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We propose functionalized graphene oxides as an effective cancer cell-targeting agent for multiphoton imaging and near-infrared photothermal therapy. The dual-functional property of functionalized graphene oxides is investigated in human breast cancer cell lines (MDA-MB-231).

Background Label-free molecular profiling, imaging, and analysis are of particular interest in cancer biology for detecting subtle biochemical changes during cancer progression and potentially during cancer treatment. Multimodal, multiphoton imaging that combines diverse molecular contrasts derived from different physical mechanisms can improve our understanding of the tumor microenvironment. Methods A label-free optical molecular profiling technique has been developed based on penta-modal multiphoton imaging to investigate mammary tumor progression in a pre-clinical rat model. Pulses from a coherent supercontinuum were tailored for two-photon (2PF) and three-photon fluorescence (3PF), second (SHG) and third harmonic generation (THG), and hyperspectral coherent anti-Stokes Raman scattering (CARS)-based imaging. A graphic multiphoton molecular profiling model was constructed to intuitively combine the co-registered quantitative, chemical, functional, and structural tissue information, enabling longitudinal in situ biomolecular analysis. Results Over a 9-week period of tumor progression, and even before the formation of solid tumor, we observed lipid-protein transitions, microenvironmental reorganization, and a shift from FAD to NAD(P)H fluorescence, which reflects the reprogramming of cellular metabolism in carcinogenesis. Conclusions Multimodal multiphoton imaging reveals and interrelates diverse carcinogenic signatures, identifying biomarkers that could serve as early molecular indicators for breast cancer diagnosis. This quantitative multimodal imaging methodology for molecular profiling of associated cancer biomarkers may have a broader impact in fundamental cancer research and future clinical applications.

Ovarian cancer is the deadliest gynecologic cancer due predominantly to late diagnosis. Early detection of ovarian cancer can increase 5-year survival rates from 40% up to 92%, yet no reliable early detection techniques exist. Multiphoton microscopy (MPM) is a relatively new imaging technique sensitive to endogenous fluorophores, which has tremendous potential for clinical diagnosis, though it is limited in its application to the ovaries. Wide-field fluorescence imaging (WFI) has been proposed as a complementary technique to MPM, as it offers high-resolution imagery of the entire organ and can be tailored to target specific biomarkers that are not captured by MPM imaging. We applied texture analysis to MPM images of a mouse model of ovarian cancer. We also conducted WFI targeting the folate receptor and matrix metalloproteinases. We find that texture analysis of MPM images of the ovary can differentiate between genotypes, which is a proxy for disease, with high statistical significance (p

The complex DZ1 displayed enhanced three-photon absorption activity and could avoid light-quenching and light-bleaching effectively due to its aggregation-induced emission feature., Multiphoton bioimaging benefits from good penetration of tissue, low phototoxicity and high resolution. Hence, development of efficient multiphoton imaging agents is highly desirable but remains challenging. Herein, a novel terpyridine-based Zn(ii) complex bearing a thiophene bridge was designed rationally and fabricated. Thanks to its aggregation-induced emission (AIE), DZ1 emitted bright yellow-green fluorescence (λem = 575 nm) under physiological conditions. The three-photon spectral changes of DZ1 when binding with RNA unambiguously reflected its RNA-specific targeting behaviour, resulting in twofold enhancement in three-photon action cross-sections located at the second near-infrared window (1700 nm).

Geosmin is one of the most recognizable and common microbial smells on the planet. Some insects, like mosquitoes, require microbial-rich environments for their progeny, whereas for other insects such microbes may prove dangerous. In the vinegar flyDrosophila melanogaster, geosmin is decoded in a remarkably precise fashion and induces aversion, presumably signaling the presence of harmful microbes. We have here investigated the effect of geosmin on the behavior of the yellow fever mosquitoAedes aegypti. In contrast to flies, geosmin is not aversive in mosquitoes but stimulates egg-laying site selection. Female mosquitoes could associate geosmin with microbes, including cyanobacteria consumed by larvae, who also find geosmin – as well as geosmin producing cyanobacteria – attractive. Usingin vivomultiphoton imaging from mosquitoes with pan-neural expression of the calcium reporter GCaMP6s, we show thatAe. aegypticode geosmin in a similar fashion to flies, i.e. with extreme sensitivity and with a high degree of selectivity. We further demonstrate that geosmin can be used as bait under field conditions, and finally we show that geosmin, which is both expensive and difficult to obtain, can be substituted by beetroot peel extract, providing a cheap and viable mean of mosquito control and surveillance in developing countries.

There is an increasing need in cosmetic research for non-invasive, high content, skin imaging techniques offering the possibility to avoid performing invasive biopsies and to supply a maximum of information on skin state throughout a study, especially before, during and after product application. Multiphoton microscopy is one of these techniques compatible with in vitro and in vivo investigations of human skin, allowing its three-dimensional (3D) structure to be characterized with sub-μm resolution. Various intra-/extra-cellular constituents present specific endogenous two-photon excited fluorescence and second harmonic generation signals enabling a non-invasive visualization of the 3D structure of epidermal and superficial dermal layers. In association with fluorescence lifetime imaging (FLIM) and specific 3D image processing, one can extract several quantitative parameters characterizing skin constituents in terms of morphology, density and function. Multiphoton FLIM applications in cosmetic research range from knowledge to efficacy evaluation studies. Knowledge studies aim at acquiring a better understanding of appearing skin differences, for example, with aging, solar exposure or between the different skin phototypes. Evaluation studies deal with efficacy assessment of cosmetic ingredients in anti-aging or whitening domains. When using other nonlinear optics phenomena such as CARS (Coherent Anti-Stokes Raman Scattering), multiphoton imaging opens up the possibility of characterizing the cosmetic ingredients distribution inside the skin and founds application in other cosmetic domains such as hydration or antiperspirants. Developments in user-friendly, ultrasensitive, compact, multimodal imaging systems, on-the-fly data analysis and the synthesis of cosmetic ingredients with non-linear optical properties will certainly allow trespassing the todays frontiers of cosmetic applications.

We used intravital multiphoton microscopy to study the recovery of hepatobiliary metabolism following carbon tetrachloride (CCl4) induced hepatotoxicity in mouse. Our images were processed by a first order kinetic model to generate rate constant resolved images of the mouse liver. At Day 14 following induction, a restoration of the mouse hepatobiliary function was found. Our approach allows the study of the response of hepatic functions to chemical agents in real time and is useful for studying pharmacokinetics of drug molecules through optical microscopic imaging.

Ovarian cancer is the deadliest gynecologic cancer due to predominantly late diagnosis. Early detection of ovarian cancer can increase 5-year survival rates from 40% up to 92%, yet no reliable early detection techniques exist. Multiphoton microscopy (MPM) is a relatively new imaging technique with tremendous potential for clinical diagnosis. A sub-modality of MPM is second harmonic generation (SHG) imaging, which generates contrast from anisotropic structures like collagen molecules, enabling the acquisition of detailed molecular structure maps. As collagen is known to change throughout the progression of cancer, MPM is a promising candidate for ovarian cancer screening. While MPM has shown favorable results in a research environment, it has not yet found broad success in a clinical setting. One major obstacle is the quantitative analysis of the image content. Recently, the application of texture analysis to MPM images has shown success for characterizing the collagen content of the tissue, making it a prime candidate for disease screening. Unfortunately, existing work is limited in its application to ovarian tissue and few texture analysis approaches have been evaluated in this context. To address these challenges, we applied texture analysis to second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) images of a mouse model (TgMISIIR-TAg) of ovarian cancer. Using features from the grey-level co-occurrence matrix, we find that texture analysis of TPEF images of the ovary can differentiate between genotype with high statistical significance (p

The clinical diagnosis of melanocytic lesions is an ongoing medical challenge. Non-invasive tools and technologies can help to distinguish equivocal lesions. The aim of the study was to improve the in vivo diagnostic possibilities for the differentiation of benign and malignant melanocytic lesions based on combination of three imaging label-free modalities (multiphoton tomography, fluorescence lifetime imaging and optical coherence angiography). Thirty-two melanocytic lesions were studied, using multiphoton tomography, fluorescence lifetime imaging and optical coherence angiography. Multiphoton tomography features of benign melanocytic nevi were characterized by the normal morphology of both the keratinocytes and the nevus cell nests surrounded by collagen. Dysplastic nevi were characterized by their increased intercellular distances and enlarged cell nuclei. Melanomas showed the presence of melanocytes and dendritic structures in all layers of the epidermis. Analysis of the metabolic state revealed that melanomas and dysplastic nevi were characterized by enhanced glycolysis. Optical coherence angiography shows that benign nevi had regular vascular networks and equal numbers of thin and thick vessels. Vascular networks of dysplastic nevi were characterized thin curved vessels. Thick irregular spiral vessels formed a dense microvascular network of melanomas.

Introduction The detection of circulating tumor cells (CTCs) is very important for cancer diagnosis. CTCs can travel from primary tumors through the circulation to form secondary tumor colonies via bloodstream extravasation. The number of CTCs has been used as an indicator of cancer progress. However, the population of CTCs is very heterogeneous. It is very challenging to identify CTC subpopulations such as cancer stem cells (CSCs) with high metastatic potential, which are very important for cancer diagnostic management. Results We report a study of real-time CTC and CSC imaging in the bloodstreams of living animals using multi-photon microscopy and antibody conjugated quantum dots. We have developed a cancer model for noninvasive imaging wherein pancreatic cancer cells expressing fluorescent proteins were subcutaneously injected into the earlobes of mice and then formed solid tumors. When the cancer cells broke away from the solid tumor, CTCs with fluorescent proteins in the bloodstream at different stages of development could be monitored noninvasively in real time. The number of CTCs observed in the blood vessels could be correlated to the tumor size in the first month and reached a maximum value of approximately 100 CTCs/min after 5 weeks of tumor inoculation. To observe CTC subpopulations, conjugated quantum dots were used. It was found that cluster of differentiation (CD)24+ CTCs can move along the blood vessel walls and migrate to peripheral tissues. CD24+ cell accumulation on the solid tumors’ sides was observed, which may provide valuable insight for designing new drugs to target cancer subpopulations with high metastatic potential. We also demonstrated that our system is capable of imaging a minor population of cancer stem cells, CD133+ CTCs, which are found in 0.7% of pancreatic cancer cells and 1%–3% of solid tumors in patients. Conclusions With the help of quantum dots, CTCs with higher metastatic potential, such as CD24+ and CD133+ CTCs, have been identified in living animals. Using our approach, it may be possible to investigate detailed metastatic mechanism such as tumor cell extravasation to the blood vessels. In addition, the number of observed CTCs in the blood stream could be correlated with tumor stage in the early stage of cancer. Electronic supplementary material The online version of this article (10.1186/s12951-019-0453-7) contains supplementary material, which is available to authorized users.

Summary How neural circuits develop in the human brain has remained almost impossible to study at the neuronal level. Here, we investigate human cortical neuron development, plasticity, and function using a mouse/human chimera model in which xenotransplanted human cortical pyramidal neurons integrate as single cells into the mouse cortex. Combined neuronal tracing, electrophysiology, and in vivo structural and functional imaging of the transplanted cells reveal a coordinated developmental roadmap recapitulating key milestones of human cortical neuron development. The human neurons display a prolonged developmental timeline, indicating the neuron-intrinsic retention of juvenile properties as an important component of human brain neoteny. Following maturation, human neurons in the visual cortex display tuned, decorrelated responses to visual stimuli, like mouse neurons, demonstrating their capacity for physiological synaptic integration in host cortical circuits. These findings provide new insights into human neuronal development and open novel avenues for the study of human neuronal function and disease. Video Abstract, Graphical Abstract, Highlights • Cell-intrinsic mechanisms of human neoteny in mouse-human chimeric cerebral cortex • Human neurons show prolonged maturation and single-cell integration in mouse cortex • Stable dendritic spines and long-term synaptic plasticity in xenotransplanted neurons • Human neurons show decorrelated activity and tuned responses to visual stimuli, Human cortical neurons integrate as single cells in the mouse cortex and display human-like prolonged development, indicating cell-intrinsic mechanisms. Following maturation in the visual cortex, xenotransplanted human neurons display decorrelated activity and tuned responses to visual stimuli that are similar to host neurons.

We describe a new strategy for wide field multiphoton imaging through turbulent media by projecting orthonormal patterns using temporal focusing and recording fluorescent signals with single pixel detection.

Ovarian cancer is the deadliest gynecologic cancer, but can be addressed with early detection. We investigate fluorescence and multiphoton imaging for imaging ovarian cancer, finding that tissue changes can be detected through quantitative analysis.

The number of people being diagnosed with end-stage renal disease is increasing globally. Therapeutic options to slow or halt the progression of kidney disease are limited and are not always successful, despite the increasing body of research and number of basic scientific reports in this field. Further studies are required to investigate new approaches to renal pathophysiology. State of the art optical imaging is a powerful tool used to non-invasively observe the pathophysiology of small animals and has the potential to elucidate the unknown mechanisms of renal disease and aid in our understanding of the disease. This paper is a brief summary of the current usefulness of intravital imaging using multiphoton microscopy and discusses possible future applications of the technique.

Intravital multiphoton microscopy is widely used to assess the structure and function of organs in live animals. Although different tissues vary in their accessibility for intravital multiphoton imaging, considerable progress has been made in the imaging quality of all tissues due to substantial technical improvements in the relevant imaging components, such as optics, excitation laser, detectors, and signal analysis software. In this review, we provide an overview of the technical background of intravital multiphoton microscopy. Then, we note a few seminal findings that were made through the use of multiphoton microscopy. Finally, we address the technical limitations of the method and provide an outlook for how these limitations may be overcome through future technical developments.

Here we present the first generation of two-photon beta cell specific in vivo imaging probes based on GLP1R targeting peptides. Among the three compounds of potential interest, we found quite unexpectedly that a squarine-rotaxane conjugate (2PEx-647) had near ideal in vivo imaging characteristics.

Glaucoma filtration surgery (GFS) commonly fails due to excessive fibrosis. As collagen structure aberrations is implicated in adverse fibrotic progression, this study aims to uncover collagen organization alterations during postoperative scarring. Via quantitative second harmonic generation/ two photon excitation multiphoton imaging, we reveal the scar development and phenotype in the mouse model of conjunctival scarring. We also show that multiphoton imaging corroborated the collagen ultrastructure anomaly characteristic of the SPARC-/- mouse postoperative conjunctiva. These data improve our understanding of postoperative conjunctival scarring and further enhance the utility of this model for the development of anti-fibrotic therapeutics for GFS.

To date, numerous studies have been performed to elucidate the complex cellular dynamics in skin diseases, but few have attempted to characterize these cellular events under conditions similar to the native environment. To address this challenge, a three-dimensional (3D) multimodal analysis platform was developed for characterizing in vivo cellular dynamics in skin, which was then utilized to process in vivo wound healing data to demonstrate its applicability. Special attention is focused on in vivo biological parameters that are difficult to study with ex vivo analysis, including 3D cell tracking and techniques to connect biological information obtained from different imaging modalities. These results here open new possibilities for evaluating 3D cellular dynamics in vivo, and can potentially provide new tools for characterizing the skin microenvironment and pathologies in the future.

In contrast to single photon excitation fluorescence imaging, laser scanning confocal imaging, and wide-field imaging, the multi-photon imaging has advantages of minimal invasion and deeper penetration by using near-infrared (NIR) laser source. Moreover, it can carry out three-dimensional high-spatial-resolution imaging of biological tissues due to its natural optical tomography capability. Since its advent, multi-photon imaging has become a powerful tool in biomedicine and achieved a series of significant discoveries in cancer pathology, neurological diseases and brain functional imaging. In the past decade, as a major form of multi-photon imaging techonoogy, two-photon excited fluorescence microscopy imaging has a great potential in biomedical applications. In order to satisfy the practical biomedical applications, multi-photon imaging technologies have made significant breakthroughs in improving the deficiencies of traditional 2PEF in multi-color imaging, functional imaging, live imaging and imaging depth, such as multicolor two-photon excitation fluorescence microscopy, two-photon fluorescence lifetime imaging microscopy, two-photon fiber endoscopic imaging, and three-photon microscopy imaging technology. For example, multicolor two-photon excitation fluorescence microscopy is demonstrated to achieve simultaneous imaging of multiple fluorophores with multiple wavelenth excitation lasers or continuous spectrum. In addition, the two-photon fluorescence lifetime microscopic imaging provides a method to achieve high-resolution three-dimensional imaging of biological tissue with multi-dimensional information including fluorescence intensity and lifetime. In addition, two-photon optical fiber endoscopic imaging with small system size and mimal invasion is developed and used to image the tissue inside the deep organ. Finally, two-photon excitation fluorescence microscopy technique still has relatively strong scattering for brain functional imaging in vivo. Therefore, the imaging depth is limited by the signal-to-background ratio. Three-photon microscopic imaging technique can achieve higher imaging depth and a desired signal-to-noise ratio by extending the wavelength from 1600 nm to 1820 nm because the attenuation of the excitation light in this wavelenth range is much smaller. In this article, we briefly introduce the principles and applications of these multi-photon imaging technologies, and finally provide our view for their future development.

Kidney proximal tubules (PTs) are densely packed with mitochondria, and defects in mitochondrial function are implicated in many kidney diseases. However, little is known about intrinsic mitochondrial function within PT cells. Here, using intravital multiphoton microscopy and live slices of mouse kidney cortex, we show that autofluorescence signals provide important functional readouts of redox state and substrate metabolism and that there are striking axial differences in signals along the PT. Mitochondrial NAD(P)H intensity was similar in both PT segment (S)1 and S2 and was sensitive to changes in respiratory chain (RC) redox state, whereas cytosolic NAD(P)H intensity was significantly higher in S2. Mitochondrial NAD(P)H increased in response to lactate and butyrate but decreased in response to glutamine and glutamate. Cytosolic NAD(P)H was sensitive to lactate and pyruvate and decreased dramatically in S2 in response to inhibition of glucose metabolism. Mitochondrial flavoprotein (FP) intensity was markedly higher in S2 than in S1 but was insensitive to changes in RC redox state. Mitochondrial FP signal increased in response to palmitate but decreased in response to glutamine and glutamate. Fluorescence lifetime decays were similar in both S1 and S2, suggesting that intensity differences are explained by differences in abundance of the same molecular species. Expression levels of known fluorescent mitochondrial FPs were higher in S2 than S1. In summary, substantial metabolic information can be obtained in kidney tissue using a label-free live imaging approach, and our findings suggest that metabolism is tailored to the specialized functions of S1 and S2 PT segments.

Background Confocal laser endomicroscopy (CLE) requires fluorescence agents, the use of which leads to blurred images and low diagnostic accuracy owing to fluorescein leakage. We aimed to explore whether multiphoton imaging (MPI) could serve as a better method of optical biopsy. Methods First, a pilot study was performed to set up the optical diagnostic criteria of MPI for benign or malignant colorectal lesions in 30 patients. Then, a blinded study was conducted to compare the sensitivity, specificity, and accuracy of MPI versus CLE imaging in 79 patients. Results In the pilot study, MPI revealed regular tissue architecture and cell morphology in the normal tissue, and irregular tubular structures, and cellular and nuclear pleomorphism in the abnormal tissue. In the blinded study, compared with CLE imaging, MPI significantly improved the diagnostic sensitivity, specificity, and accuracy of the optical biopsy (89.74 % vs. 61.54 %, P = 0.008; 92.5 % vs. 67.5 %, P = 0.009; and 91.14 % vs. 64.56 %, P = 0.001, respectively). Conclusions MPI can provide a superior optical biopsy to that of CLE imaging for colorectal lesions.

Multiphoton imaging commonly relies on laser-scanning setups which quickly image horizontal sections (x y images) by pixelwise scanning a sample region with focused laser pulses. Different horizontal planes are imaged by adjusting the distance relative between focusing optics and sample. However, in many cases actually a visualization of vertical sections is desired that then can only be obtained indirectly from time-consuming acquisition and processing of complete volume scans. We present a modified multiphoton tomograph for clinical in vivo and ex vivo tissue imaging with direct and fast x-z-imaging capability and exemplify different applications spanning from visualizing anatomic structures to substance penetration studies. The fast x-z imaging is realized by synchronizing the scanning-mirror movement with the tuning of the relative distance between sample and focusing optics

C.M.J. and D.A. contributed equally to this work. The grant “Chercheur d'Avenir Languedoc-Roussillon” attributed to M.G.-B. and the ANR nanoptPDT are gratefully acknowledged. The authors thank L. Lichon for technical assistance. DFT calculations were performed using HPC resources from the CINES (projects x2016087394 and x2015087394), and from the CALMIP (Grant 2016-[P16042]). The authors thank MRI (Montpellier RIO Imaging platform) for confocal and multiphoton imaging facilities.

Highlights • We concurrently imaged the activity of individual synapses and surrounding astroglia in live animals. • Perisynaptic astrocytic regions display localiased, context-dependent Ca2+ elevations in vivo. • Such elevations may precede ‘spontaneous’ spike-evoked Ca2+ entry at presynaptic boutons. • This method paves the way for optical investigation of glutamate release probability in vivo., All-optical registration of neuronal and astrocytic activities within the intact mammalian brain has improved significantly with recent advances in optical sensors and biophotonics. However, relating single-synapse release events and local astroglial responses to sensory stimuli in an intact animal has not hitherto been feasible. Here, we present a multiplexed multiphoton excitation imaging approach for assessing the relationship between presynaptic Ca2+ entry at thalamocortical axonal boutons and perisynaptic astrocytic Ca2+ elevations, induced by whisker stimulation in the barrel cortex of C57BL/6 mice. We find that, unexpectedly, Ca2+ elevations in the perisynaptic astrocytic regions consistently precede local presynaptic Ca2+ signals during spontaneous brain activity associated with anaesthesia. The methods described here can be adapted to a variety of optical sensors and are compatible with experimental designs that might necessitate repeated sampling of single synapses over a longitudinal behavioural paradigm.

The combination of a tunable acoustic gradient (TAG) lens and a resonant mirror were integrated into a multiphoton excited (MPE) microscope that is rapidly and precisely controlled by an embedded field programmable gate array (FPGA) to acquire images at high frame rate.

We demonstrate a new approach, temporal focusing microscopy with single-pixel detection (TEMPPIX), for wide-field multiphoton imaging through scattering media without any a priori knowledge or requirement to determine the properties of the media.

Fluorescent imaging combined with H&E-rendering algorithms can provide histological imaging of surgical specimens more rapidly than conventional histology by avoiding the need to formalin fix, freeze or physically section specimens. However, validation of histology performed on live tissue is challenging because fixation and sectioning frequently result in changes to tissue appearance, while histological cutting planes are often substantially different than the true tissue surface. We present validation studies aiming to understand the relationship between features visualized using multiphoton fluorescence and conventional histology.

Semiconductor quantum dots (QDs) are characterized by orders of magnitude higher multiphoton linear absorption cross-sections compared with conventional organic dyes. Combined with the QD photoluminescence quantum yield approaching 100% and their rock-solid photostability, this fact opens great prospects for the two-photon functional tumor imaging with QDs tagged with highly specific recognition molecules. Single-domain antibodies (sdAbs) or “nanobodies” derived from lamas are the smallest high-affinity recognition molecules, which may be tagged with the QDs thus permitting not only solid tumors multiphoton imaging but also rare disseminated cancer cells and micrometastases in the depth of the tissue to be detected.

We propose and demonstrate an all-fiber Raman laser based on phosphosilicate fiber. It produces ∼5 ps chirped dissipative soliton pulses with energy up to 8 nJ, average power of 0.3 W at 1230 nm center wavelength.

In plants, transcription factors often act as cell-to-cell trafficking mobile proteins and specify cell fate. Thus, to visualize spatiotemporal expression pattern and localization of transcription factors are essential to understand their functions during development. Several protocols have been developed to observe fluorescent protein. However, plant-specific autofluorescent compounds and various tissue components with different refractive indexes interfere with detection of fluorescent signals of your interest. Furthermore, cell fate specification often occurs in a limited number of cells covered by lateral/layers of organs. To overcome those issues, the plant clearing method, ClearSee, was recently developed for high-resolution imaging inside tissues by making background transparent. In this chapter, we provide three-dimensional imaging of fluorescent-protein-fused transcription factors by two-photon excitation microscopy in Arabidopsis and rice. Complex cell patterning with gene expression could be observed from any direction three-dimensionally. This method could be applicable to visualize any protein of your interest or it can readily be adapted in various other plants.

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.

The purpose of this chapter is to introduce the theoretical fundamentals responsible for nonlinear optical (NLO) processes. Fascinating key processes such as second harmonic generation (SHG), third harmonic generation (THG), and two-photon absorption (TPA) are the consequences of light–matter interaction described by higher order corrections. The use of these techniques is the most popular for multiphoton imaging which is a powerful microscopy technique that has found growing application in fields ranging from fundamental physics to biomedicine.

We report a directly blue diode pumped Ti:Sapphire oscillator that generates 5 nJ pulses. This is five times higher pulse energy than previously reported for a directly diode pumped Ti:sapphire laser. With 460 mW of average power at 92 MHz and 82 fs pulses, its peak power reaches 61 kW, also several times higher the value than previously published. Direct diode pumping significantly reduces the complexity and therefore the footprint and the cost of the laser, while SESAM modelocking ensures reliable selfstarting and robust operation. Such a laser is ideally suited for biomedical imaging and nanostructuring applications. As a demonstration of sufficient peak power for microscopy applications, we perform different modalities of nonlinear microscopy of biological samples.

Hypericin (Hyp) has received attention due to its high phototoxicity against viruses and anti-tumor photoactivity. Using two-photon imaging, we demonstrated that Hyp induced photosensitized modification of collagen fibers in native tissues. Dynamics of photo-processes was monitored by time-lapse multiphoton imaging. We showed that Hyp–mediated processes in collagen tissues may be used for the selective modification of collagen fibers.

Multicolor two-photon fluorescence imaging is performed using blue-diode-pumped SESAM-modelocked Ti:Sapphire oscillator generating 5 nJ pulse energy, 82 fs pulse duration, at 780 nm central wavelength, with 92 MHz pulse repetition rate.

Generation and programmable shaping of widely-coherent supercontinuum pulses enables multimodal multiphoton imaging for basic science investigations of carcinogenesis and novel biomarkers for clinical cancer diagnostics and monitoring.

We develop a high-peak-power coherent supercontinuum source to systemically overcome some critical limitations of clinical multiphoton imaging. This design can tailor the performance parameters of multiphoton imaging to adapt to varying applications.

Using custom and adaptive optics, we developed multiphoton calcium imaging systems for large volumes (~ 20 cubic millimeters), with subcellular and subsecond resolution, and independent, temporally multiplexed beams for neuroscience research.

We demonstrate 1600–2520 nm wavelength-tunable, high energy soliton generation using a large-mode-area fiber pumped by a compact fiber source. Using second harmonic generation, we show their applications for in vivo multi-photon mouse brain imaging.