hepatic ischemia and reperfusion injury (IRI) in liver surgery and transplantation. 3D optical cryoimaging was used to measure the levels of mitochondrial coenzymes NADH and FAD, and their redox ratio (NADH/FAD) gave a quantitative marker for hepatocyte oxidative stress during IRI. Using a rat model, five groups were compared: control, ischemia for 60 or 90 minutes (Isc60, Isc90), ischemia for 60 or 90 minutes followed by reperfusion of 24 hours (IRI60, IRI90). Ischemia alone did not cause a significant increase in the redox ratio; however, the redox ratio in both IRI60 and IRI90 groups was significantly decreased by 29% and 71%, respectively. A significant correlation was observed between the redox ratio and other markers of injury such as serum aminotransferase levels and the tissue ATP level. The mitochondrial redox state can be successfully measured using optical cryoimaging as a quantitative marker of hepatic IR injury.

The mechanism by which epithelial cells undergo directed rearrangement is central to morphogenesis, yet the regulation of these movements remains poorly understood. We have investigated epithelial cell rearrangement (intercalation) in the dorsal hypodermis, or embryonic epidermis, of the C. elegans embryo by analyzing the die-1(w34) mutant, which fails to undergo normal intercalation. Dorsal hypodermal cells of die-1(w34) homozygous embryos initiate but fail to complete the process of intercalation. Multiphoton microscopy reveals that intercalating cells extend monopolar, basolateral protrusions in their direction of migration; posterior dorsal hypodermal cells in die-1(w34) mutants appear to extend protrusions normally, but fail to translocate their cell bodies to complete rearrangement. Despite abnormal intercalation, the subsequent morphogenetic movements that enclose the embryo with epithelial cells and the process of dorsal cell fusion still occur. However, elongation of the embryo into a wormlike shape is disrupted in die-1(w34) embryos, suggesting that intercalation may be necessary for subsequent elongation of the embryo. Actin filaments are not properly organized within the dorsal hypodermis of die-1(w34) embryos, consistent with intercalation's being a necessary prerequisite for elongation. The die-1 gene encodes a C2H2 zinc finger protein containing four fingers, which likely acts as a transcriptional regulator. DIE-1 is present in the nuclei of hypodermal, muscle, gut, and pharyngeal cells; its distribution suggests that DIE-1 acts in each of these tissues to regulate morphogenetic movements. die-1(w34) mutants display morphogenetic defects in the pharynx, gut, and muscle quadrants, in addition to the defects in the dorsal hypodermis, consistent with the DIE-1 expression pattern. Mosaic analysis indicates that DIE-1 is autonomously required in the posterior dorsal hypodermis for intercalation. Our analysis documents for the first time the dynamics of protrusive activity during epithelial cell rearrangement. Moreover, our analysis of die-1 shows that the events of epithelial cell rearrangement are under transcriptional control, and that early and later phases of epithelial cell rearrangement are genetically distinguishable.

Dendritic cells (DCs), macrophages (Mf), and T cells are major components of the skin immune system, but their interstitial spatial organization is poorly characterized. Using four-channel whole-mount immunofluorescence staining of the human dermis, we demonstrated the three-dimensional distribution of CD31 þ blood capillaries, LYVE-1 þ lymphatics, discrete populations of CD11c þ myeloid DCs, FXIIIa þ Mf, and lymphocytes. We showed phenotypic and morphological differences in situ between DCs and Mf. DCs formed the first dermal cellular layer (0-20 mm beneath the dermoepidermal junction), Mf were located deeper (40-60 mm), and CD3 þ lymphocytes were observed throughout (0-60 mm). Below this level, DCs, T cells, and the majority of Mf formed stable perivascular sheaths. Whole-mount imaging revealed the true extent of dermal leukocytes previously underestimated from cross-section views. The total area of apical dermis (0-30 mm) contained approximately 10-fold more myeloid DCs than the entire blood volume of an average individual. Surprisingly, o1% of dermal DCs occupied lymphatics in freshly isolated skin. Dermal DCs rapidly accumulated within lymphatics, but Mf remained fixed in skin explants cultured ex vivo. The leukocyte architecture observed in normal skin was distorted in inflammation and disease. These studies illustrate the micro-anatomy of dermal leukocytes and provide further insights into their functional organization.

Received: Revised: Accepted: November 06, 2014 April 22, 2015 June 24, 2015 A four-year old Husky dog suffering of chronic diarrhea was radiologically diagnosed with megacolon and consequently, partial resection of the colon wall was performed. Histological examination of the colonic wall revealed the presence of Sarcocystis spp. oocysts. Chronic inflammation of colonic mucosa resulted in local amyloidosis, overexpression of Bcl-2 in the goblet cells, which suggested decreased apoptosis and increased mucous cells population in Lieberkuhn’s glands. The expression of immunoglobulin A (IgA) was reduced intra-epithelially and increased in blood capillaries, periglandulary and in the submucosal stroma consequently to parasitic infection. The colonic mucosa showed plasma cells, eosinophilic, mast cells and lymphocytic infiltrations. The Sarcocystis chronic parasitism may cause colon chronic dilatation due to persistent inflammation of the mucosa, characterized by infiltration of lymphocyt...

Colloid response is a type of tumor response that occurs after preoperative radiochemotherapy for rectal carcinoma. Given its important influence on survival, the colloid response should be considered when estimating histopathological reactions. Here, multiphoton microscopy (MPM) was applied to evaluate the colloid response ex vivo. This study demonstrated that MPM has the capability to visualize the colloid response in the absence of labels and can, in particular, identify rare residual carcinomatous cells in mucin pools. These results highlight the potential of this nonlinear optical technique as a diagnostic tool for tumor response after neoadjuvant treatment.

The tongue consists of a complex, multiscale array of myofibers that comprise the anatomical underpinning of lingual mechanical function. 3-D myoarchitecture was imaged in mouse tongues with diffusion spectrum magnetic resonance imaging ͑DSI͒ at 9.4 T ͑b max 7000 s / mm, 150-m isotropic voxels͒, a method that derives the preferential diffusion of water/voxel, and high-throughput ͑10 fps͒ two-photon microscope ͑TPM͒. Net fiber alignment was represented for each method in terms of the local maxima of an orientational distribution function ͑ODF͒ derived from the local diffusion ͑DSI͒ and 3-D structural autocorrelation ͑TPM͒, respectively. Mesoscale myofiber tracts were generated by alignment of the principal orientation vectors of the ODFs. These data revealed a consistent relationship between the properties of the respective ODFs and the virtual superimposition of the distributed mesoscale myofiber tracts. The identification of a mesoscale anatomical construct, which specifically links the microscopic and macroscopic spatial scales, provides a method for relating the orientation and distribution of cells and subcellular components with overall tissue morphology, thus contributing to the development of multiscale methods for mechanical analysis.

Radioimmunotherapy using a radiolabeled monoclonal antibody that targets tumor cells has been shown to be efficient for the treatment of many malignant cancers, with reduced side effects. However, the blood-brain barrier (BBB) inhibits the transport of intravenous antibodies to tumors in the brain. Recent studies have demonstrated that focused ultrasound (FUS) combined with microbubbles (MBs) is a promising method to transiently disrupt the BBB for the drug delivery to the central nervous system. To find the optimal FUS and MBs that can induce reversible increase in the BBB permeability, we employed minimally invasive multiphoton microscopy to quantify the BBB permeability to dextran-155 kDa with similar molecular weight to an antibody by applying different doses of FUS in the presence of MBs with an optimal size and concentration. The cerebral microcirculation was observed through a section of frontoparietal bone thinned with a micro-grinder. About 5 minutes after applying the FUS on the thinned skull in the presence of MBs for 1 minute, TRITC (tetramethylrhodamine isothiocyanate)-dextran-155 kDa in 1% bovine serum albumin in mammalian Ringer's solution was injected into the cerebral circulation via the ipsilateral carotid artery by a syringe pump. Simultaneously, the temporal images were collected from the brain parenchyma ~100-200 μm below the pia mater. Permeability was determined from the rate of tissue solute accumulation around individual microvessels. After several trials, we found the optimal dose of FUS. At the optimal dose, permeability increased by ~14-fold after 5 minutes post-FUS, and permeability returned to the control level after 25 minutes. FUS without MBs or MBs injected without FUS did not change the permeability. Our method provides an accurate in vivo assessment for the transient BBB permeability change under the treatment of FUS. The optimal FUS dose found for the reversible BBB permeability increase without BBB disruption is reliable and can be applied to future clinical trials.

Development of an optimal systemic drug delivery strategy to the brain will require noninvasive or minimally invasive methods to quantify the permeability of the cerebral microvessel wall or blood-brain barrier (BBB) to various therapeutic agents and to measure their transport in the brain tissue. To address this problem, we used laser-scanning multiphoton microscopy to determine BBB permeability to solutes (P) and effective solute diffusion coefficients (Deff) in rat brain tissue 100–250 μm below the pia mater. The cerebral microcirculation was observed through a section of frontoparietal bone thinned with a microgrinder. Sodium fluorescein, fluorescein isothiocyanate (FITC)-dextrans, or Alexa Fluor 488-immunoglobulin G (IgG) in 1% bovine serum albumin (BSA) mammalian Ringer's solution was injected into the cerebral circulation via the ipsilateral carotid artery by a syringe pump at a constant rate of ∼3 ml/min. P and Deff were determined from the rate of tissue solute accumula...

There is a need for biochemical contrast mediators with high signal-to-noise ratios enabling noninvasive biomedical sensing, for example, for neural sensing and protein−protein interactions, in addition to cancer diagnostics. The translational challenge is to develop a biocompatible approach ensuring high biochemical contrast while avoiding a raise of the background signal. We here present a concept where gold nanoparticles (AuNPs) can be utilized as a stimuli responsive contrast medium by chemically triggering their ability to exhibit multiphoton-induced luminescence (MIL) when performing multiphoton laser scanning microscopy (MPM). Proof-of-principle is demonstrated using peptidefunctionalized AuNPs sensitive to zinc ions (Zn 2+). Dispersed particles are invisible in the MPM until addition of millimolar concentrations of Zn 2+ upon which MIL is enabled through particle aggregation caused by specific peptide interactions and folding. The process can be reversed by removal of the Zn 2+ using a chelator, thereby resuspending the AuNPs. In addition, the concept was demonstrated by exposing the particles to matrix metalloproteinase-7 (MMP-7) causing peptide digestion resulting in AuNP aggregation, significantly elevating the MIL signal from the background. The approach is based on the principle that aggregation shifts the plasmon resonance, elevating the absorption cross section in the near-infrared wavelength region enabling onset of MIL. This Letter demonstrates how biochemical sensing can be obtained in far-field MPM and should be further exploited as a future tool for noninvasive optical biosensing.

Although proinflammatory cell signaling in the alveolo-capillary region predisposes to acute lung injury, key cell-signaling mechanisms remain inadequately understood. Alveolo-capillary inflammation is likely to involve coordinated signaling among cells of different phenotypes. For example, migration of inflammatory cells into the alveolus might entail coordinated signaling between adjoining alveolar epithelial and microvascular endothelial cells. The popular cultured cell experimental strategy fails to replicate this multicellular environment. Cultured lung cells, both alveolar and endothelial, undergo phenotypic transformations; hence they might inadequately reflect innate responses of native cells. Consequently, new approaches are required for the investigation of cell signaling in the native setting. Here we summarize new developments in classical intravital microscopy and discuss real-time fluorescence imaging as a novel technique for studying second-messenger mechanisms in the alveolo-capillary region.

In this study, a microscope based on spatiotemporal focusing offering widefield multiphoton excitation has been developed to provide fast optical sectioning images. Key features of this microscope are the integrations of a 10 kHz repetition rate ultrafast amplifier featuring high instantaneous peak power (maximum 400 J/pulse at a 90 fs pulse width) and a TE-cooled, ultra-sensitive photon detecting, electron multiplying charge-coupled camera into a spatiotemporal focusing microscope. This configuration can produce multiphoton images with an excitation area larger than 200 × 100 m 2 at a frame rate greater than 100 Hz (current maximum of 200 Hz). Brownian motions of fluorescent microbeads as small as 0.5 m were observed in real-time with a lateral spatial resolution of less than 0.5 m and an axial resolution of approximately 3.5 m. Furthermore, second harmonic images of chicken tendons demonstrate that the developed widefield multiphoton microscope can provide high resolution z-sectioning for bioimaging.

Third harmonic generation microscopy is shown to be a robust method for obtaining structural information on a variety of biological specimens. Its nature allows depth-resolved imaging of inhomogeneities with virtually no background from surrounding homogeneous media. With an appropriate illumination geometry, third harmonic generation microscopy is shown to be particularly suitable for imaging of biogenic crystals, enabling extraction of the crystal orientation.

Emission of energy from any source is known as 'radiation'. High frequency radiation, e.g., ionizing radiation and ultraviolet (UV) radiation may cause genetic damage, leading to cancer. The ionizing radiations include x-rays, γ-rays, cosmic rays and particles given off by radioactive materials, such as α and β particles, protons and neutrons. Radiant energy (UV rays of sunlight, ionizing electromagnetic or particulate radiation) induced the neoplasm (cancer) in both human beings and experimental animals. The ionized molecules are unstable and quickly undergo chemical changes, thereby forming the 'free radicals' that can damage the DNA molecule or other molecules around it. Hence, the ionizing radiations can cause mutation (change) in a cell's DNA, resulting into cancer. Thyroid gland and bone marrow are most sensitive to radiations; while kidney, bladder and ovary are least affected. Some forms of leukemia (bone marrow cancer) appear to be the most common radiation-induced cancers. The atomic blasts in Japan showed that high-dose radiation increases the risk of developing several cancers, including leukemia. Breast cancer can be developed in elderly women by a much more extensive use of personal computers (more than 3 hours a day), mobile telephones, TV sets and other household electrical appliances. Children appear to be twice as sensitive as adults to the leukemia-causing effects of radiation, and unborn children exposed to radiation in the uterus are even more sensitive. The breast cancer risk is more than twice as high as normal; however, the risk of developing lung cancer is 50% higher and the risk for multiple myeloma is more than twice as high as in the general population. The radiation therapy at high doses can cause DNA mutations in cells that survive the radiation, which may lead to a second primary cancer. Of all the types of non-ionizing radiations, only UV rays are cancer-causing agents. The most skin cancers are a direct result of sunlight exposure as sun is the major source of UV radiation.

lex C. T. Ko,* Andrew Ridsdale, Michael S. D. Smith, eila B. Mostaço-Guidolin, Mark D. Hewko, drian F. Pegoraro, Elicia K. Kohlenberg, ernie Schattka, Masashi Shiomi, Albert Stolow, and ichael G. Sowa National Research Council Canada, Institute for iodiagnostics, 435 Ellice Avenue, Winnipeg, Manitoba, anada R3B 1Y6 National Research Council Canada, Steacie Institute for olecular Sciences, 100 Sussex Drive, Ottawa, ntario, Canada K1A 0R6 Kobe University School of Medicine, Institute of xperimental Animals, 7-5-1, Kusunoki-cho, Chuo-ku, obe 650-0017, Japan

Recent advances in molecular biology and pathology have opened new opportunities for refining our knowledge of pathophysiologic events and biomarkers. Particular interest in applying these novel methods to current and archived tissues collected in experimental and epidemiological/clinical studies is evident. Until now, it has not always been possible to use archived alcohol-fixed paraffin-embedded (AFPE) tissues for immunohistochemisty (IHC), because AFPE slices and blocks were not often amenable to standard IHC methods. In order to solve this problem, we developed a simple method of post-fixation, which allows to use, on AFPE slices, standard IHC protocols already used for formalin-fixed paraffin-embedded (FFPE) samples. For the assessment of post-fixation processing and to test the feasibility of IHC, we selected the spleen from Sprague-Dawley rats as a demonstrative tissue. Antibodies to PAX5, CD3, CD68 and Ki-67, were tested on FFPE, AFPE and AFPE post-fixed spleen samples. The ...

Infrared neural stimulation is a promising tool for stimulating the brain because it can be used to excite with high spatial precision without the need of delivering or inserting any exogenous agent into the tissue. Very few studies have explored its use in the brain, as most investigations have focused on sensory or motor nerve stimulation. Using intravital calcium imaging with the genetically encoded calcium indicator GCaMP6f, here we show that the application of infrared neural stimulation induces intracellular calcium signals in Layer 2/3 neurons in mouse cortex in vivo. The number of neurons exhibiting infrared-induced calcium response as well as the amplitude of those signals are shown to be both increasing with the energy density applied. By studying as well the spatial extent of the stimulation, we show that reproducibility of the stimulation is achieved mainly in the central part of the infrared beam path. Stimulating in vivo at such a degree of precision and without any ex...

We demonstrate Z stack imaging of a fly (Musca domestica) with 289 Z planes of 2.5 μm Z step width, 1024 x 1024 pixels in X and Y, and 1024
channels in time by using existing functions of the Becker & Hickl DCS-120 confocal FLIM system. Doubleexponential decay analysis was performed by the batch processing function of Becker & Hickl SPCImage NG. 3D reconstructions on the basis of the SPCImage results were performed by ImageJ / FIJI.

We demonstrate two-photon FLIM with a FemtoFibre smart 780 laser of Toptica Photonics AG in combination with a Becker & Hickl DCS-120 MP TCSPC / FLIM system. The laser delivers femtosecond pulses at 783 nm, 80 MHz repetition rate, and 125 mW average power. We show that the laser power is sufficient to obtain highresolution images in all the commonly performed FLIM applications. We demonstrate the performance of the system for label-free (autofluorescence) imaging of tissue, label-free imaging of cells, FLIM of ultrafast fluorescence decay in biological material, and FLIM of fluorophore-labelled cells and tissues.

In this article we present the development of a multibeam two‐photon laser scanning microscope. A new type of beam splitter to create the multitude of laser beams is described. This type of beam splitter has higher transmission and generates more uniform beams than can be achieved with the microlens approach used by other groups. No crosstalk exists between the different foci due to small temporal delays between the individual beams. The importance of dispersion compensation to obtain maximum efficiency of the microscope is discussed. With optimum compensation the fluorescence signal was raised by a factor of 14. Different modes of detecting the fluorescence signals and their effect on imaging speed and resolution are discussed.

Second Harmonic Generation Microscopy (SHG) is generally acknowledged as a powerful tool for the label-free 3D visualization of tissues and advanced materials, with one of its most popular applications being collagen imaging. Although the great need, progress in superresolved SHG imaging lags behind the developments reported over the past years in fluorescencebased optical nanoscopy. In this work, we quantitatively show on collagenous tissues that by combining SHG imaging with re-scan microscopy resolutions that surpass the diffraction limit with ~1.4x become available. Besides Re-scan Second Harmonic Generation Microscopy (rSHG), we demonstrate as well super-resolved Re-scan Two-Photon Excited Fluorescence Microscopy (rTPEF). These two techniques are implemented by modifying a Re-scan Confocal Microscope (RCM), retaining its initial function, resulting thus in a multimodal rSHG/rTPEF/RCM system. Given the simplicity and flexibility of re-scan microscopy, we consider that the reported results are likely to augment the number and nature of applications relying on super-resolved non-linear optical imaging.

Two-Photon Microscopy has become an invaluable tool for biological and medical research, providing high sensitivity, molecular specificity, inherent three-dimensional sub-cellular resolution and deep tissue penetration. In terms of imaging speeds, however, mechanical scanners still limit the acquisition rates to typically 10-100 frames per second. Here we present a high-speed non-linear microscope achieving kilohertz frame rates by employing pulse-modulated, rapidly wavelength-swept lasers and inertia-free beam steering through angular dispersion. In combination with a high bandwidth, single-photon sensitive detector, this enables recording of fluorescent lifetimes at speeds of 88 million pixels per second. We show high resolution, multi-modal -two-photon fluorescence and fluorescence lifetime (FLIM)microscopy and imaging flow cytometry with a digitally reconfigurable laser, imaging system and data acquisition system. These high speeds should enable high-speed and high-throughput image-assisted cell sorting.

Second Harmonic Generation Microscopy (SHG) is generally acknowledged as a powerful tool for the label-free 3D visualization of tissues and advanced materials, with one of its most popular applications being collagen imaging. Although the great need, progress in superresolved SHG imaging lags behind the developments reported over the past years in fluorescencebased optical nanoscopy. In this work, we quantitatively show on collagenous tissues that by combining SHG imaging with re-scan microscopy resolutions that surpass the diffraction limit with ~1.4x become available. Besides Re-scan Second Harmonic Generation Microscopy (rSHG), we demonstrate as well super-resolved Re-scan Two-Photon Excited Fluorescence Microscopy (rTPEF). These two techniques are implemented by modifying a Re-scan Confocal Microscope (RCM), retaining its initial function, resulting thus in a multimodal rSHG/rTPEF/RCM system. Given the simplicity and flexibility of re-scan microscopy, we consider that the reported results are likely to augment the number and nature of applications relying on super-resolved non-linear optical imaging.

Structural proteins like collagen and elastin are major constituents of the extracellular matrix (ECM). ECM degradation and remodeling in diseases significantly impact the microorganization of these structural proteins. Therefore, tracking the changes of collagen and elastin fiber morphological features within ECM impacted by disease progression could provide valuable insight into pathological processes such as tissue fibrosis and atherosclerosis. Benefiting from its intrinsic high-resolution imaging power and superior biochemical specificity, nonlinear optical microscopy (NLOM) is capable of providing information critical to the understanding of ECM remodeling. In this study, alterations of structural fibrillar proteins such as collagen and elastin in arteries excised from atherosclerotic rabbits were assessed by the combination of NLOM images and textural analysis methods such as fractal dimension (FD) and directional analysis (DA). FD and DA were tested for their performance in t...

Nonlinear optical (NLO) microscopy is used to follow key structural and biochemical changes associated with the progression of atherosclerosis. Arteries from WHHL-MI rabbits are examined using a 3 channel NLO microscope that can simultaneously monitor the coherent anti-stokes Raman scattered light (CARS), the two-photon excited fluorescence (TPEF) and the second harmonic generation (SHG) from a sample. Distinct differences in the nonlinear optical signals are observed that correlate with the age of the vessel and the presence of atherosclerotic plaque. These differences are attributed to the changing extracellular matrix and the increased lipid deposition associated with plaque development. The capability of NLO to perform 3D sectioning in thick highly scattering vessels in order to visualize structural details of the artery wall and highlight vessel pathology is demonstrated. These features make NLO a potentially valuable tool to help understand the progression of atherosclerosis.

We demonstrate a novel multiphoton imaging modality based on the detection of four-wave mixing (FWM) from colloidal nanoparticles. This third-order signal is excited and detected in resonance with the ground-state excitonic transition of CdSe∕ZnS quantum dots. We measure transversal and axial resolutions of 140 and 590nm, respectively, significantly improved compared to the one-photon diffraction limit. The coherent FWM signal is detected using a heterodyne technique which rejects incoherent background for improved image contrast compared to fluorescence methods. As sensitivity limit, we estimate a minimum number of 10dots∕Hz detectable in the focal volume.

Our study aimed to examine and quantify age-related structural alterations in the healthy mouse bladder using ex vivo two-photon laser scanning microscopy (TPLSM). Freshly dissected bladders from 25-, 52-, and 85-week-old C57bl/6J mice were examined, and morphological analyses and quantification of cell layers and nerves were performed. The numbers of stretched, curled, branched, and total number of nerves in volume units of the stained muscle layer were quantified. We observed differences in the bladder wall architecture and innervation with age. Especially in 85week-old mice, age-related changes were found, including detachment of urothelial cells and an increase in connective tissue, intermingled with the smooth muscle fibers in the muscle layer (collagen-smooth muscle ratio of 1.15 ± 0.29). In 25-and 52-week-old mice, the collagen-smooth muscle ratios were 0.20 ± 0.04 and 0.31 ± 0.11, respectively, and a clear separation of collagen and muscle was observed. The overall number of nerves and the number of curled nerves were significantly higher in the 85-week-old mice (74.0 ± 13.0 and 25.9 ± 4.8, respectively), when comparing to 25-weekold mice (26.0 ± 2.7 and 6.7 ± 1.2, respectively) and 52week-old mice (43.8 ± 4.3 and 22.1 ± 3.3, respectively). Significant age-related alterations in bladder morphology and innervation were found, when comparing freshly dissected bladder tissue from 25-, 52-, and 85-week-old mice. The higher number of curled nerves might be an indication of an increased neurotransmitter release, resulting in a higher nerve activity, with a part of the nerves being possibly mechanically impaired. This study shows that two-photon laser scanning microscopy of healthy aging male mice is a useful method to investigate and quantify the age-related changes in the bladder wall.

Recently, using two-photon imaging it has been found that the movement of B and T cells in lymph nodes can be described by a random walk with persistence of orientation in the range of 2 minutes. We interpret this new class of lymphocyte motility data within a theoretical model. The model considers cell movement to be composed of the movement of subunits of the cell membrane. In this way movement and deformation of the cell are correlated to each other. We find that, indeed, the lymphocyte movement in lymph nodes can best be described as a random walk with persistence of orientation. The assumption of motility induced cell elongation is consistent with the data. Within the framework of our model the two-photon data suggest that T and B cells are in a single velocity state with large stochastic width. The alternative of three different velocity states with frequent changes of their state and small stochastic width is less likely. Two velocity states can be excluded.

Background/purpose: Multiphoton microscopy has emerged in the past decade as a useful non invasive imaging technique for in vivo human skin characterization. However, it has not been used until now in routine clinical practice, mainly because of the lack of specific image processing tools that would allow the investigator to extract pertinent quantitative three-dimensional (3D) information from the different skin components. Methods: We propose a 3D automatic segmentation method of multiphoton images which is a key step for epidermis and dermis quantification. This method, based on the morphological watershed and graph cuts algorithms, takes into account the real shape of the skin surface and of the dermal-epidermal junction and allows separating in 3D the epidermis and the superficial dermis. Results: The automatic segmentation method and the associated quantitative measurements have been developed and validated on a clinical database designed for aging characterization. The segmentation achieves its goals for epidermis-dermis separation and allows quantitative measurements inside the different skin compartments with sufficient relevance. Conclusions: This study shows that multiphoton microscopy associated with specific image processing tools provides access to new quantitative measurements on the various skin components. The proposed 3D automatic segmentation method will contribute to build a powerful tool for characterizing human skin condition. To our knowledge, this is the first 3D approach to the segmentation and quantification of these original images.

Background/purposeMultiphoton microscopy has emerged in the past decade as a useful noninvasive imaging technique for in vivo human skin characterization. However, it has not been used until now in evaluation clinical trials, mainly because of the lack of specific image processing tools that would allow the investigator to extract pertinent quantitative three‐dimensional (3D) information from the different skin components.MethodsWe propose a 3D automatic segmentation method of multiphoton images which is a key step for epidermis and dermis quantification. This method, based on the morphological watershed and graph cuts algorithms, takes into account the real shape of the skin surface and of the dermal–epidermal junction, and allows separating in 3D the epidermis and the superficial dermis.ResultsThe automatic segmentation method and the associated quantitative measurements have been developed and validated on a clinical database designed for aging characterization. The segmentation ...

Despite neuroblastoma being the most common extracranial solid cancer in childhood, it is still a rare disease. Consequently, the unavailability of tissue for research limits the statistical power of studies. Pathology archives are possible sources of rare tissue, which, if proven to remain consistent over time, could prove useful to research of rare disease types. We applied immunohistochemistry to investigate whether long term storage caused any changes to antigens used diagnostically for neuroblastoma. We constructed and quantitatively assessed a tissue microarray containing neuroblastoma archival material dating between 1950 and 2007. A total of 119 neuroblastoma tissue cores were included spanning 6 decades. Fourteen antibodies were screened across the tissue microarray (TMA). These included seven positive neuroblastoma diagnosis markers (NB84, Chromogranin A, NSE, Ki-67, INI1, Neurofilament Protein, Synaptophysin), two anticipated to be negative (S100A, CD99), and five research antibodies (IL-7, IL-7R, JAK1, JAK3, STAT5). The staining of these antibodies was evaluated using Aperio ImageScope software along with novel pattern recognition and quantification algorithms.

Retinal cell remodelling has been reported as a consistent feature of ageing. However the degree to which this results in trans-retinal degeneration is unclear. To address this, we used multiphoton microscopy to quantify retinal degeneration in post-mortem human eyes of two age groups. Methods: Retinas from six young subjects (18-33 years old) and six aged subjects (74-90 years old) were prepared as wholemount preparations. All retinae were stained with DAPI and imaged by multiphoton confocal microscopy to quantify neuron densities in the retinal ganglion cell layer (RGCL), inner nuclear layer (INL) and outer nuclear layer (ONL). Neurons were counted using automated cell identification algorithms. All retinas were imaged hydrated to minimise tissue artefacts. Results: In both groups, 56% of the area within the central 4 mm eccentricity, and 27% of the area with eccentricity between 4 mm and 7 mm were imaged. Compared with young subjects, the peak RGCL neuron loss in the aged subjects (25.5%) was at 1mm eccentricity. INL and ONL, neuron densities significantly decreased at 1-2 mm eccentricity (8.7%) and 0.5-4 mm eccentricity (15.6%) respectively (P < 0.05). The reduction in neuron density in the INL corresponded, spatially, to the region with the greatest neuron loss in the RGCL and ONL. Conclusions: This is the first study to correlate neurodegeneration in different populations of cells in the ageing retinas. Our data confirm that the greatest neuronal loss occurs in the RGCL and ONL in human ageing retinas, whereas the INL is relatively preserved.