Temporal focusing‐based widefield multiphoton microscopy with spatially modulated illumination for biotissue imaging

A developed temporal focusing‐based multiphoton excitation microscope (TFMPEM) has a digital micromirror device (DMD) which is adopted not only as a blazed grating for light spatial dispersion but also for patterned illumination simultaneously. Herein, the TFMPEM has been extended to implement spatially modulated illumination at structured frequency and orientation to increase the beam coverage at the back‐focal aperture of the objective lens. The axial excitation confinement (AEC) of TFMPEM can be condensed from 3.0 μm to 1.5 μm for a 50 % improvement. By using the TFMPEM with HiLo technique as two structured illuminations at the same spatial frequency but different orientation, reconstructed biotissue images according to the condensed AEC structured illumination are shown obviously superior in contrast and better scattering suppression. Picture : TPEF images of the eosin‐stained mouse cerebellar cortex by conventional TFMPEM (left), and the TFMPEM with HiLo technique as 1.09 μm^?1 spatially modulated illumination at 90° (center) and 0° (right) orientations.

     

Multicolor multiphoton microscopy based on a nanosecond supercontinuum laser source

Multicolor multiphoton microscopy is experimentally demonstrated for the first time on a spectral bandwidth of excitation of 300 nm (full width half maximum) thanks to the implementation a nanosecond supercontinuum (SC) source compact and simple with a low repetition rate. The interest of such a wide spectral bandwidth, never demonstrated until now, is highlighted in vivo: images of glioma tumor cells stably expressing eGFP grafted on the brain of a mouse and its blood vessels network labelled with Texas Red^® are obtained. These two fluorophores have a spectral bandwidth covering the whole 300 nm available. In parallel, a similar image quality is obtained on a sample of mouse muscle in vitro when excited with this nanosecond SC source or with a classical high rate, femtosecond and quasi monochromatic laser. This opens the way for (i) a simple and very complete biological characterization never performed to date with multiphoton processes, (ii) multiple means of contrast in nonlinear imaging allowed by the use of numerous fluorophores and (iii) other multiphoton processes like three‐photon ones.

     

A novel multiphoton microscopy images segmentation method based on superpixel and watershed

Multiphoton microscopy (MPM) imaging technique based on two‐photon excited fluorescence (TPEF) and second harmonic generation (SHG) shows fantastic performance for biological imaging. The automatic segmentation of cellular architectural properties for biomedical diagnosis based on MPM images is still a challenging issue. A novel multiphoton microscopy images segmentation method based on superpixels and watershed (MSW) is presented here to provide good segmentation results for MPM images. The proposed method uses SLIC superpixels instead of pixels to analyze MPM images for the first time. The superpixels segmentation based on a new distance metric combined with spatial, CIE Lab color space and phase congruency features, divides the images into patches which keep the details of the cell boundaries. Then the superpixels are used to reconstruct new images by defining an average value of superpixels as image pixels intensity level. Finally, the marker‐controlled watershed is utilized to segment the cell boundaries from the reconstructed images. Experimental results show that cellular boundaries can be extracted from MPM images by MSW with higher accuracy and robustness.

     

Volumetric imaging of erythrocytes using label‐free multiphoton photoacoustic microscopy

Photoacoustic microscopy (PAM) is an imaging modality well suited to mapping vasculature and other strong absorbers in tissue. However, one of the primary drawbacks to PAM when used for high‐resolution imaging is the relatively poor axial resolution due to the inverse dependence on the transducer bandwidth. While submicron lateral resolution PAM can be achieved by tightly focusing the excitation light, the axial resolution is fundamentally limited to 10s of microns for typical transducer frequencies. Here we present a multiphoton PAM technique called transient absorption ultrasonic microscopy (TAUM), which results in a completely optically resolved voxel with an experimentally measured axial resolution of 1.5 microns. This technique is demonstrated by imaging individual red blood cells in three dimensions in blood smear and ex vivo tissues. To the best of our knowledge, this is the first demonstration of fully resolved, volumetric photoacoustic imaging of erythrocytes. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Detection of pathological response of axillary lymph node metastasis after neoadjuvant chemotherapy in breast cancer using multiphoton microscopy

Neoadjuvant treatment is often considered in breast cancer patients with axillary lymph node involvement, but most of patients do not have a pathologic complete response to therapy. The detection of residual nodal disease has a significant impact on adjuvant therapy recommendations which may improve survival. Here, we investigate whether multiphoton microscopy (MPM) could identify the pathological changes of axillary lymphatic metastasis after neoadjuvant chemotherapy in breast cancer. And furthermore, we find that there are obvious differences in seven collagen morphological features between normal node and residual axillary disease by combining with a semi-automatic image processing method, and also find that there are significant differences in four collagen features between the effective and no-response treatment groups. These research results indicate that MPM may help estimate axillary treatment response in the neoadjuvant setting and thereby tailor more appropriate and personalized adjuvant treatments for breast cancer patients.     

Self‐referenced axial chromatic dispersion measurement in multiphoton microscopy through 2‐color third‐harmonic generation imaging

With tunable excitation light, multiphoton microscopy is widely used for imaging biological structures at subcellular resolution. Axial chromatic dispersion, present in virtually every transmissive optical system including the multiphoton microscope, leads to focal (and the resultant image) plane separation. Here, we experimentally demonstrate a technique to measure the axial chromatic dispersion in a multiphoton microscope, using simultaneous 2‐color third‐harmonic generation imaging excited by a 2‐color soliton source with tunable wavelength separation. Our technique is self‐referenced, eliminating potential measurement error when 1‐color tunable excitation light is used which necessitates reciprocating motion of the mechanical translation stage. Using this technique, we demonstrate measured axial chromatic dispersion with 2 different objective lenses in a multiphoton microscope. Further measurement in a biological sample also indicates that this axial chromatic dispersion, in combination with 2‐color imaging, may open up opportunity for simultaneous imaging of 2 different axial planes.      

Automated classification of hepatocellular carcinoma differentiation using multiphoton microscopy and deep learning

In the case of hepatocellular carcinoma (HCC) samples, classification of differentiation is crucial for determining prognosis and treatment strategy decisions. However, a label‐free and automated classification system for HCC grading has not been yet developed. Hence, in this study, we demonstrate the fusion of multiphoton microscopy and a deep‐learning algorithm for classifying HCC differentiation to produce an innovative computer‐aided diagnostic method. Convolutional neural networks based on the VGG‐16 framework were trained using 217 combined two‐photon excitation fluorescence and second‐harmonic generation images; the resulting classification accuracy of the HCC differentiation grade was over 90%. Our results suggest that a combination of multiphoton microscopy and deep learning can realize label‐free, automated methods for various tissues, diseases and other related classification problems.      

Quantitative assessment of microenvironment characteristics and metabolic activity in glioma via multiphoton microscopy

Tumor microenvironment and metabolic activity in gliomas are the important biomarkers to evaluate the progression of gliomas. Many evidences have suggested that the targeting of metabolic activity and tumor microenvironment simultaneously can be more effective to take the tumor therapy. Therefore, the noninvasive, accurate assessment of tumor microenvironment and metabolic activity is quite important in clinical practice. Multiphoton microscopy (MPM), based on two‐photon‐excited fluorescence and second harmonic generation was performed on unstained glioma tissues. With our combined image analysis approaches, our research findings indicate that MPM is able to qualitatively and quantitatively describe the microenvironment characteristics in gliomas, such as collage deposition in extracellular matrix, lymphocyte infiltration and tumor angiogenesis, etc. Meanwhile, the metabolic activity can also be quantitatively evaluated by optical redox ratio, NADH and FAD intensity. With the microendoscope and fiberscope are portable, MPM technique can be used to perform in‐vivo studies and clinical examinations in gliomas.      

Label-free detection of invasive micropapillary carcinoma of the breast using multiphoton microscopy

Invasive micropapillary carcinoma of the breast (IMPC) is a rare form of breast cancer with unique histological features, and is associated with high axillary lymph node metastasis and poor clinical prognosis. Thus, IMPC should be diagnosed in time to improve the treatment and management of patients. In this study, multiphoton microscopy (MPM) is used to label-free visualize the morphological features of IMPC. Our results demonstrate that MPM images are well in agreement with hematoxylin and eosin staining and epithelial membrane antigen staining, indicating MPM is comparable to traditional histological analysis in identifying the tissue structure and cell morphology. Statistical analysis shows significant differences in the circumference and area of the glandular lumen and cancer nest between the different IMPC cell clusters with complete glandular lumen morphology, and also shows difference in collagen length, width, and orientation, indicating the invasive ability of different morphologies of IMPC may be different.     

High contrast imaging and flexible photomanipulation for quantitative in vivo multiphoton imaging with polygon scanning microscope

In this study, we introduce two key improvements that overcome limitations of existing polygon scanning microscopes while maintaining high spatial and temporal imaging resolution over large field of view (FOV). First, we proposed a simple and straightforward means to control the scanning angle of the polygon mirror to carry out photomanipulation without resorting to high speed optical modulators. Second, we devised a flexible data sampling method directly leading to higher image contrast by over 2‐fold and digital images with 100 megapixels (10 240 × 10 240) per frame at 0.25 Hz. This generates sub‐diffraction limited pixels (60 nm per pixels over the FOV of 512 μm) which increases the degrees of freedom to extract signals computationally. The unique combined optical and digital control recorded fine fluorescence recovery after localized photobleaching (r ~10 μm) within fluorescent giant unilamellar vesicles and micro‐vascular dynamics after laser‐induced injury during thrombus formation in vivo. These new improvements expand the quantitative biological‐imaging capacity of any polygon scanning microscope system.

     

Perfectly registered multiphoton and reflectance confocal video rate imaging of in vivo human skin

We present a multimodal in vivo skin imaging instrument that is capable of simultaneously acquiring multiphoton and reflectance confocal images at up to 27 frames per second with 256 × 256 pixel resolution without the use of exogenous contrast agents. A single femtosecond laser excitation source is used for all channels ensuring perfect image registration between the two‐photon fluorescence (TPF), second harmonic generation (SHG), and reflectance confocal microscopy (RCM) images. Images and videos acquired with the system show that the three imaging channels provide complementary information in in vivo human skin measurements. In the epidermis, cell boundaries are clearly seen in the RCM channel, while cytoplasm is better seen in the TPF imaging channel, whereas in the dermis, SHG and TPF channels show collagen bundles and elastin fibers, respectively. The demonstrated fast imaging speed and multimodal imaging capabilities of this MPM/RCM instrument are essential features for future clinical application of this technique. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Tunable fibre‐coupled multiphoton microscopy with a negative curvature fibre

Negative curvature fibre (NCF) guides light in its core by inhibiting the coupling of core and cladding modes. In this work, an NCF was designed and fabricated to transmit ultrashort optical pulses for multiphoton microscopy with low group velocity dispersion (GVD) at 800 nm. Its attenuation was measured to be <0.3 dB m^–1 over the range 600–850 nm and the GVD was –180 ± 70 fs² m^–1 at 800 nm. Using an average fibre output power of ～20 mW and pulse repetition rate of 80 MHz, the NCF enabled pulses with a duration of <200 fs to be transmitted through a length of 1.5 m of fibre over a tuning range of 180 nm without the need for dispersion compensation. In a 4 m fibre, temporal and spectral pulse widths were maintained to within 10% of low power values up to the maximum fibre output power achievable with the laser system used of 278 mW at 700 nm, 808 mW at 800 nm and 420 mW at 860 nm. When coupled to a multiphoton microscope, it enabled imaging of ex vivo tissue using excitation wavelengths from 740 nm to 860 nm without any need for adjustments to the set‐up.

     

In vivo multiphoton microscopy of melasma

Melasma is a skin disorder characterized by hyperpigmented patches due to increased melanin production and deposition. In this pilot study, we evaluate the potential of multiphoton microscopy (MPM) to characterize non‐invasively the melanin content, location, and distribution in melasma and assess the elastosis severity. We employed a clinical MPM tomograph to image in vivo morphological features in melasma lesions and adjacent normal skin in 12 patients. We imaged dermal melanophages in most dermal melasma lesions and occasionally in epidermal melasma. The melanin volume fraction values measured in epidermal melasma (14% ± 4%) were significantly higher (p < 0.05) than the values measured in perilesional skin (11% ± 3%). The basal keratinocytes of melasma and perilesions showed different melanin distribution. Elastosis was predominantly more severe in lesions than in perilesions and was associated with changes in melanin distribution of the basal keratinocytes. These results demonstrate that MPM may be a non‐invasive imaging tool for characterizing melasma.     

In vivo multiphoton microscopy using a handheld scanner with lateral and axial motion compensation

This paper reports a handheld multiphoton fluorescence microscope designed for clinical imaging that incorporates axial motion compensation and lateral image stabilization. Spectral domain optical coherence tomography is employed to track the axial position of the skin surface, and lateral motion compensation is realised by imaging the speckle pattern arising from the optical coherence tomography beam illuminating the sample. Our system is able to correct lateral sample velocities of up to approximately 65 μm s^?1. Combined with the use of negative curvature microstructured optical fibre to deliver tunable ultrafast radiation to the handheld multiphoton scanner without the need of a dispersion compensation unit, this instrument has potential for a range of clinical applications. The system is used to compensate for both lateral and axial motion of the sample when imaging human skin in vivo.      

Intravital multiphoton photoconversion with a cell membrane dye

Photoconversion, an irreversible shift in a fluorophore emission spectrum after light exposure, is a powerful tool for marking cellular and subcellular compartments and tracking their dynamics in vivo. This paper reports on the photoconversion properties of Di‐8‐ANEPPS, a commercially available membrane dye. When illuminated with near‐infrared femtosecond laser pulses, Di‐8‐ANEPPS undergoes multiphoton photoconversion as indicated by the supralinear dependence of the conversion rate ρ_pc on the incident power (), and by the ability to photoconvert a thin optical section in a three‐dimensional matrix. The characteristic emission spectrum changed from red to blue, and ratiometric analysis on single cells in vitro revealed a 65‐fold increase in the blue to red wavelength ratio after photoconversion. The spectral shift is preserved in vivo for hours, making Di‐8‐ANEPPS a useful dye for intravital cell marking and tracking applications.

     

Live cell imaging by multifocal multiphoton microscopy

Multifocal multiphoton microscopy (MMM) permits parallel multiphoton excitation by scanning an array of high numerical aperture foci across a plane in the sample. MMM is particularly suitable for live cell investigations since it combines advantages of standard multiphoton microscopy such as optical sectioning and suppression of out-of-focus phototoxicity with high recording speeds. Here we describe several applications of MMM to live cell imaging using the neuroendocrine cell line PC12 and bovine chromaffin cells. Stainings were performed with the acidophilic dye acridine orange and the lipophilic dyes FM1-43 and Fast DiA as well as by transfection of the cells with GFP. In both bovine chromaffin and PC12 cells structural elements of nuclear chromatin and the 3-D distribution of acidic organelles inside the cells were visualized. In PC12 cells differentiated by nerve growth factor examples of neurites were monitored. Stainings of membranes were used to reconstruct the morphology of cells and neurites in three dimensions by volume-rendering and by isosurface plots. 3-D reconstructions were composed from stacks of about 50 images each with a diameter of 30-100 microm that were acquired within a few seconds. We conclude that MMM proves to be a technically simple and very effective method for fast 3-D live cell imaging at high resolution.     

Automatic and label‐free identification of blood vessels in gliomas using the combination of multiphoton microscopy and image analysis

Currently, the targeted treatment of tumor based on the tumor microenvironment is newly developed. Blood vessels are the key parts in the tumor microenvironment, which is taken as a new visible target for tumor therapy. Multiphoton microscopy (MPM), based on the second harmonic generation and two‐photon excited fluorescence, is available to make the label‐free analysis on the blood vessels in human gliomas. MPM can reveal the vascular morphological characteristics in gliomas, including vascular malformation, intense vascular proliferation, perivascular collagen deposition, perivascular lymphocytes aggregation and microvascular proliferation. In addition, the image analysis algorithms were developed to automatically calculate the perivascular collagen content, vascular cavity area, lumen area, wall area and vessel number. Thus, the vascular morphology, the perivascular collagen deposition and intense vascular proliferation degree can be further quantitatively characterized. Compared with the pathological analysis, the combination of MPM and image analysis has potential advantages in making a quantitative and qualitative analyzing on vascular morphology in glioma microenvironment. As micro‐endoscope and two‐photon fiberscope are technologically improved, this combined method will be a useful imaging way to make the real‐time research on the targeting tumor microenvironment in gliomas.     

Dynamic multiphoton imaging of acellular dermal matrix scaffolds seeded with mesenchymal stem cells in diabetic wound healing

Significantly effective therapies need to be developed for chronic nonhealing diabetic wounds. In this work, the topical transplantation of mesenchymal stem cell (MSC) seeded on an acellular dermal matrix (ADM) scaffold is proposed as a novel therapeutic strategy for diabetic cutaneous wound healing. GFP‐labeled MSCs were cocultured with an ADM scaffold that was decellularized from normal mouse skin. These cultures were subsequently transplanted as a whole into the full‐thickness cutaneous wound site in streptozotocin‐induced diabetic mice. Wounds treated with MSC‐ADM demonstrated an increased percentage of wound closure. The treatment of MSC‐ADM also greatly increased angiogenesis and rapidly completed the reepithelialization of newly formed skin on diabetic mice. More importantly, multiphoton microscopy was used for the intravital and dynamic monitoring of collagen type I (Col‐I) fibers synthesis via second harmonic generation imaging. The synthesis of Col‐I fibers during diabetic wound healing is of great significance for revealing wound repair mechanisms. In addition, the activity of GFP‐labeled MSCs during wound healing was simultaneously traced via two‐photon excitation fluorescence imaging. Our research offers a novel advanced nonlinear optical imaging method for monitoring the diabetic wound healing process while the ADM and MSCs interact in situ. Schematic of dynamic imaging of ADM scaffolds seeded with mesenchymal stem cells in diabetic wound healing using multiphoton microscopy. PMT, photo‐multiplier tube.      

Clinical multiphoton tomography

Clinical multiphoton tomography and two‐photon microendoscopy provide clinicians and researchers with high‐resolution in vivo optical biopsies based on two‐photon autofluorescence, second harmonic generation, and fluorescence lifetime imaging. This review reflects state of the art technology and reports on applications in the fields of early stage melanoma detection, skin aging, nanoparticle imaging, tissue engineering, and in situ screening of pharmaceutical and cosmetical products. So far, more than 500 patients and volunteers in Europe, Asia, and Australia have been investigated with these novel molecular imaging tools. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Epithelium segmentation and automated Gleason grading of prostate cancer via deep learning in label‐free multiphoton microscopic images

In the current clinical care practice, Gleason grading system is one of the most powerful prognostic predictors for prostate cancer (PCa). The grading system is based on the architectural pattern of cancerous epithelium in histological images. However, the standard procedure of histological examination often involves complicated tissue fixation and staining, which are time‐consuming and may delay the diagnosis and surgery. In this study, label‐free multiphoton microscopy (MPM) was used to acquire subcellular‐resolution images of unstained prostate tissues. Then, a deep learning architecture (U‐net) was introduced for epithelium segmentation of prostate tissues in MPM images. The obtained segmentation results were then merged with the original MPM images to train a classification network (AlexNet) for automated Gleason grading. The developed method achieved an overall pixel accuracy of 92.3% with a mean F1 score of 0.839 for epithelium segmentation. By merging the segmentation results with the MPM images, the accuracy of Gleason grading was improved from 72.42% to 81.13% in hold‐out test set. Our results suggest that MPM in combination with deep learning holds the potential to be used as a fast and powerful clinical tool for PCa diagnosis.