The tumor microenvironment (TME) is now recognized as an important participant of tumor progression. As the most abundant extracellular matrix component in TME, collagen plays an important role in tumor development. The imaging study of collagen morphological feature in TME is of great significance for understanding the state of tumor. Multiphoton microscopy (MPM), based on second harmonic generation (SHG) and two-photon excitation fluorescence (TPEF), can be used to monitor the morphological changes of biological tissues without labeling. In this study, we used MPM for large-scale imaging of early invasive breast cancer from the tumor center to normal tissues far from the tumor. We found that there were significant differences in collagen morphology between breast cancer tumor boundary, near tumor transition region and normal tissues far from the tumor. Furthermore, the morphological feature of eight collagen fibers was extracted to quantify the variation trend of collagen in three regions. These results may provide a new perspective for the optimal negative margin width of breast-conserving surgery and the understanding of tumor metastasis.

Multi-photon microscopy (MPM) and coherent anti-Stokes Raman scattering (CARS) are two advanced nonlinear optical imaging techniques, which provide complementary information and have great potential in combination for noninvasive in vivo biomedical applications. This paper provides a detailed discussion of the basics, development and applications of these technologies for in vivo skin research, covering the following topics: The principle and advantage of MPM and CARS, instrumentation development for in vivo applications, MPM and CARS of normal skin, application of MPM and CARS in skin cancer and disease diagnosis; application of MPM in skin disease intervention, i.e., imaging guided two-photon photothermolysis.Multi-photon microscopy (MPM) and coherent anti-Stokes Raman scattering (CARS) are two advanced nonlinear optical imaging techniques, which provide complementary information and have great potential in combination for noninvasive in vivo biomedical applications. This paper provides a detailed discussion of the basics, development and applications of these technologies for in vivo skin research, covering the following topics: The principle and advantage of MPM and CARS, instrumentation development for in vivo applications, MPM and CARS of normal skin, application of MPM and CARS in skin cancer and disease diagnosis; application of MPM in skin disease intervention, i.e., imaging guided two-photon photothermolysis.

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 di±cult 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.

Simultaneous metabolic and oxygen imaging is promising to follow up therapy response, disease development and to determine prognostic factors. FLIM of metabolic coenzymes is now widely accepted to be the most reliable method to determine cellular bioenergetics. Also, oxygen consumption has to be taken into account to understand treatment responses. The phosphorescence lifetime of oxygen sensors is able to indicate local oxygen changes. For phosphorescence lifetime imaging (PLIM) dyes based on ruthenium (II) coordination complexes are useful, in detail TLD1433 which possesses a variety of different triplet states, enables complex photochemistry and redox reactions. PLIM is usually reached by two photon excitation of the drug with a femtosecond (fs) pulsed Ti:Sapphire laser working at 80 MHz repetition rate and (time-correlated single photon counting) (TCSPC) detection electronics. The interesting question was whether it is possible to follow up PLIM using faster repetition rates. Faster repetition rates could be advantageous for the induction of specific photochemical reactions because of similar light doses used normally in standard CW light treatments. For this, a default 2p-FLIM–PLIM system was expanded by adding a second fs pulsed laser (“helixx") which provides 50 fs pulses at a repetition rate of 250MHz, more than 2.3W average power and tunable from 720 nm to 920 nm. The laser beam was coupled into the AOM instead of the default 80MHz laser. We demonstrated successful applications of the 250MHz laser for PLIM which correlates well with measurements done by excitation with the conventional 80 MHz laser source.

Using the combination of a reflective blazed grating and a reflective phase-only diffractive spatial light modulator (SLM), scanless multitarget-matching multiphoton excitation fluorescence microscopy (SMTM-MPM) was achieved. The SLM shaped an incoming mode-locked, near-infrared Ti:sapphire laser beam into an excitation pattern with addressable shapes and sizes that matched the samples of interest in the field of view. Temporal and spatial focusing were simultaneously realized by combining an objective lens and a blazed grating. The fluorescence signal from illuminated areas was recorded by a two-dimensional sCMOS camera. Compared with a conventional temporal focusing multiphoton microscope, our microscope achieved effective use of the laser power and decreased photodamage with higher axial resolution.

相比于传统的光学成像技术, 近年来获得快速发展的新型多光子成像技术具有穿透深度大, 组织光损伤小, 信噪比高, 且可方便进行光学层析成像的特点, 故而被广泛应用于包括脑、肿瘤、胚胎在内的多种活体组织成像中。本综述回顾了新型多光子成像技术的诞生与发展历程, 包括微型化双光子成像技术、双光子内窥技术和三光子成像技术, 概括分析了其基本原理与成像特点, 讨论了这一领域具有代表性的最新研究成果, 重点总结了其在生物学基础研究领域和临床医学诊断中的主要应用, 并展望了其未来的应用与发展前景。可以预见, 随着激光器和光探测技术的不断进步, 多光子成像技术将会得到更大的发展与更加广泛的应用。

多光子显微(MPM)技术通过探测由飞秒激光与生物组织内在成分相互作用而产生的双光子激发荧光和二次谐波等光信号，可实现对组织的无损、非标记成像。MPM具有对组织微结构灵敏度度和可实现高空间分辨成像、对生物组织杀伤性低和成像深度深、能够获取组织的生化信息等优点，在疾病诊断中具有很大的应用潜力。简要介绍MPM的基本原理，总结其在消化道肿瘤、皮肤疾病，以及角膜疾病诊断中的应用，并对MPM的发展前景进行展望。

This review summarizes the historical and more recent developments of multiphoton microscopy, as applied to dermatology. Multiphoton microscopy offers several advantages over competing microscopy techniques: there is an inherent axial sectioning, penetration depths that compete well with confocal microscopy on account of the use of near-infrared light, and many two-photon contrast mechanisms, such as second-harmonic generation, have no analogue in one-photon microscopy. While the penetration depths of photons into tissue are typically limited on the order of hundreds of microns, this is of less concern in dermatology, as the skin is thin and readily accessible. As a result, multiphoton microscopy in dermatology has generated a great deal of interest, much of which is summarized here. The review covers the interaction of light and tissue, as well as the various considerations that must be made when designing an instrument. The state of multiphoton microscopy in imaging skin cancer and various other diseases is also discussed, along with the investigation of aging and regeneration phenomena, and finally, the use of multiphoton microscopy to analyze the transdermal transport of drugs, cosmetics and other agents is summarized. The review concludes with a look at potential future research directions, especially those that are necessary to push these techniques into widespread clinical acceptance.