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Multiphoton laser scanning microscopy commonly relies on bulky and expensive femtosecond lasers. We integrated a novel minimal-footprint Ti:sapphire oscillator, pumped by a frequency-doubled distributed Bragg reflector tapered diode laser, into a clinical multiphoton tomograph and evaluated its imaging capability using different biological samples, i.e. cell monolayers, corneal tissue, and human skin. With the novel laser, the realization of very compact Ti:sapphire-based systems for high-quality multiphoton imaging at a significantly size and weight compared to current systems will become possible., (© 2015 Wiley Periodicals, Inc.)

We have successfully introduced a proton-induced controlled reaction of HAuCl4 and poly(styrene-alt-maleic acid) (PSMA) sodium salt to prepare triangular and multicore Au@polymer nanoparticles (NPs). The interparticle interactions in the core gave rise to an absorption band at the near-infrared wavelength. The near-infrared optical properties of the resulting Au-polymer nanostructures are highly stable in a physiological environment, which offered strong photo-to-thermal conversion by a moderate continuous-wave 808 nm laser and exhibited multiphoton fluorescence for imaging using a 1230 nm light excitation (femtosecond laser). Exposure of the carboxylate groups at the polymer shell made the surface structure of the Au multicore @polymer NPs directly conjugate Pt(II)-/Pt(IV)-based drugs, which possessed the elimination of the immediate toxicity over the short time and resulted in an anticancer effect after 3 days. A synergistic effect of the chemo-photothermal therapy showed a moderate hyperthermia assistance (<1 W/cm(2)) and better anticancer performance over time compared with the individual treatments. We demonstrated that such PSMA-based methodology not only enables a broad range of chemical material synthesis in the kinetic control to form Au nano-octahedrons and nanotriangles using Br(-)/I(-) ions additives but also could be extended to form Au/Fe3O4@polymer nanocomposites via proton-assisted PSMA self-assembly.

Previous studies of spinal motoneurons in the SOD1 mouse model of amyotrophic lateral sclerosis have shown alterations long before disease onset, including increased dendritic branching, increased persistent Na(+) and Ca(2+) currents, and impaired axonal transport. In this study dendritic Ca(2+) entry was investigated using two photon excitation fluorescence microscopy and whole-cell patch-clamp of juvenile (P4-11) motoneurons. Neurons were filled with both Ca(2+) Green-1 and Texas Red dextrans, and line scans performed throughout. Steps were taken to account for different sources of variability, including (1) dye filling and laser penetration, (2) dendritic anatomy, and (3) the time elapsed from the start of recording. First, Ca(2+) Green-1 fluorescence was normalized by Texas Red; next, neurons were reconstructed so anatomy could be evaluated; finally, time was recorded. Customized software detected the largest Ca(2+) transients (area under the curve) from each line scan and matched it with parameters above. Overall, larger dendritic diameter and shorter path distance from the soma were significant predictors of larger transients, while time was not significant up to 2 h (data thereafter was dropped). However, Ca(2+) transients showed additional variability. Controlling for previous factors, significant variation was found between Ca(2+) signals from different processes of the same neuron in 3/7 neurons. This could reflect differential expression of Ca(2+) channels, local neuromodulation or other variations. Finally, Ca(2+) transients in SOD1(G93A) motoneurons were significantly smaller than in non-transgenic motoneurons. In conclusion, motoneuron processes show highly variable Ca(2+) transients, but these transients are smaller overall in SOD1(G93A) motoneurons.

Two-photon excitation microscopy (2PEM) analysis of large explanted organs is still laborious, principally because of tissue movements inducing lateral and axial drifts during extended imaging sessions. Here, we describe a two-step approach to track motile T cells in murine dorsal explanted skin with the best accuracy. First, we compared various explanted skin mounting methods for 2PEM analysis to define the setup allowing for minimal sample drift over time. Second, we developed two algorithms with the ImageJ software (National Institute of Health, Bethesda, MD) to correct the residual drift using lateral and axial registration of the collagen network. Finally, we applied the macro we developed to track fluorescent T cells in explanted skin. We found that our newly developed macro is more efficient than freely or commercially available software for shift correction, leading to more accurate velocity calculations. Our work provides a practical guide for investigators interested to employ skin-imaging approaches and offers a free alternative to commercial software for correcting lateral and axial drifts., (© 2015 Wiley Periodicals, Inc.)

Hair-follicle-associated pluripotent (HAP) stem cells can differentiate into many cell types, including neurons and heart muscle cells, and have been shown to repair peripheral nerves and the spinal cord in mice. HAP stem cells can be obtained from each individual patient for regenerative medicine which overcomes problems with immune rejection. Previously, we have demonstrated that genetically-encoded protein markers such as GFP in transgenic mice can be used to visualize HAP stem cells in vivo by multiphoton tomography. Detection and visualization of stem cells in vivo without exogenous labels such as GFP would be important for human application. In the present report, we demonstrate label-free visualization of hair follicle stem cells in mouse whiskers by multiphoton tomography due to the intrinsic fluorophores such as NAD(P)H/flavins. We compared multiphoton tomography of GFP-labeled HAP stem cells and unlabeled stem cells in isolated mouse whiskers. We show that observation of HAP stem cells by label-free multiphoton tomography is comparable to detection using GFP-labeled stem cells. The results described here have important implications for detection and isolation of human HAP stem cells for regenerative medicine.

An ultracompact high-resolution multiphoton cryomicroscope with a femtosecond near infrared fiber laser has been utilized to study the cellular autofluorescence during freezing and thawing of cells. Cooling resulted in an increase of the intracellular fluorescence intensity followed by morphological modifications at temperatures below -10 °C, depending on the application of the cryoprotectant DMSO and the cooling rate. Furthermore, fluorescence lifetime imaging revealed an increase of the mean lifetime with a decrease in temperature. Non-destructive, label-free optical biopsies of biomaterial in ice can be obtained with sub-20 mW mean powers., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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., (© 2014 Elsevier Inc. All rights reserved.)

Currently, hematoxylin-eosin (H-E) stained histopathology is the golden standard for diagnosing lung cancer. This time-consuming procedure needs tissue biopsy, sample fixation, slicing, and labeling. Therefore, the availability of a noninvasive optical diagnosis that can obtain real-time analysis comparable to golden standard H-E stained histopathology will be of extraordinary benefit to the medical community. In this study, we investigated whether multiphoton imaging can make real-time optical diagnosis for normal and cancerous lung tissue, compared with H-E stained histopathology. In the normal lung tissue, we found that multiphoton imaging could display normal lung parenchyma composed of alveolar spaces separated by thin septa. In the cancerous lung tissue, multiphoton imaging clearly illustrated that cancer cells displayed marked cellular and nuclear pleomorphism. These cancer cells were characterized by irregular size and shape, enlarged nuclei, and increased nuclear-cytoplasmic ratio. All of these histopathological features of tissue architecture and cell morphology identified by multiphoton images were readily correlated with H-E staining images. All together, multiphoton imaging can make real-time optical diagnosis for lung cancer. This study provides the groundwork for further using multiphoton imaging to perform real-time noninvasive "optical biopsy" for lung cancer in the near future., (© Wiley Periodicals, Inc.)

Thermally-induced changes in Arabidopsis thaliana leaves were investigated with a novel cryo microscope by multiphoton, fluorescence lifetime and spectral imaging as well as micro spectroscopy. Samples were excited with fs pulses in the near-infrared range and cooled/heated in a cryogenic chamber. The results show morphological changes in the chloroplast distribution as well as a shift from chlorophyll to cell-wall fluorescence with decreasing temperature. At temperatures below -40 °C, also second harmonic generation was observed. The measurements illustrate the suitability of multiphoton imaging to investigate thermally-induced changes at temperatures used for cryopreservation as well as for basic investigations of thermal effects on plant tissue in general., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: Second-harmonic generation microendoscopy is a minimally invasive technique to image sarcomeres and measure their lengths in humans, but motion artifact and low signal have limited the use of this novel technique., Methods: We discovered that an excitation wavelength of 960 nm maximized image signal; this enabled an image acquisition rate of 3 frames/s, which decreased motion artifact. We then used microendoscopy to measure sarcomere lengths in the human extensor carpi radialis brevis with the wrist at 45° extension and 45° flexion in 7 subjects. We also measured the variability in sarcomere lengths within single fibers., Results: Average sarcomere lengths in 45° extension were 2.93±0.29 μm (±SD) and increased to 3.58±0.19 μm in 45° flexion. Within single fibers the standard deviation of sarcomere lengths in series was 0.20 μm., Conclusions: Microendoscopy can be used to measure sarcomere lengths at different body postures. Lengths of sarcomeres in series within a fiber vary substantially., (© 2013 Wiley Periodicals, Inc.)

This review focuses on modern nonlinear optical microscopy (NLOM) methods that are increasingly being used in the field of tissue engineering (TE) to image tissue non-invasively and without labelling in depths unreached by conventional microscopy techniques. With NLOM techniques, biomaterial matrices, cultured cells and their produced extracellular matrix may be visualized with high resolution. After introducing classical imaging methodologies such as µCT, MRI, optical coherence tomography, electron microscopy and conventional microscopy two-photon fluorescence (2-PF) and second harmonic generation (SHG) imaging are described in detail (principle, power, limitations) together with their most widely used TE applications. Besides our own cell encapsulation, cell printing and collagen scaffolding systems and their NLOM imaging the most current research articles will be reviewed. These cover imaging of autofluorescence and fluorescence-labelled tissue and biomaterial structures, SHG-based quantitative morphometry of collagen I and other proteins, imaging of vascularization and online monitoring techniques in TE. Finally, some insight is given into state-of-the-art three-photon-based imaging methods (e.g. coherent anti-Stokes Raman scattering, third harmonic generation). This review provides an overview of the powerful and constantly evolving field of multiphoton microscopy, which is a powerful and indispensable tool for the development of artificial tissues in regenerative medicine and which is likely to gain importance also as a means for general diagnostic medical imaging.

Metastasis is the main cause of death in breast cancer patients. Cell migration is an essential component of almost every step of the metastatic cascade, especially the early step of invasion inside the primary tumor. In this report, we have used intravital multiphoton microscopy to visualize the different migration patterns of human breast tumor cells in live primary tumors. We used xenograft tumors of MDA-MB-231 cells as well as a low passage xenograft tumor from orthotopically injected patient-derived breast tumor cells. Direct visualization of human tumor cells in vivo shows two patterns of high-speed migration inside primary tumors: (1) single cells and (2) multicellular streams (i.e., cells following each other in a single file but without cohesive cell junctions). Critically, we found that only streaming and not random migration of single cells was significantly correlated with proximity to vessels, with intravasation and with numbers of elevated circulating tumor cells in the bloodstream. Finally, although the two human tumors were derived from diverse genetic backgrounds, we found that their migratory tumor cells exhibited coordinated gene expression changes that led to the same end-phenotype of enhanced migration involving activating actin polymerization and myosin contraction. Our data are the first direct visualization and assessment of in vivo migration within a live patient-derived breast xenograft tumor.

Some monoaminergic neurons can release neurotransmitters by exocytosis from their cell bodies. The amount of monoamine released by somatic exocytosis can be comparable to that released by synaptic exocytosis, though neither the underlying mechanisms nor the functional significance of somatic exocytosis are well understood. A detailed examination of these characteristics may provide new routes for therapeutic intervention in mood disorders, substance addiction, and neurodegenerative diseases. The relatively large size of the cell body provides a unique opportunity to understand the mechanism of this mode of neuronal exocytosis in microscopic detail. Here we used three photon and total internal reflection fluorescence microscopy to focus on the dynamics of the pre-exocytotic events and explore the nature of somatic vesicle storage, transport, and docking at the membrane of serotonergic neurons from raphe nuclei of the rat brain. We find that the vesicles (or unresolved vesicular clusters) are quiescent (mean square displacement, MSD ∼0.04 μm(2)/s) before depolarization, and they move minimally (<1 μm) from their locations over a time-scale of minutes. However, within minutes of depolarization, the vesicles become more dynamic (MSD ∼0.3 μm(2)/s), and display larger range (several μm) motions, though without any clear directionality. Docking and subsequent exocytosis at the membrane happen at a timescale (∼25 ms) that is slower than most synaptic exocytosis processes, but faster than almost all somatic exocytosis processes observed in endocrine cells. We conclude that, (A) depolarization causes de-tethering of the neurotransmitter vesicles from their storage locations, and this constitutes a critical event in somatic exocytosis; (B) their subsequent transport kinetics can be described by a process of constrained diffusion, and (C) the pre-exocytosis kinetics at the membrane is faster than most other somatic exocytosis processes reported so far.

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 multiphoton fluorescence laser-scanning microscopy ultrafast laser pulses, i.e. light pulses having pulse-width ≤ 1picosecond (1 p s = 10 -12 s ), are commonly used to circumvent the low multiphoton absorption cross-sections of common fluorophores. Starting with a discussion on how amplitude modulation of ultrashort pulse-train enhances the two-photon fluorescence providing deep insight into laser-induced photo-thermal damage, the effect of controlling time lag between phase-locked laser pulses on imaging is described. In addition, the prospects of laser pulse-shaping in signal enhancement (by temporal pulse-compression at the sample) and selective excitation of fluorophores (by manipulating the phase and/or amplitude of different frequency components within the pulse) are discussed with promising future applications lying ahead.