Label-free multi-photon imaging using a compact femtosecond fiber laser mode-locked by carbon nanotube saturable absorber

We demonstrate label-free multi-photon imaging of biological samples using a compact Er³⁺-doped femtosecond fiber laser mode-locked by a single-walled carbon nanotube (CNT). These compact and low cost lasers have been developed by various groups but they have not been exploited for multiphoton microscopy. Here, it is shown that various multiphoton imaging modalities (e.g. second harmonic generation (SHG), third harmonic generation (THG), two-photon excitation fluorescence (TPEF), and three-photon excitation fluorescence (3PEF)) can be effectively performed on various biological samples using a compact handheld CNT mode-locked femtosecond fiber laser operating in the telecommunication window near 1560nm. We also show for the first time that chlorophyll fluorescence in plant leaves and diatoms can be observed using 1560nm laser excitation via three-photon absorption.OCIS codes: (170.5810) Scanning microscopy, (180.6900) Three-dimensional microscopy, (140.7090) Ultrafast lasers, (190.4180) Multiphoton processes     

Multimodal microscopy with sub-30 fs Yb fiber laser oscillator

Nonlinear optical microscopy with sub-30 fs pulses from an Yb-fiber laser, approximately three times shorter than typical fiber laser pulses, leads to an order of magnitude brighter third harmonic generation imaging. Multiphoton fluorescence, second and third harmonic generation modalities are compared on stained microspheres and unstained biological tissues.     

Multiphoton morpho-functional imaging of healthy colon mucosa,adenomatous polyp and adenocarcinoma

Two-photon spectral resolved imaging was used to image fresh human biopsies of colon tissue and to characterize healthy colon mucosa, adenomatous polyp and adenocarcinoma by means of a morpho-functional analysis. Morphological examination, performed using endogenous tissue fluorescence, discriminated adenomatous and adenocarcinoma tissues from normal mucosa in terms of cellular asymmetry and nucleus-to-cytoplasm ratio. Good agreement was found between multiphoton images and histological examination performed on the same samples. Further characterization, performed by means of spectral-resolved analysis of NADH and FAD fluorescence, demonstrated an altered metabolic activity in both adenomatous and adenocarcinoma tissues compared to healthy mucosa. This morpho-functional approach may represent a powerful method to be used in combination with endoscopy for in vivo optical diagnosis of colon cancer and may be extended to other tissues.OCIS codes: (180.4315) Nonlinear microscopy, (170.3880) Medical and biological imaging     

Long-term two-photon neuroimaging with a photostable AIE luminogen

In neuroscience, fluorescence labeled two-photon microscopy is a promising tool to visualize ex vivo and in vivo tissue morphology, and track dynamic neural activities. Specific and highly photostable fluorescent probes are required in this technology. However, most fluorescent proteins and organic fluorophores suffer from photobleaching, so they are not suitable for long-term imaging and observation. To overcome this problem, we utilize tetraphenylethene-triphenylphosphonium (TPE-TPP), which possesses aggregation-induced emission (AIE) and two-photon fluorescence characteristics, for neuroimaging. The unique AIE feature of TPE-TPP makes its nanoaggregates resistant to photobleaching, which is useful to track neural cells and brain-microglia for a long period of time. Two-photon fluorescence of TPE-TPP facilitates its application in deep in vivo neuroimaging, as demonstrated in the present paper.OCIS codes: (160.4890) Organic materials, (160.2540) Fluorescent and luminescent materials, (190.4180) Multiphoton processes, (170.3880) Medical and biological imaging, (180.2520) Fluorescence microscopy, (180.4315) Nonlinear microscopy     

Fluorescent labeling of renal cells in vivo

In vivo fluorescence imaging, using confocal or multiphoton microscopes, provides a powerful method to analyze kidney function in experimental animals. In this review, the preparation used for physiological studies in rats is described. A variety of fluorescent probes are available to study glomerular permeability, renal blood flow, peritubular capillary permeability, cell ion concentrations, tubule transport properties, and the functional status of renal cells. We have recently used micropuncture techniques and an adenovirus vector to accomplish gene transfer into kidney tubule and endothelial cells; this new methodology will allow the dynamic study of fluorescently-labeled proteins. Two examples of the use of two-photon fluorescence microscopy to study renal pathophysiology, namely polycystic kidney disease and renal ischemia, are presented. Software is available to quantify data collected from in vivo imaging experiments and to construct 3-dimensional images of renal structures. Two-photon or confocal microscopy offers many opportunities for a better understanding of kidney function in health and disease.     

胃肠道肿瘤的多光子成像与光学活检

胃肠道恶性肿瘤手术术式的选择及手术切除范围的确定急需一种原位的、实时的诊断技术来评估肿瘤的浸润深度、转移情况以及外科手术切缘有无癌残留。利用多光子成像技术,多光子显微镜能够提供实时的胃肠道组织结构和细胞形态学信息。多光子成像技术具备无需外源标记组织、对胶原极其敏感、对组织的光损伤小和穿透深度深等特点,其可能应用于胃肠道肿瘤的光学活检。本综述从肿瘤浸润深度、转移情况和外科手术切缘有无癌残留等相关研究角度,旨在综合概述多光子成像技术用于评估胃肠道肿瘤光学活检的可行性以及探讨多光子成像技术可观的发展前景。      

Focal switching of photochromic fluorescent proteins enables multiphoton microscopy with superior image contrast

Probing biological structures and functions deep inside live organisms with light is highly desirable. Among the current optical imaging modalities, multiphoton fluorescence microscopy exhibits the best contrast for imaging scattering samples by employing a spatially confined nonlinear excitation. However, as the incident laser power drops exponentially with imaging depth into the sample due to the scattering loss, the out-of-focus background eventually overwhelms the in-focus signal, which defines a fundamental imaging-depth limit. Herein we significantly improve the image contrast for deep scattering samples by harnessing reversibly switchable fluorescent proteins (RSFPs) which can be cycled between bright and dark states upon light illumination. Two distinct techniques, multiphoton deactivation and imaging (MPDI) and multiphoton activation and imaging (MPAI), are demonstrated on tissue phantoms labeled with Dronpa protein. Such a focal switch approach can generate pseudo background-free images. Conceptually different from wave-based approaches that try to reduce light scattering in turbid samples, our work represents a molecule-based strategy that focused on imaging probes.     

Stimulated emission reduced fluorescence microscopy: a concept for extending the fundamental depth limit of two-photon fluorescence imaging

Two-photon fluorescence microscopy has become an indispensable tool for imaging scattering biological samples by detecting scattered fluorescence photons generated from a spatially confined excitation volume. However, this optical sectioning capability breaks down eventually when imaging much deeper, as the out-of-focus fluorescence gradually overwhelms the in-focal signal in the scattering samples. The resulting loss of image contrast defines a fundamental imaging-depth limit, which cannot be overcome by increasing excitation efficiency. Herein we propose to extend this depth limit by performing stimulated emission reduced fluorescence (SERF) microscopy in which the two-photon excited fluorescence at the focus is preferentially switched on and off by a modulated and focused laser beam that is capable of inducing stimulated emission of the fluorophores from the excited states. The resulting image, constructed from the reduced fluorescence signal, is found to exhibit a significantly improved signal-to-background contrast owing to its overall higher-order nonlinear dependence on the incident laser intensity. We demonstrate this new concept by both analytical theory and numerical simulations. For brain tissues, SERF is expected to extend the imaging depth limit of two-photon fluorescence microscopy by a factor of more than 1.8.     

A CANDLE for a deeper in vivo insight

A new Collaborative Approach for eNhanced Denoising under Low-light Excitation (CANDLE) is introduced for the processing of 3D laser scanning multiphoton microscopy images. CANDLE is designed to be robust for low signal-to-noise ratio (SNR) conditions typically encountered when imaging deep in scattering biological specimens. Based on an optimized non-local means filter involving the comparison of filtered patches, CANDLE locally adapts the amount of smoothing in order to deal with the noise inhomogeneity inherent to laser scanning fluorescence microscopy images. An extensive validation on synthetic data, images acquired on microspheres and in vivo images is presented. These experiments show that the CANDLE filter obtained competitive results compared to a state-of-the-art method and a locally adaptive optimized non-local means filter, especially under low SNR conditions (PSNR<8dB). Finally, the deeper imaging capabilities enabled by the proposed filter are demonstrated on deep tissue in vivo images of neurons and fine axonal processes in the Xenopus tadpole brain.     

Comparison of objective lenses for multiphoton microscopy in turbid samples

Optimization of illumination and detection optics is pivotal for multiphoton imaging in highly scattering tissue and the objective lens is the central component in both of these pathways. To better understand how basic lens parameters (NA, magnification, field number) affect fluorescence collection and image quality, a two-detector setup was used with a specialized sample cell to separate measurement of total excitation from epifluorescence collection. Our data corroborate earlier findings that low-mag lenses can be superior at collecting scattered photons, and we compare a set of commonly used multiphoton objective lenses in terms of their ability to collect scattered fluorescence, providing guidance for the design of multiphoton imaging systems. For example, our measurements of epi-fluorescence beam divergence in the presence of scattering reveal minimal beam broadening, indicating that often-advocated over-sized collection optics are not as advantageous as previously thought. These experiments also provide a framework for choosing objective lenses for multiphoton imaging by relating the results of our measurements to various design parameters of the objectives lenses used.OCIS codes: (110.0113) Imaging through turbid media, (180.2520) Fluorescence microscopy, (180.4315) Nonlinear microscopy, (180.6900) Three-dimensional microscopy     

Two-photon excited hemoglobin fluorescence

We discovered that hemoglobin emits high energy Soret fluorescence when two-photon excited by the visible femtosecond light sources. The unique spectral and temporal characteristics of hemoglobin fluorescence were measured by using a time-resolved spectroscopic detection system. The high energy Soret fluorescence of hemoglobin shows the spectral peak at 438 nm with extremely short lifetime. This discovery enables two-photon excitation fluorescence microscopy to become a potentially powerful tool for in vivo label-free imaging of blood cells and vessels.OCIS codes: (170.0170) Medical optics and biotechnology, (300.6500) spectroscopy, time-resolved, (300.6410) Spectroscopy, multiphoton, (170.2520) Fluorescence microscopy, (180.4315) Nonlinear microscopy     

Imaging carious dental tissues with multiphoton fluorescence lifetime imaging microscopy

In this study, multiphoton excitation was utilized to image normal and carious dental tissues noninvasively. Unique structures in dental tissues were identified using the available multimodality (second harmonic, autofluorescence, and fluorescence lifetime analysis) without labeling. The collagen in dentin exhibits a strong second harmonic response. Both dentin and enamel emit strong autofluorescence that reveals in detail morphological features (such as dentinal tubules and enamel rods) and, despite their very similar spectral profiles, can be differentiated by lifetime analysis. Specifically, the carious dental tissue exhibits a greatly reduced autofluorescence lifetime, which result is consistent with the degree of demineralization, determined by micro-computed tomography. Our findings suggest that two-photon excited fluorescence lifetime imaging may be a promising tool for diagnosing and monitoring dental caries.OCIS codes: (170.1850) Dentistry, (170.3880) Medical and biological imaging, (170.6935) Tissue characterization, (170.6920) Time-resolved imaging     

Nonlinear structured-illumination enhanced temporal focusing multiphoton excitation microscopy with a digital micromirror device

In this study, the light diffraction of temporal focusing multiphoton excitation microscopy (TFMPEM) and the excitation patterning of nonlinear structured-illumination microscopy (NSIM) can be simultaneously and accurately implemented via a single high-resolution digital micromirror device. The lateral and axial spatial resolutions of the TFMPEM are remarkably improved through the second-order NSIM and projected structured light, respectively. The experimental results demonstrate that the lateral and axial resolutions are enhanced from 397 nm to 168 nm (2.4-fold) and from 2.33 μm to 1.22 μm (1.9-fold), respectively, in full width at the half maximum. Furthermore, a three-dimensionally rendered image of a cytoskeleton cell featuring ~25 nm microtubules is improved, with other microtubules at a distance near the lateral resolution of 168 nm also able to be distinguished.OCIS codes: (170.3880) Medical and biological imaging, (180.4315) Nonlinear microscopy, (190.4180) Multiphoton processes     

Multi-photon excited luminescence of magnetic FePt core-shell nanoparticles

We present magnetic FePt nanoparticles with a hydrophilic, inert, and biocompatible silico-tungsten oxide shell. The particles can be functionalized, optically detected, and optically manipulated. To show the functionalization the fluorescent dye NOPS was bound to the FePt core-shell nanoparticles with propyl-triethoxy-silane linkers and fluorescence of the labeled particles were observed in ethanol (EtOH). In aqueous dispersion the NOPS fluorescence is quenched making them invisible using 1-photon excitation. However, we observe bright luminescence of labeled and even unlabeled magnetic core-shell nanoparticles with multi-photon excitation. Luminescence can be detected in the near ultraviolet and the full visible spectral range by near infrared multi-photon excitation. For optical manipulation, we were able to drag clusters of particles, and maybe also single particles, by a focused laser beam that acts as optical tweezers by inducing an electric dipole in the insulated metal nanoparticles. In a first application, we show that the luminescence of the core-shell nanoparticles is bright enough for in vivo multi-photon imaging in the mouse neocortex down to cortical layer 5.OCIS codes: (160.4236) Nanomaterials, (300.6410) Spectroscopy, multiphoton, (170.3880) Medical and biological imaging, (170.6280) Spectroscopy, fluorescence and luminescence, (170.2520) Fluorescence microscopy, (020.4180) Multiphoton processes     

Linear diattenuation imaging of biological tissues with near infrared Mueller scanning microscopy

Among the multitude of optical polarization contrasts that can be observed in complex biological specimens, linear diattenuation (LD) imaging has received little attention. It is indeed challenging to image LD with basic polarizing microscopes because it is often relatively small in comparison with linear retardance (LR). In addition, interpretation of LD images is not straightforward when experiments are conducted in the visible range because LD can be produced by both dichroism and anisotropic scattering. Mueller polarimetry is a powerful implementation of polarization sensing able to differentiate and measure the anisotropies of specimens. In this article, near infrared transmission Mueller scanning microscopy is used to image LD in thin biological specimen sections made of various proteins with unprecedented resolution and sensitivity. The near infrared spectral range makes it possible to lower the contribution of dichroism to the total linear diattenuation in order to highlight anisotropic scattering. Pixel-by-pixel comparison of LD images with LR and multiphoton images demonstrates that LD is produced by under-resolved structures that are not revealed by other means, notably within the sarcomere of skeletal muscles. LD microscopy appears as a powerful tool to provide new insights into the macro-molecular organization of biological specimens at the sub-microscopic scale without labelling.     

Intravital imaging of the kidney using multiparameter multiphoton microscopy

Intravital optical microscopy provides a powerful means of studying the cell biology in the most physiologically relevant setting. The ability of multiphoton microscopy to collect optical sections deep into biological tissues has opened up the field of intravital microscopy to high-resolution studies of multiple organs. Presented here are examples of how two-photon microscopy can be applied to intravital studies of kidney physiology and the study of disease processes. These include studies of cell vitality and apoptosis, fluid transport, receptor-mediated endocytosis, blood flow, and leukocyte trafficking. Efficient two-photon excitation of multiple fluorophores permits comparison of multiple probes and simultaneous characterization of multiple parameters. Two-photon microscopy can now provide a level of investigation previously unattainable in intravital microscopy, enabling kinetic analyses and physiological studies of the organs of living animals with subcellular resolution. Therefore, application of this technology will provide direct visualization of organ-specific and cell-specific responses to an array of stimuli and therapeutic approaches, enhancing our understanding and treatment of disease processes.     

应用超高灵敏度荧光显微成像及共聚焦显微成像观察光敏剂细胞内分布的对比研究

背景光敏剂亚细胞分布研究多采用激光共聚焦显微镜来开展.激光共聚焦显微成像系统能够获得细节清晰的细胞断层荧光图像,可以进行精确的光敏剂亚细胞定位.但其样品准备过程复杂,费用不菲,而且对于荧光效率较低的光敏剂探测难度很大.目的应用带像增强器的冷电感耦合装置(intensified charge-coupled device,ICCD)的超高灵敏度荧光显微成像系统及共聚焦显微成像进行光敏剂细胞内分布的对比研究,深入探讨光动力疗法的损伤机制,为拓展其在临床康复治疗领域的适应证奠定实验基础.设计非随机非对照的实验研究.地点、材料和干预实验地点为北京理工大学光电工程系实验室.传代培养内皮细胞、食管癌细胞和肺癌细胞,将不同浓度血卟啉单甲醚(hematoporphrin monomethyl ether,HMME)与细胞共同孵育不同时间.采用荧光显微镜及ICCD组成的荧光显微成像系统采集不同浓度及不同孵育时间条件下HMME的荧光图像,并采用计算机图像处理技术进行图像增强、滤波后计算其细胞浆与细胞核的平均荧光强度比值.同时应用激光共聚焦显微镜图像采集进行对比.主要观察指标不同荧光显微成像系统采集到的细胞的光敏剂荧光图像的细节特点及胞浆与胞核区域光强比值的对比观察.结果HMME浓度为5 mg/L时,荧光显微镜采集到HMME的荧光图像;HMME浓度升高到160 mg/L,激光共聚焦显微镜获得HMME的荧光图像.两组图像的特点都为胞浆中荧光强度较高,细胞核区荧光较弱;细胞浆与细胞核的比值约为2～31.结论荧光显微镜和ICCD采集细胞内光敏剂的荧光图像灵敏度高,方法可靠、实用,可用来对荧光效率较低的光敏剂进行细胞内分布研究.HMME较多分布在细胞质中,细胞核吸收较少.从而得出HMME介导的内皮细胞及肺癌细胞光动力损伤的初次损伤位点可能是细胞浆内多种细胞器.     

Shot noise limits on binary detection in multiphoton imaging

Much of fluorescence-based microscopy involves detection of if an object is present or absent (i.e., binary detection). The imaging depth of three-dimensionally resolved imaging, such as multiphoton imaging, is fundamentally limited by out-of-focus background fluorescence, which when compared to the in-focus fluorescence makes detecting objects in the presence of noise difficult. Here, we use detection theory to present a statistical framework and metric to quantify the quality of an image when binary detection is of interest. Our treatment does not require acquired or reference images, and thus allows for a theoretical comparison of different imaging modalities and systems.     

Deep in vivo two-photon microscopy with a low cost custom built mode-locked 1060 nm fiber laser

Here we demonstrate that a mode-locked ytterbium fiber laser for two-photon fluorescence microscopy can be built for $13,000. The laser emits at a wavelength of 1060 nm with a usable average power of 1 W at a repetition rate of 40 MHz and a compressed pulse width of 81 fs at the sample. The laser is used to obtain deep in vivo two-color images of layer-V pyramidal neurons expressing YFP and vasculature labelled with Texas Red at depths up to 900 µm. The sub-1 µm features of dendritic spines can be resolved at a 200 µm depth.OCIS codes: (140.4050) Mode-locked lasers, (170.3880) Medical and biological imaging, (190.4180) Multiphoton processes     

Imaging of targeted lipid microbubbles to detect cancer cells using third harmonic generation microscopy

The use of receptor-targeted lipid microbubbles imaged by ultrasound is an innovative method of detecting and localizing disease. However, since ultrasound requires a medium between the transducer and the object being imaged, it is impractical to apply to an exposed surface in a surgical setting where sterile fields need be maintained and ultrasound gel may cause the bubbles to collapse. Multiphoton microscopy (MPM) is an emerging tool for accurate, label-free imaging of tissues and cells with high resolution and contrast. We have recently determined a novel application of MPM to be used for detecting targeted microbubble adherence to the upregulated plectin-receptor on pancreatic tumor cells. Specifically, the third-harmonic generation response can be used to detect bound microbubbles to various cell types presenting MPM as an alternative and useful imaging method. This is an interesting technique that can potentially be translated as a diagnostic tool for the early detection of cancer and inflammatory disorders.OCIS codes: (020.4180) Multiphoton processes, (170.1610) Clinical applications, (190.1900) Diagnostic applications of nonlinear optics, (320.7090) Ultrafast lasers