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Light attenuation in thick biological tissues, caused by a combination of absorption and scattering, limits the penetration depth in multiphoton microscopy (MPM). Both tissue scattering and absorption are dependent on wavelengths, which makes it essential to choose the excitation wavelength with minimum attenuation for deep imaging. Although theoretical models have been established to predict the wavelength dependence of light attenuation in brain tissues, the accuracy of these models in experimental settings needs to be verified. Furthermore, the water absorption contribution to the tissue attenuation, especially at 1450 nm where strong water absorption is predicted to be the dominant contributor in light attenuation, has not been confirmed. Here we performed a systematic study of in vivo three-photon imaging at different excitation wavelengths, 1300 nm, 1450 nm, 1500 nm, 1550 nm, and 1700 nm, and quantified the tissue attenuation by calculating the effective attenuation length at each wavelength. The experimental data show that the effective attenuation length at 1450 nm is significantly shorter than that at 1300 nm or 1700 nm. Our results provide unequivocal validation of the theoretical estimations based on water absorption and tissue scattering in predicting the effective attenuation lengths for long wavelength in vivo imaging.
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The clear and accurate understanding of the degree of hepatocellular-carcinoma (HCC) differentiation plays a key role in the determination of the patient prognosis and development of a treatment plan by the clinician. However, label-free and automated classification of the HCC grading is challenging. Here, we demonstrate second-harmonic generation (SHG) microscopy for label-free classification of HCC grading in paraffin-embedded specimens. A total of 217 images from 113 patients were obtained using SHG microscopy, and the SHG signals from the collagen within the tumor were analyzed using feature extraction and selection, the Mann-Whitney test, and the receiver operating characteristic curves. The results exhibit good correlation between the software analysis and the diagnosis by experienced pathologists. Combining the image features and clinical information, an adaptive quantification algorithm is generated for automatically determining the HCC grade. The results suggest that SHG microscopy might be a promising automated diagnostic method for clinical use, without requiring time for tissue processing and staining.
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Quantitative second harmonic generation microscopy was used to investigate collagen organization in the fibrillar capsules of human benign and malignant thyroid nodules. We demonstrate that the combination of texture analysis and second harmonic generation images of collagen can be used to differentiate between capsules surrounding the thyroid follicular adenoma and papillary carcinoma nodules. Our findings indicate that second harmonic generation microscopy can provide quantitative information about the collagenous capsule surrounding both the thyroid and thyroid nodules, which may complement traditional histopathological examination.
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We developed a compact stimulated emission depletion (STED) two-photon excitation microscopy that utilized electrically controllable components. Transmissive liquid crystal devices inserted directly in front of the objective lens converted the STED light into an optical vortex while leaving the excitation light unaffected. Light pulses of two different colors, 1.06 and 0.64 µm, were generated by laser diode-based light sources, and the delay between the two pulses was flexibly controlled so as to maximize the fluorescence suppression ratio. In our experiments, the spatial resolution of this system was up to three times higher than that obtained without STED light irradiation, and we successfully visualize the fine microtubule network structures in fixed mammalian cells without causing significant photo-damage.
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[This corrects the article on p. 3735 in vol. 8, PMID: 28856046.].
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The femtosecond laser ablation in biological tissue produces highly fluorescent compounds that are of great significance for intrinsically labelling ablated tissue in vivo and achieving imaging-guided laser microsurgery. In this study, we analyzed the molecular structures of femtosecond laser-ablated tissues using Raman spectroscopy and transmission electron microscopy. The results showed that though laser ablation caused carbonization, no highly fluorescent nanostructures were found in the ablated tissues. Further, we found that the fluorescence properties of the newly formed compounds were spatially heterogeneous across the ablation site and the dominant fluorescent signals exhibited close similarity to the tissue directly heated at a temperature of 200 °C. The findings of our study indicated that the new fluorescent compounds were produced via the laser heating effect and their formation mechanism likely originated from the Maillard reaction, a chemical reaction between amino acids and reducing sugars in tissue.
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A fluorescence lifetime imaging microscopy (FLIM) integrated with two-photon excitation technique was developed. A wavelength-tunable femtosecond pulsed laser with nominal pulse repetition rate of 76-MHz was used to acquire FLIM images with a high pixel rate of 3.91 MHz by processing the pulsed two-photon fluorescence signal. Analog mean-delay (AMD) method was adopted to accelerate the lifetime measurement process and to visualize lifetime map in real-time. As a result, rapid tomographic visualization of both structural and chemical properties of the tissues was possible with longer depth penetration and lower photo-damage compared to the conventional single-photon FLIM techniques.
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Deep tissue multiphoton imaging requires high peak power to enhance signal and low average power to prevent thermal damage. Both goals can be advantageously achieved through laser repetition rate tuning instead of simply adjusting the average power. We show that the ideal repetition rate for deep two-photon imaging in the mouse brain is between 1 and 10 MHz, and we present a fiber-based source with an arbitrarily tunable repetition rate within this range. The performance of the new source is compared to a mode-locked Ti:Sapphire (Ti:S) laser for in vivo imaging of mouse brain vasculature. At 2.5 MHz, the fiber source requires 5.1 times less average power to obtain the same signal as a standard Ti:S laser operating at 80 MHz.
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Ischemic stroke is a leading cause of death and permanent disability worldwide. Middle cerebral artery occlusion (MCAO) of variable duration times could be anticipated to result in varying degrees of injury that evolve spatially over time. Therefore, investigations following strokes require information concerning the spatiotemporal dimensions of the ischemic core as well as of perilesional areas. In the present study, multiphoton microscopy (MPM) based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) was applied to image such pathophysiological events. The ischemic time-points for evaluation were set at 6, 24, 48, and 72 hours after MCAO. Our results demonstrated that MPM has the ability to not only identify the normal and ischemic brain regions, but also reveal morphological changes of the cortex and striatum at various times following permanent MCAO. These findings corresponded well with the hematoxylin and eosin (H&E) stained tissue images. With the technologic progression of miniaturized imaging devices, MPM can be developed into an effective diagnostic and monitoring tool for ischemic stroke.
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We present the design, implementation and performance analysis of a compact multi-photon endoscope based on a piezo electric scanning tube. A miniature objective lens with a long working distance and a high numerical aperture (≈ 0.5) is designed to provide a diffraction limited spot size. Furthermore, a 1700 nm wavelength femtosecond fiber laser is used as an excitation source to overcome the scattering of biological tissues and reduce water absorption. Therefore, the novel optical system along with the unique wavelength allows us to increase the imaging depth. We demonstrate that the endoscope is capable of performing third and second harmonic generation (THG/SHG) and three-photon excitation fluorescence (3PEF) imaging over a large field of view (> 400 µm) with high lateral resolution (2.2 µm). The compact and lightweight probe design makes it suitable for minimally-invasive in-vivo imaging as a potential alternative to surgical biopsies.
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Acquiring images of biological tissues and cells without the assistance of exogenous labels with a fast repetition rate and chemical specificity is what coherent anti-Stokes Raman Scattering (CARS) imaging offers. Nonresonant background (NRB) is one of the main drawbacks of the CARS microscopy technique because it limits the detection of weak Raman lines and the detection of low-concentration molecules. We show that a six-wave mixing process with two beams, which is a cascade effect of CARS, show better signal/NRB ratio and can be utilized for biological tissues imaging. The cascade CARS (CCARS) depends on chi-3 to the fourth power, instead of chi-3 squared as in the usual CARS signal; therefore, the contrast ratio with NRB is higher for CCARS than for CARS. We present analytic calculations showing that CCARS have better contrast over CARS in any situation. Comparison of the signals of both techniques generated on water-ethanol solutions confirm these results. Finally, we acquired CCARS images of fresh biological tissues, attesting that it is a useful tool for biological studies.
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The ability to histologically assess surgical specimens in real-time is a long-standing challenge in cancer surgery, including applications such as breast conserving therapy (BCT). Up to 40% of women treated with BCT for breast cancer require a repeat surgery due to postoperative histological findings of close or positive surgical margins using conventional formalin fixed paraffin embedded histology. Imaging technologies such as nonlinear microscopy (NLM), combined with exogenous fluorophores can rapidly provide virtual H&E imaging of surgical specimens without requiring microtome sectioning, facilitating intraoperative assessment of margin status. However, the large volume of typical surgical excisions combined with the need for rapid assessment, make comprehensive cellular resolution margin assessment during surgery challenging. To address this limitation, we developed a multiscale, real-time microscope with variable magnification NLM and real-time, co-registered position display using a widefield white light imaging system. Margin assessment can be performed rapidly under operator guidance to image specific regions of interest located using widefield imaging. Using simulated surgical margins dissected from human breast excisions, we demonstrate that multi-centimeter margins can be comprehensively imaged at cellular resolution, enabling intraoperative margin assessment. These methods are consistent with pathology assessment performed using frozen section analysis (FSA), however NLM enables faster and more comprehensive assessment of surgical specimens because imaging can be performed without freezing and cryo-sectioning. Therefore, NLM methods have the potential to be applied to a wide range of intra-operative applications.
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Super-resolution microscopy has become a powerful tool for biological research. However, its spatial resolution and imaging depth are limited, largely due to background light. Interferometric temporal focusing (ITF) microscopy, which combines structured illumination microscopy and three-photon excitation fluorescence microscopy, can overcome these limitations. Here, we demonstrate ITF microscopy using three-photon excitation fluorescence, which has a spatial resolution of 106 nm at an imaging depth of 100 µm with an excitation wavelength of 1060 nm.
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Super-resolution fluorescence microscopy is an important tool in biomedical research for its ability to discern features smaller than the diffraction limit. However, due to its difficult implementation and high cost, the super-resolution microscopy is not feasible in many applications. In this paper, we propose and demonstrate a saturation-based super-resolution fluorescence microscopy technique that can be easily implemented and requires neither additional hardware nor complex post-processing. The method is based on the principle of stepwise optical saturation (SOS), where M steps of raw fluorescence images are linearly combined to generate an image with a [Formula: see text]-fold increase in resolution compared with conventional diffraction-limited images. For example, linearly combining (scaling and subtracting) two images obtained at regular powers extends the resolution by a factor of 1.4 beyond the diffraction limit. The resolution improvement in SOS microscopy is theoretically infinite but practically is limited by the signal-to-noise ratio. We perform simulations and experimentally demonstrate super-resolution microscopy with both one-photon (confocal) and multiphoton excitation fluorescence. We show that with the multiphoton modality, the SOS microscopy can provide super-resolution imaging deep in scattering samples.
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Preoperative neoadjuvant treatment in locally advanced breast cancer is recognized as an effective adjuvant therapy, as it improves treatment outcomes. However, the potential complications remain a threat, so there is an urgent clinical need to assess both the tumor response and changes in its microenvironment using non-invasive and precise identification techniques. Here, two-photon microscopy was employed to detect morphological alterations in breast cancer progression and recession throughout chemotherapy. The changes in structure were analyzed based on the autofluorescence and collagen of differing statuses. Parameters, including optical redox ratio, the ratio of second harmonic generation and auto-fluorescence signal, collagen density, and collagen shape orientation, were studied. Results indicate that these parameters are potential indicators for evaluating breast tumors and their microenvironment changes during progression and chemotherapy. Combined analyses of these parameters could provide a quantitative, novel method for monitoring tumor therapy.
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Early identification of premalignant and malignant gastric mucosa is crucial to decrease the incidence and mortality of stomach cancer. Spectrum- and time-resolved multiphoton microscopy are capable of providing not only structural but also biochemical information at the subcellular level. Based on this multidimensional imaging technique, we performed a systematic investigation on fresh human tissue specimens at the typical stages of gastric carcinogenesis, including normal, chronic gastritis with erosion, chronic gastritis with intestinal metaplasia, and intestinal-type adenocarcinoma. The results demonstrate that this technique is available to characterize the three-dimensional subcellular morphological and biochemical properties of gastric mucosa and further provide quantitative indicators of different gastric disorders, by using endogenous contrast. With advances in multiphoton endoscopy, it has the potential to allow noninvasive, label-free, real-time histological and functional diagnosis of premalignant and malignant lesions of stomach in the future.
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Femtosecond laser microsurgery has become an advanced method for clinical procedures and biological research. The tissue treated by femtosecond laser can become highly fluorescent, indicating the formation of new fluorescent compounds that can naturally label the treated tissue site. We systematically characterized the fluorescence signals produced by femtosecond laser ablation in biological tissues in vivo. Our findings showed that they possess unique fluorescence properties and can be clearly differentiated from endogenous signals and major fluorescent proteins. We further demonstrated that the new fluorescent compounds can be used as in vivo labelling agent for biological imaging and guided laser microsurgery.
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Owing to its near infrared (NIR) absorption, graphene oxide (GO) is promising for both photothermal (PT) therapy and multiphoton (MP) imaging. Novel therapy/imaging modality switching is proposed here based on the selected excitation wavelength of femtosecond (FS) laser. GO-based destruction of cancer cells is demonstrated when the laser power of 800-nm-wavelength FS laser is increased above 7 mW. However, GO-based imaging is mainly monitored without damaging the sample when using 1200-nm wavelength FS laser in the same laser power range. Folic acid (FA) conjugated graphene oxide (FA-GO) was synthesized for selective cancer cell targeting. Dual-function FA-GO-based cancer cell targeting agents were experimentally optimized to enable therapy/imaging modality switching.
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In situ fluorescence lifetime imaging microscopy (FLIM) in an endoscopic configuration of the endogenous biomarker nicotinamide adenine dinucleotide (NADH) has a great potential for malignant tissue diagnosis. Moreover, two-photon nonlinear excitation provides intrinsic optical sectioning along with enhanced imaging depth. We demonstrate, for the first time to our knowledge, nonlinear endogenous FLIM in a fibered microscope with proximal detection, applied to NADH in cultured cells, as a first step to a nonlinear endomicroscope, using a double-clad microstructured fiber with convenient fiber length (> 3 m) and excitation pulse duration (≈50 fs). Fluorescence photons are collected by the fiber inner cladding and we show that its contribution to the impulse response function (IRF), which originates from its intermodal and chromatic dispersions, is small (< 600 ps) and stable for lengths up to 8 m and allows for short lifetime measurements. We use the phasor representation as a quick visualization tool adapted to the endoscopy speed requirements.
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Many optical and biomechanical properties of the cornea, specifically the transparency of the stroma and its stiffness, can be traced to the degree of order and direction of the constituent collagen fibers. To measure the degree of order inside the cornea, a new metric, the order coefficient, was introduced to quantify the organization of the collagen fibers from images of the stroma produced with a custom-developed second harmonic generation microscope. The order coefficient method gave a quantitative assessment of the differences in stromal collagen arrangement across the cornea depths and between untreated stroma and cross-linked stroma.