Nonlinear Optical Imaging Modalities Research Articles

Fourier Ptychographic Coherent Anti-Stokes Raman Scattering Microscopy with Point-Scanning for Super-Resolution Imaging.

Fourier ptychography (FP) is a high resolution wide-field imaging method based on the extended aperture in the Fourier space, which is synthesized from raw images with varying illumination angles. If FP is extended to coherent nonlinear optical imaging, the resolution could be further improved due to the increase of the cutoff frequency of the synthesized coherent optical transfer function (C-OTF) with respect to the order of nonlinear optical processes. However, there is a fundamental conflict between wide-field FP and nonlinear optical imaging, whereby the nonlinear optical imaging typically requires a focused excitation laser beam with high power density. To tackle the problem, in this work, a unique point-scanning FP (PS-FP) method is presented for super-resolution nonlinear optical imaging, in which the nonlinear optical signal is obtained by using focused laser beam, while the conventional FP algorithm can still be used to retrieve the super-resolution image. PS-FP coherent anti-Stokes Raman scattering (PS-FP-CARS) imaging on a variety of samples, where a 1.8-fold expansion of the OTF is achieved experimentally for enhancing vibrational imaging. Further theoretical calculation shows that the C-OTF of PS-FP higher-order CARS (PS-FP-HO-CARS) can be expanded up to ≈4.9-fold, thereby improving the spatial resolution by ≈3-fold in comparison with conventional point-scanning CARS with under tightly focused beams. The generality of PS-FP method developed in this work can be adapted to other coherent nonlinear optical imaging modalities for super-resolution imaging in tissue and cells.

Nonlinear Optical Imaging Modalities in Biomedicine

Nonlinear Optical Imaging Modalities in Biomedicine

Time-of-flight resolved stimulated Raman scattering microscopy using counter-propagating ultraslow Bessel light bullets generation

We present a novel time-of-flight resolved Bessel light bullet-enabled stimulated Raman scattering (B2-SRS) microscopy for deeper tissue 3D chemical imaging with high resolution without a need for mechanical z-scanning. To accomplish the tasks, we conceive a unique method to enable optical sectioning by generating the counter-propagating pump and Stokes Bessel light bullets in the sample, in which the group velocities of the Bessel light bullets are made ultraslow (e.g., vg ≈ 0.1c) and tunable by introducing programmable angular dispersions with a spatial light modulator. We theoretically analyze the working principle of the collinear multicolor Bessel light bullet generations and velocity controls with the relative time-of-flight resolved detection for SRS 3D deep tissue imaging. We have also built the B2-SRS imaging system and present the first demonstration of B2-SRS microscopy with Bessel light bullets for 3D chemical imaging in a variety of samples (e.g., polymer bead phantoms, biological samples such as spring onion tissue and porcine brain) with high resolution. The B2-SRS technique provides a > 2-fold improvement in imaging depth in porcine brain tissue compared to conventional SRS microscopy. The method of optical sectioning in tissue using counter-propagating ultraslow Bessel light bullets developed in B2-SRS is generic and easy to perform and can be readily extended to other nonlinear optical imaging modalities to advance 3D microscopic imaging in biological and biomedical systems and beyond.

Super‐Resolution Two‐Photon Fluorescence Tomography Through the Phase‐Shifted Optical Beatings of Bessel Beams for High‐Resolution Deeper Tissue 3D Imaging

AbstractA novel z‐scanning‐free epi‐detected super‐resolution two‐photon fluorescence tomography (TPFT) technique enabling super‐resolution deeper tissue 3D imaging is reported. To accomplish this, a unique method is conceived by generating the phase‐shifted optical beatings of Bessel beams (PS‐OB3) with a spatial light modulator (SLM) to break the diffraction limit for enhancing both the lateral and axial resolutions as well as improving the penetration depth in TPFT for super‐resolution deeper tissue imaging. By electronically varying the optical beating frequency and the phase shifts of the beating patterns through SLM, the depth‐resolved TPF signals about the volumetric tissue are encoded in the spatial frequency domain and hence, a series of depth‐resolved TPF images can be retrieved by implementing inverse fast Fourier transform without a need of mechanical depth‐scanning. PS‐OB3 TPFT provides ≈1.3‐ and 2‐fold improvements in lateral and axial resolutions in comparison with conventional point‐scan TPF imaging. It is also illustrated that the epi‐detected PS‐OB3 TPFT imaging with inherent scattering‐resilient properties of the Bessel beams employed gives over 2‐fold improvement in imaging depth in porcine brain tissue compared to conventional point‐scan Gaussian beam TPF imaging. The z‐scanning‐free optical sectioning ability of PS‐OB3 method developed in TPFT is universal, which can be readily extended to practically any other nonlinear optical imaging modalities for super‐resolution deeper 3D imaging in biological and biomedical tissues.

Phototoxic effects of nonlinear optical microscopy on cell cycle, oxidative states, and gene expression

Nonlinear optical imaging modalities, such as stimulated Raman scattering (SRS) microscopy, use pulsed-laser excitation with high peak intensity that can perturb the native state of cells. In this study, we used bulk RNA sequencing, quantitative measurement of cell proliferation, and fluorescent measurement of the generation of reactive oxygen species to assess phototoxic effects of near-IR pulsed laser radiation, at different time scales, for laser excitation settings relevant to SRS imaging. We define a range of laser excitation settings for which there was no significant change in mouse Neuro2A cells after laser exposure. This study provides guidance for imaging parameters that minimize photo-induced perturbations in SRS microscopy to ensure accurate interpretation of experiments with time-lapse imaging or with paired measurements of imaging and sequencing on the same cells.

Label-free multimodal nonlinear optical imaging of needle biopsy cores for intraoperative cancer diagnosis.

.Significance: Needle biopsy (NB) procedures are important for the initial diagnosis of many types of cancer. However, the possibility of NB specimens being unable to provide diagnostic information, (i.e., non-diagnostic sampling) and the time-consuming histological evaluation process can cause delays in diagnoses that affect patient care.Aim: We aim to demonstrate the advantages of this label-free multimodal nonlinear optical imaging (NLOI) technique as a non-destructive point-of-procedure evaluation method for NB tissue cores, for the visualization and characterization of the tissue microenvironment.Approach: A portable, label-free, multimodal NLOI system combined second-harmonic generation (SHG) and third-harmonic generation and two- and three-photon autofluorescence (2PF, 3PF) microscopy. It was used for intraoperative imaging of fresh NB tissue cores acquired during canine cancer surgeries, which involved liver, lung, and mammary tumors as well as soft-tissue sarcoma; in total, eight canine patients were recruited. An added tissue culture chamber enabled the use of this NLOI system for longitudinal imaging of fresh NB tissue cores taken from an induced rat mammary tumor and healthy mouse livers.Results: The intraoperative NLOI system was used to assess fresh canine NB specimens during veterinary cancer surgeries. Histology-like morphological features were visualized by the combination of four NLOI modalities at the point-of-procedure. The NLOI results provided quantitative information on the tissue microenvironment such as the collagen fiber orientation using Fourier-domain SHG analysis and metabolic profiling by optical redox ratio (ORR) defined by 2PF/(2PF + 3PF). The analyses showed that the canine mammary tumor had more randomly oriented collagen fibers compared to the tumor margin, and hepatocarcinoma had a wider distribution of ORR with a lower mean value compared to the liver fibrosis and the normal-appearing liver. Moreover, the loss of metabolic information during tissue degradation of fresh murine NB specimens was shown by overall intensity decreases in all channels and an increase of mean ORR from 0.94 (standard deviation 0.099) to 0.97 (standard deviation 0.077) during 1-h longitudinal imaging of a rat mammary tumor NB specimen. The tissue response to staurosporine (STS), an apoptotic inducer, from fresh murine liver NB specimens was also observed. The mean ORR decreased from 0.86 to 0.74 in the first 40 min and then increased to 0.8 during the rest of the hour of imaging, compared to the imaging results without the addition of STS, which showed a continuous increase of ORR from 0.72 to 0.75.Conclusions: A label-free, multimodal NLOI platform reveals microstructural and metabolic information of the fresh NB cores during intraoperative cancer imaging. This system has been demonstrated on animal models to show its potential to provide a more comprehensive histological assessment and a better understanding of the unperturbed tumor microenvironment. Considering tissue degradation, or loss of viability upon fixation, this intraoperative NLOI system has the advantage of immediate assessment of freshly excised tissue specimens at the point of procedure.

Stimulated Raman Scattering Tomography Enables Label‐Free Volumetric Deep Tissue Imaging

AbstractA novel z‐scan‐free stimulated Raman scattering tomography (SRST) is presented which is enabled by using optical beating technique (OBT) associated with Bessel beams to achieve deeper penetration for label‐free volumetric chemical imaging with subcellular resolution. In SRST, the pump beam is modulated with a spatial light modulator to convert to Bessel beam with optical beating, which is overlapped with the Bessel Stokes beam in the sample. By electronically varying the beating frequency, the depth‐resolved SRS signals about the volumetric tissue are encoded in the spatial frequency domain and thus, SRST can be rapidly retrieved by implementing inverse fast Fourier transform without a need of mechanical depth‐scan. It is demonstrated that SRST imaging using Bessel beams with self‐reconstructing properties provides at least twofold improvement in imaging depth in highly scattering polymer beads phantom as compared to conventional SRS microscopy. The capability of SRST for label‐free volumetric deeper imaging on a variety of imaging targets (e.g., Raman‐active crystals, plant cells, and biological tissue) is also proved. The generality of optical sectioning ability of Bessel beam‐OBT in SRST can be readily extended to practically any other nonlinear optical imaging modalities for deep tissue 3D imaging in biological and biomedical systems.

Real-time three-dimensional histology-like imaging by label-free nonlinear optical microscopy.

The current gold-standard formalin-fixed and paraffin-embedded (FFPE) histology typically requires several days for tissue fixing, embedding, sectioning, and staining to provide depth-resolved tissue feature visualization. During these time- and labor- intense processes, the in vivo tissue dynamics and three-dimensional structures undergo inevitable loss and distortion. A simultaneous label-free autofluorescence multiharmonic (SLAM) microscope is used to conduct ex vivo and in vivo imaging of fresh human and rat tissues. Four nonlinear optical imaging modalities are integrated into this SLAM microscope, including second harmonic generation (SHG), two-photon fluorescence (2PF), third harmonic generation (THG), and three-photon fluorescence (3PF). By imaging fresh human and rat tissues without any tissue processing or staining, various biological tissue features are effectively visualized by one or multiple imaging modalities of the SLAM microscope. In particular, some of the most essential features in hematoxylin and eosin (H&E)-stained histology, such as collagen fibers and nuclei, are also present in the SLAM microscopy images with good contrast. Because nuclei are evident from negative contrast, the nuclei are segmented from the SLAM images using deep learning. Finally, a color-transforming algorithm is developed to convert the grey-scale images acquired by the SLAM microscope to the virtually H&E-stained histology-like images. The converted histology-like images are later compared with the FFPE histology at the same tissue site. In addition, the nuclear-to-cytoplasmic ratios (N/C ratios) of the cells in the SLAM image are quantified, which has diagnostic relevance for cancer. Various histological correlations are identified with high similarities for the color-converted histology-like SLAM microscopy images. By applying the color transforming algorithm on real-time SLAM image sequences and 3D SLAM image stacks, we report, for the first time and to the best our knowledge, real-time 3D histology-like imaging. Furthermore, the quantified N/C ratio of the cells in the SLAM image are overlaid on the converted histology-like image as a new image contrast. We demonstrated real-time 3D histology-like imaging and its future potential using SLAM microscopy aided by color remapping and deep-learning-based feature segmentation.

Cell-specific three-photon-fluorescence brain imaging: neurons, astrocytes, and gliovascular interfaces.

We present brain imaging experiments on rat cortical areas, demonstrating that, when combined with a suitable high-brightness, cell-specific genetically encoded fluorescent marker, three-photon-excited fluorescence (3PEF), enables subcellular-resolution, cell-specific 3D brain imaging that is fully compatible and readily integrable with other nonlinear-optical imaging modalities, including two-photon-fluorescence and harmonic-generation microscopy. With laser excitation provided by sub-100-fs, 1.25-µm laser pulses, cell-specific 3PEF from astrocytes and their processes detected in parallel with a three-photon-resonance-enhanced third harmonic from blood vessels is shown to enable a high-contrast 3D imaging of gliovascular interfaces.

Highly longitudinally polarized coherent anti-Stokes Raman scattering microscopy with improved spatial resolution

We proposed a method to enhance the longitudinally polarized component and improve the spatial resolution of radially polarized coherent anti-Stokes Raman scattering (CARS) microscopy by phase modulation. A specially designed phase pattern is applied onto the pump beam to suppress the radially polarized field component at the focal region. With this modulation, the calculated intensity ratio between the longitudinally and radially polarized CARS field is increased from 2.57 to 14.7, and the simulation of CARS imaging on a 120 nm polystyrene bead shows more than 3-fold spatial resolution improvement for both forward and backward detection. It is expected that this method could also be applied to other nonlinear optical imaging modalities for enhancing the longitudinally polarized component.

Advances in Intravital Non-Linear Optical Imaging of the Central Nervous System inRodents.

Purpose of review: Highly coordinated cellular interactions occur in the healthy or pathologic adult rodent central nervous system (CNS). Until recently, technical challenges have restricted the analysis of these events to largely static modes of study such as immuno-fluorescence and electron microscopy on fixed tissues. The development of intravital imaging with subcellular resolution is required to probe the dynamics of these events in their natural context, the living brain.Recent findings: This review focuses on the recently developed live non-linear optical imaging modalities, the core principles involved, the identified technical challenges that limit their use and the scope of their applications. We highlight some practical applications for these modalities with a specific attention given to Experimental Autoimmune Encephalomyelitis (EAE), a rodent model of a chronic inflammatory disease of the CNS characterized by the formation of disseminated demyelinating lesions accompanied by axonal degeneration.Summary: We conclude that label-free nonlinear optical imaging combined to two photon imaging will continue to contribute richly to comprehend brain function and pathogenesis and to develop effective therapeutic strategies.

Carcinogenic damage to deoxyribonucleic acid is induced by near-infrared laser pulses in multiphoton microscopy via combination of two- and three-photon absorption

Nonlinear optical imaging modalities (multiphoton excited fluorescence, second and third harmonic generation) applied in vivo are increasingly promising for clinical diagnostics and the monitoring of cancer and other disorders, as they can probe tissue with high diffraction-limited resolution at near-infrared (IR) wavelengths. However, high peak intensity of femtosecond laser pulses required for two-photon processes causes formation of cyclobutane-pyrimidine-dimers (CPDs) in cellular deoxyribonucleic acid (DNA) similar to damage from exposure to solar ultraviolet (UV) light. Inaccurate repair of subsequent mutations increases the risk of carcinogenesis. In this study, we investigate CPD damage that results in Chinese hamster ovary cells in vitro from imaging them with two-photon excited autofluorescence. The CPD levels are quantified by immunofluorescent staining. We further evaluate the extent of CPD damage with respect to varied wavelength, pulse width at focal plane, and pixel dwell time as compared with more pronounced damage from UV sources. While CPD damage has been expected to result from three-photon absorption, our results reveal that CPDs are induced by competing twoand three-photon absorption processes, where the former accesses UVA absorption band. This finding is independently confirmed by nonlinear dependencies of damage on laser power, wavelength, and pulse width.

Non-Linear Optical Imaging of Obesity-Related Health Risks: Review.

This review highlights the recent applications of non-linear optical (NLO) microscopy to study obesity-related health risks. A strong emphasis is given to the applications of coherent anti-Stokes Raman scattering (CARS) microscopy where multiple non-linear optical imaging modalities including CARS, sum-frequency generation (SFG), and two-photon fluorescence are employed simultaneously on a single microscope platform. Specific examples on applications of NLO microscopy to study lipid-droplet biology, obesity-cancer relationship, atherosclerosis, and lipid-rich biological structures are discussed.

Multimodal Nonlinear Optical Microscopy and Applications to Central Nervous System Imaging

Multimodal nonlinear optical (NLO) imaging is poised to become a powerful tool in bioimaging given its ability to capitalize on the unique advantages possessed by different NLO imaging modalities. The integration of different imaging modalities such as two-photon-excited fluorescence, sum frequency generation, and coherent anti-Stokes Raman scattering on the same platform can facilitate simultaneous imaging of different biological structures. Parameters to be considered in constructing a multimodal NLO microscope are discussed with emphasis on achieving a compromise in these parameters for efficient signal generation with each imaging modality. As an example of biomedical applications, multimodal NLO imaging is utilized to investigate the central nervous system in healthy and diseased states.