Measurements of multiphoton action cross sections for multiphoton microscopy

We report quantitative measurements of two-, three-, and four-photon excitation action cross sections of several commonly used fluorophores and fluorescent proteins at three different excitation wavelengths of 800 nm, 1300 nm, and 1680 nm. The measured cross section values are consistent with simple quantum mechanic estimations. These values indicate that the optimum repetition rate for deep tissue 3-photon microscopy is approximately 1 to 2 MHz. We further demonstrate that it is feasible to perform 4-photon fluorescence microscopy of GFP labeled microglia in mouse brain in vivo at 1700 nm. 4-photon excitation increases the accessibility of fluorophores at the long wavelength spectral window of 1700 nm.OCIS codes: (190.0190) Nonlinear optics, (190.4180) Multiphoton processes     

Principles of multiphoton microscopy

Multiphoton fluorescence microscopy is a powerful, important tool in biomedical research that offers low photon toxicity and higher spatial and temporal resolution than other in vivo imaging modalities. The capability to collect images hundreds of micrometers into biological tissues provides an invaluable tool for studying cellular and subcellular processes in the context of tissues and organs in living animals. Multiphoton microscopy is based upon two-photon excitation of fluorescence that occurs only in a sub-femtoliter volume at the focus; by scanning the focus through a sample, 2- and 3-dimensional images can be collected. The complex 3-dimensional organization of the kidney makes it especially appropriate for multiphoton microscopic analysis, which has been used to characterize numerous aspects of renal physiology and pathophysiology in living rats and mice. However, the ability to collect fluorescence images deep into biological tissues raises unique problems not encountered in other forms of optical microscopy, including issues of probe access, and tissue optics. Future improvements in multiphoton fluorescence microscopy will involve optimizing objectives for the unique characteristics of multiphoton fluorescence imaging, improving the speed at which images may be collected and extending the depth to which imaging may be conducted.     

Remote focusing for programmable multi-layer differential multiphoton microscopy

We present the application of remote focusing to multiphoton laser scanning microscopy and utilize this technology to demonstrate simultaneous, programmable multi-layer imaging. Remote focusing is used to independently control the axial location of multiple focal planes that can be simultaneously imaged with single element detection. This facilitates volumetric multiphoton imaging in scattering specimens and can be practically scaled to a large number of focal planes. Further, it is demonstrated that the remote focusing control can be synchronized with the lateral scan directions, enabling imaging in orthogonal scan planes.     

Femtosecond diode-based time lens laser for multiphoton microscopy

We demonstrate a near-infrared, femtosecond, diode laser-based source with kW peak power for two-photon microscopy. At a wavelength of 976 nm, the system produces sub-ps pulses operating at a repetition rate of 10 MHz with kilowatt class peak powers suitable for deep tissue two-photon microscopy. The system, integrated with a laser-scanning microscope, images to a depth of 900 µm in a fixed sample of PLP-eGFP labeled mouse brain tissue. This represents a significant development that will lead to more efficient, compact, and accessible laser sources for biomedical imaging.     

A coherent model for turbid imaging with confocal microscopy

We present an engineering model of coherent imaging within a turbid volume, such as human tissues, with a confocal microscope. The model is built to analyze the statistical effect of aberrations and multiply scattered light on the resulting image. Numerical modeling of theory is compared with experimental results. We describe the construction of a stable phantom that represents the statistical effect of object turbidity on the image recorded. The model and phantom can serve as basis for system optimization in turbid imaging.OCIS codes: (110.0113) Imaging through turbid media, (170.1790) Confocal microscopy, (170.3660) Light propagation in tissues     

Three-dimensional-generator U-net for dual-resonant scanning multiphoton microscopy image inpainting and denoising

A dual-resonant scanning multiphoton (DRSM) microscope incorporating a tunable acoustic gradient index of refraction lens and a resonant mirror is developed for rapid volumetric bioimaging. It is shown that the microscope achieves a volumetric imaging rate up to 31.25 volumes per second (vps) for a scanning volume of up to 200 × 200 × 100 µm³ with 256 × 256 × 128 voxels. However, the volumetric images have a severe negative signal-to-noise ratio (SNR) as a result of a large number of missing voxels for a large scanning volume and the presence of Lissajous patterning residuals. Thus, a modified three-dimensional (3D)-generator U-Net model trained using simulated microbead images is proposed and used to inpaint and denoise the images. The performance of the 3D U-Net model for bioimaging applications is enhanced by training the model with high-SNR in-vitro drosophila brain images captured using a conventional point scanning multiphoton microscope. The trained model shows the ability to produce clear in-vitro drosophila brain images at a rate of 31.25 vps with a SNR improvement of approximately 20 dB over the original images obtained by the DRSM microscope. The training convergence time of the modified U-Net model is just half that of a general 3D U-Net model. The model thus has significant potential for 3D in-vivo bioimaging transfer learning. Through the assistance of transfer learning, the model can be extended to the restoration of in-vivo drosophila brain images with a high image quality and a rapid training time.     

Light-field microscopy with temporal focusing multiphoton illumination for scanless volumetric bioimaging

A temporal focusing multiphoton illumination (TFMI) method is proposed for achieving selective volume illumination (SVI) (i.e., illuminating only the volume of interest) in light-field microscopy (LFM). The proposed method minimizes the background noise of the LFM images and enhances the contrast, and thus improves the imaging quality. Three-dimensional (3D) volumetric imaging is achieved by reconstructing the LFM images using a phase-space deconvolution algorithm. The experimental results obtained using 100-nm fluorescent beads show that the proposed TFMI-LFM system achieves lateral and axial resolutions of 1.2 µm and 1.1 µm, respectively, at the focal plane. Furthermore, the TFMI-LFM system enables 3D images of the single lobe of the drosophila mushroom body with GFP biomarker (OK-107) to be reconstructed in a one-snapshot record.     

Diamond Raman laser and Yb fiber amplifier for in vivo multiphoton fluorescence microscopy

Here we introduce a fiber amplifier and a diamond Raman laser that output high powers (6.5 W, 1.3 W) at valuable wavelengths (1060 nm, 1250 nm) for two-photon excitation of red-shifted fluorophores. These custom excitation sources are both simple to construct and cost-efficient in comparison to similar custom and commercial alternatives. Furthermore, they operate at a repetition rate (80 MHz) that allows fast image acquisition using resonant scanners. With our system we demonstrate compatibility with fast resonant scanning, the ability to acquire neuronal images, and the capability to image vasculature at deep locations (>1 mm) within the mouse cerebral cortex.     

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.     

Label-free monitoring of colonic cancer progression using multiphoton microscopy

Real-time histology or virtual biopsy for the diagnosis of colonic cancer is of great medical significance. In this work, we show that label-free multiphoton imaging is feasible and effective in monitoring colonic cancer progression by providing cellular and subcellular details in fresh, unfixed, unstained colonic specimens. Our results also demonstrate the capability of using tissue quantitative analysis of the redox ratio for quantifying colonic cancer progression. These results suggest that multiphoton microscopy has potential to become an in situ histological tool, which is free from the labeling requirement of conventional methods, for the early diagnosis and detection of malignant lesions in the colon.     

Retinal cell imaging in myopic chickens using adaptive optics multiphoton microscopy

Abnormal eye growth induced by visual deprivation can modify the structure and density of the retinal cells. We have used an adaptive optics multiphoton microscope to image photoreceptors (PRs) and ganglion cells (GCs) at different retinal locations in unstained retinas of chicken eyes with about 10D of myopia and their normal-sighted fellow eyes. In all samples, the local averaged inter-PR distance increased with eccentricity. No significant differences in PR density were found between control and myopic eyes. GC density declined in myopic eyes compared to control eyes and the inter-cell distance increased. In normal eyes, the size of the GC cell bodies increased approximately two-fold between the area centralis and the peripheral retina. In myopic eyes, this trend was preserved but the GC bodies were larger at each retinal location, compared to control eyes. Obviously, GC morphology is changing when the retinal area is enlarged in myopic eyes.OCIS codes: (170.3880) Medical and biological imaging, (180.4315) Nonlinear microscopy, (110.1080) Active or adaptive optics, (170.4470) Ophthalmology     

Characterization of multiphoton microscopy in the bone marrow following intravital laser osteotomy

The bone marrow is an important site where all blood cells are formed from hematopoietic stem cells and where hematologic malignancies such as leukemia emerge. It is also a frequent site for metastasis of solid tumors such as breast cancer and prostate cancer. Intravital microscopy is a powerful tool for studying the bone marrow with single cell and sub-cellular resolution. To improve optical access to this rich biological environment, plasma-mediated laser ablation with sub-microjoule femtosecond pulses was used to thin cortical bone. By locally removing a superficial layer of bone (local laser osteotomy), significant improvements in multiphoton imaging were observed in individual bone marrow compartments in vivo. This work demonstrates the utility of scanning laser ablation of hard tissue with sub-microjoule pulses as a preparatory step to imaging.OCIS codes: (120.0120) Instrumentation, measurement, and metrology; (170.1020) Ablation of tissue; (180.5810) Scanning microscopy     

Photo-induced processes in collagen-hypericin system revealed by fluorescence spectroscopy and multiphoton microscopy

Collagen is the main structural protein and the key determinant of mechanical and functional properties of tissues and organs. Proper balance between synthesis and degradation of collagen molecules is critical for maintaining normal physiological functions. In addition, collagen influences tumor development and drug delivery, which makes it a potential cancer therapy target. Using second harmonic generation, two-photon excited fluorescence microscopy, and spectrofluorimetry, we show that the natural pigment hypericin induces photosensitized destruction of collagen-based tissues. We demonstrate that hypericin–mediated processes in collagen fibers are irreversible and may be used for the treatment of cancer and collagen-related disorders.OCIS codes: (190.1900) Diagnostic applications of nonlinear optics, (170.3880) Medical and biological imaging, (180.4315) Nonlinear microscopy     

Real-time histology in liver disease using multiphoton microscopy with fluorescence lifetime imaging

Conventional histology with light microscopy is essential in the diagnosis of most liver diseases. Recently, a concept of real-time histology with optical biopsy has been advocated. In this study, live mice livers (normal, with fibrosis, steatosis, hepatocellular carcinoma and ischemia-reperfusion injury) were imaged by MPM-FLIM for stain-free real-time histology. The acquired MPM-FLIM images were compared with conventional histological images. MPM-FLIM imaged subsurface cellular and subcellular histopathological hallmarks of live liver in mice models at high resolution. Additional information such as distribution of stellate cell associated autofluorescence and fluorescence lifetime changes was also gathered by MPM-FLIM simultaneously, which cannot be obtained from conventional histology. MPM-FLIM could simultaneously image and quantify the cellular morphology and microenvironment of live livers without conventional biopsy or fluorescent dyes. We anticipate that in the near future MPM-FLIM will be evaluated from bench to bedside, leading to real-time histology and dynamic monitoring of human liver diseases.OCIS codes: (180.4315) Nonlinear microscopy, (170.3880) Medical and biological imaging, (170.6510) Spectroscopy, tissue diagnostics     

Femtosecond infrared intrastromal ablation and backscattering-mode adaptive-optics multiphoton microscopy in chicken corneas

The performance of femtosecond (fs) laser intrastromal ablation was evaluated with backscattering-mode adaptive-optics multiphoton microscopy in ex vivo chicken corneas. The pulse energy of the fs source used for ablation was set to generate two different ablation patterns within the corneal stroma at a certain depth. Intrastromal patterns were imaged with a custom adaptive-optics multiphoton microscope to determine the accuracy of the procedure and verify the outcomes. This study demonstrates the potential of using fs pulses as surgical and monitoring techniques to systematically investigate intratissue ablation. Further refinement of the experimental system by combining both functions into a single fs laser system would be the basis to establish new techniques capable of monitoring corneal surgery without labeling in real-time. Since the backscattering configuration has also been optimized, future in vivo implementations would also be of interest in clinical environments involving corneal ablation procedures.     

In-vivo imaging of psoriatic lesions with polarization multispectral dermoscopy and multiphoton microscopy

Psoriasis is a skin autoimmune disease characterized by hyperkeratosis, hyperproliferation of the epidermis and dilatation of dermal papillary blood vessels. Healthy skin (5 volunteers) and psoriatic lesions (3 patients) were visualized in vivo, with high contrast and resolution, with a Polarization Multispectral Dermoscope and a Multiphoton Microscope. Psoriatic features were identified and quantified. The effective diameter of the superficial blood vessels was measured at 35.2 ± 7.2 μm and the elongated dermal papillae had an effective diameter of 64.2 ± 22.6 μm. The methodologies developed could be employed for quantitative diagnostic purposes and furthermore serve as a monitoring method of the effect of personalized treatments.OCIS codes: (180.4315) Nonlinear microscopy, (170.1870) Dermatology, (100.2980) Image enhancement, (170.6900) Three-dimensional microscopy, (170.6510) Spectroscopy, tissue diagnostics     

Active image optimization for lattice light sheet microscopy in thick samples

Lattice light-sheet microscopy (LLSM) is a very efficient technique for high resolution 3D imaging of dynamic phenomena in living biological samples. However, LLSM imaging remains limited in depth due to optical aberrations caused by sample-based refractive index mismatch. Here, we propose a simple and low-cost active image optimization (AIO) method to recover high resolution imaging inside thick biological samples. AIO is based on (1) a light-sheet autofocus step (AF) followed by (2) an adaptive optics image-based optimization. We determine the optimum AIO parameters to provide a fast, precise and robust aberration correction on biological samples. Finally, we demonstrate the performances of our approach on sub-micrometric structures in brain slices and plant roots.     

Deep-skin multiphoton microscopy of lymphatic vessels excited at the 1700-nm window in vivo

Visualization of lymphatic vessels is key to the understanding of their structure, function, and dynamics. Multiphoton microscopy (MPM) is a potential technology for imaging lymphatic vessels, but tissue scattering prevents its deep penetration in skin. Here we demonstrate deep-skin MPM of the lymphatic vessels in mouse hindlimb in vivo, excited at the 1700 nm window. Our results show that with contrast provided by indocyanine green (ICG), 2-photon fluorescence (2PF) imaging enables noninvasive imaging of lymphatic vessels 300 μm below the skin surface, visualizing both its structure and contraction dynamics. Simultaneously acquired second-harmonic generation (SHG) and third-harmonic generation (THG) images visualize the local environment in which the lymphatic vessels reside. After removing the surface skin layer, 2PF and THG imaging visualize finer structures of the lymphatic vessels: most notably, the label-free THG imaging visualizes lymphatic valves and their open-and-close dynamics in real time. MPM excited at the 1700-nm window thus provides a promising technology for the study of lymphatic vessels.     

Label-free imaging of age-related cardiac structural changes in non-human primates using multiphoton nonlinear microscopy

Heart failure is one of the most common causes of morbidity and mortality. Both maturational abnormalities and age-associated cardiac pathologies contribute to heart failure. Imaging-based assessment to discern detailed cardiac structure at various maturational stages is imperative for understanding mechanisms behind cardiac growth and aging. Using multiphoton nonlinear optical microscopy (NLOM) based label-free imaging, we investigated cardiac structural composition in a human-relevant aging model, the common marmoset monkey (Callithrix jacchus). Animals were divided into three different age groups including neonatal, young adult and old. By devising a unique strategy for segregating collagen and myosin emitted second harmonic generation (SHG) signals, we performed a volumetric assessment of collagen and total scattering tissue (collagen + myosin). Aged marmoset hearts exhibited an increase in collagen and total scattering tissue volume at the sites of severe tissue remodelling indicating age-related cardiac fibrosis. Significantly low scattering tissue volume in neonatal marmoset hearts was attributed to a lack of binding between the myofibrils in maturing cardiac tissue. Comprehensive quantitative assessment of structural composition during maturation and aging of marmoset hearts revealed significant differences in myofibril length, alignment, curvature and angular distribution. In conclusion, label-free high-resolution NLOM facilitates visualization and quantification of subcellular structural features for understanding vital age-related morphological alterations in the marmoset heart.