Optical Sectioning Ability Research Articles

A spatial carrier dynamic quantitative differential phase imaging method

Differential interference contrast (DIC) microscopy is an important method for quantitative phase imaging (QPI) due to its high compatibility regarding to partial coherent light illumination and excellent optical sectioning ability. However, limited by the on-axis interferometric configuration of Nomarski DIC microscopic imaging system, it is difficult to achieve single-shot phase retrieval. Here, we propose a simple and efficient method for dynamic quantitative differential phase imaging (QDPI) by introducing a spatial carrier device into DIC microscope. A Wollaston prism (WP) is placed on the imaging plane of DIC microscope and then projected by a 4f imaging system, so two orthogonally polarized imaging beams with a certain shearing amount will interfere on the conjugate plane of the 4f imaging system and form a spatial carrier DIC image, thus the QDPI can be conveniently realized. Importantly, the system not only possesses high temporal phase stability provided by the common-path geometries and low spatial phase noise due to partially coherent light illumination, but also the advantages of simple optical implementation, no additional phase distortion, high light energy utilization efficiency, and high signal-to-noise ratio. The experimental result demonstrates that using the proposed method, the phase measurement sensitivity reaches 0.003 rad, the corresponding applicability is verified by a polystyrene microsphere crown and biological cells.

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.

Development of a confocal line-scan laser scattering probe for dark-field surface defects detection of transmissive optics.

Dark-field detection has long been used to identify micron/submicron-sized surface defects benefiting from the broadening effect of the actual defect size caused by light scattering. However, the back-side scattering of a transmissive optical slab is inevitably confused with the front-side scattering phenomenon, resulting in deterioration of the signal-to-noise ratio (SNR) of the scattering signal and false alarms for real defect detection. To this end, a confocal line-scan laser scattering probe equipped with optical sectioning ability is proposed to separate the back-side scattering from the front-side scattering. The optical sectioning ability is realized through a confocal light scattering collector, which overcomes the restriction imposed on the numerical aperture (NA) and the field of view (FOV), reaching an FOV length of 90mm and NA of 0.69. The line-scan principle of the probe protects itself from crosstalk because it produces only a laser spot on the tested surface in an instant. Experimental results verified that the probe has a line-scan length of 90mm with a uniformity better than 98%, an rms electronic noise of 3.4 mV, and an rms background noise of 6.4 mV with laser on. The probe can reject the false back-side scattering light for a 2mm thick fused silica slab at 17.1 dB SNR and operate at a high imaging efficiency of 720mm2/s with a minimum detectability limit of 1.4 µm at 12 dB SNR. This work put forward an effective method with great application value for submicron-sized defect detection in transmissive optics.

Image improvement of temporal focusing multiphoton microscopy via superior spatial modulation excitation and Hilbert–Huang transform decomposition

Temporal focusing-based multiphoton excitation microscopy (TFMPEM) just provides the advantage of widefield optical sectioning ability with axial resolution of several micrometers. However, under the plane excitation, the photons emitted from the molecules in turbid tissues undergo scattering, resulting in complicated background noise and an impaired widefield image quality. Accordingly, this study constructs a general and comprehensive numerical model of TFMPEM utilizing Fourier optics and performs simulations to determine the superior spatial frequency and orientation of the structured pattern which maximize the axial excitation confinement. It is shown experimentally that the optimized pattern minimizes the intensity of the out-of-focus signal, and hence improves the quality of the image reconstructed using the Hilbert transform (HT). However, the square-like reflection components on digital micromirror device leads to pattern residuals in the demodulated image when applying high spatial frequency of structured pattern. Accordingly, the HT is replaced with Hilbert–Huang transform (HHT) in order to sift out the low-frequency background noise and pattern residuals in the demodulation process. The experimental results obtained using a kidney tissue sample show that the HHT yields a significant improvement in the TFMPEM image quality.

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.

Axially overlapped multi-focus light sheet with enlarged field of view

Light sheet fluorescence microscopy provides optical sectioning and is widely used in volumetric imaging of large specimens. However, the axial resolution and the lateral Field of View (FoV) of the system, defined by the light sheet, typically limit each other due to the spatial band product of the excitation objective. Here, we develop a simple multi-focus scheme to extend the FoV, where a Gaussian light sheet can be focused at three or more consecutive positions. Axially overlapped multiple light sheets significantly enlarge the FoV with improved uniformity and negligible loss in axial resolution. By measuring the point spread function of fluorescent beads, we demonstrated that the obtained light sheet has a FoV of 450 μm and a maximum axial FWHM of 7.5 μm. Compared with the conventional single-focus one, the multi-focus Gaussian light sheet displays a significantly improved optical sectioning ability over the full FoV when imaging cells and zebrafish.

Multi-plane confocal microscopy with multiplexed volume holographic gratings [Invited

A volume holographic (VHG) grating-based multi-plane differential confocal microscopy (DCM) is proposed for axial scan-free imaging. Also, we briefly reviewed our previous works on volume holographic-based confocal imaging. We show that without degrading imaging performance, it is possible to simultaneously obtain two depth-resolved optically sectioned images with improved axial resolution using multi-plane DCM. The performance of our multi-plane DCM was evaluated by measuring the surface profile of a silicon micro-hole array with depths separation around 10 µm. The axial sensitivity of the system is around 25 nm. Our system has the advantages of multi-plane imaging with high axial sensitivity and high optical sectioning ability. Our method can be used for reflective surface profiling and multi-plane fluorescence imaging. The present methods may find important applications in surface metrology for label-free biological samples, as well as industrial applications.

Two-Photon Fluorescence Imaging.

Two-photon fluorescence imaging is a powerful tool for observing the dynamics of cells in vivo in intact tissue and is well suitable for imaging neuronal activity for neuroscience research. Due to the nonlinear two-photon absorption, the optical sectioning ability is inherent, resulting in two-photon images with high signal contrast and signal-to-noise ratio with efficient illumination. In addition, the longer wavelength excitation light in two-photon imaging compared to one-photon imaging suffers less scattering and absorption by tissue, which allows deeper penetration. Today, two-photon microscopy is being rapidly developed to adapt to various biological applications for high-speed, high-resolution, large-volume, long-term imaging in freely behaving animals.

Two-photon fluorescence imaging of subsurface tissue structures with volume holographic microscopy.

.Significance: Two-photon (2P) fluorescence imaging can provide background-free high-contrast images from the scattering tissues. However, obtaining a multiplane image is not straightforward. We present a two-photon volume holographic imaging (2P-VHI) system for multiplane imaging.Aim: Our goal was to design and implement a 2P-VHI system that can provide the high-contrast optically sectioned images at multiple planes.Approach: A 2P-VHI system is presented that incorporates angularly multiplexed volume holographic gratings and a femtosecond laser source for fluorescence excitation for multiplane imaging. A volume hologram with multiplexed gratings provides multifocal observation, whereas nonlinear excitation using a femtosecond laser helps in significantly enhancing both depth resolution and contrast of images.Results: Standard fluorescent beads are used to demonstrate the imaging performance of the 2P-VHI system. Two-depth resolved optical-sectioning images of fluorescently labeled thick mice intestine samples were obtained. In addition, the optical sectioning capability of our system is measured and compared with that of a conventional VHI system.Conclusions: Results demonstrated that 2P excitation in VHI systems provided the optical sectioning ability that helps in reducing background noise in the images. Integration of nonlinear fluorescence excitation in the VHI provides some unique advantages to the system and has potential to design multidepth optical sectioned spatial–spectral imaging systems.

A Versatile Tiling Light Sheet Microscope for Imaging of Cleared Tissues

We present a tiling light sheet microscope compatible with all tissue clearing methods for rapid multicolor 3D imaging of cleared tissues with micron-scale (4× 4× 10μm3) to submicron-scale (0.3× 0.3× 1μm3) spatial resolution. The resolving ability is improved to sub-100nm (70× 70× 200nm3) via tissue expansion. The microscope uses tiling light sheets to achieve higher spatial resolution and better optical sectioning ability than conventional light sheet microscopes. The illumination light is phase modulated to adjust the position and intensity profile of the light sheet based on the desired spatial resolution and imaging speed and to keep the microscope aligned. The ability of the microscope to align via phase modulation alone also ensures its accuracy for multicolor 3D imaging and makes the microscope reliable and easy to operate. Here we describe the working principle and design of the microscope. We demonstrate its utility by imaging various cleared tissues.

Harmonically decoupled gradient light interference microscopy (HD-GLIM).

Differential phase sensitive methods, such as Nomarski microscopy, play an important role in quantitative phase imaging due to their compatibility with partially coherent illumination and excellent optical sectioning ability. In this Letter, we propose a new system, to the best of our knowledge, to retrieve differential phase information from transparent samples. It is based on a 4f optical system with an amplitude-type spatial light modulator (SLM), which removes the need for traditional differential interference contrast (DIC) optics and specialized phase-only SLMs. We demonstrate the principle of harmonically decoupled gradient light interference microscopy using standard samples, as well as static and dynamic biospecimens.

Quadrature demodulation in line-scan focal modulation microscopy for imaging three-dimensional zebrafish neural structure.

Visualizing biological processes in neuroscience requires in vivo functional imaging at single-neuron resolution, high image acquisition speed and strong optical sectioning ability. However, due to light scattering of in tissue, very often conventional wide-field fluorescence microscopes are unable to resolve cells in the presence of a strong out-of-focus background. Line-scan focal modulation microscopy enables high temporal resolution and good optical sectioning ability at the same time. Here we demonstrate a quadrature demodulation method to extract the focal information with an extended frequency bandwidth and therefore higher spatial resolution. The performance of the demodulation scheme in line-scan focal modulation microscope has been evaluated by performing imaging experiments with fluorescence beads and zebrafish neural structure. Reduced background, reduced artifacts and more detailed morphological information are evident in the obtained images.

Accurate aberration correction in confocal microscopy based on modal sensorless method.

Confocal microscopy has the advantages of high resolution and optical sectioning ability over conventional microscopy. However, aberration induced by the optical system can compromise these advantages and considerably reduce the energy reaching the pointlike detector. We propose an accurate aberration correction method with a liquid-crystal spatial light modulator (LCSLM) in the confocal system. Each coefficient of Zernike aberration modes is calculated by directly measuring the variance of the images with different bias aberration modes. Large-coefficient (>0.7 rad) aberration is compensated first by LCSLM, following which aberrations with small coefficients are measured precisely, minimizing the cross talk between different kinds of aberrations. With this predistortion strategy, the aberration correction is much more accurate, and maximum image intensity in the normal and nonconjugated systems is improved by 2.5 times and 4 times compared to the normal correction method, respectively, demonstrating the effectiveness of our method.

Enhanced photon collection enables four dimensional fluorescence nanoscopy of living systems

The theoretically unlimited spatial resolution of fluorescence nanoscopy often comes at the expense of time, contrast and increased dose of energy for recording. Here, we developed MoNaLISA, for Molecular Nanoscale Live Imaging with Sectioning Ability, a nanoscope capable of imaging structures at a scale of 45–65 nm within the entire cell volume at low light intensities (W-kW cm−2). Our approach, based on reversibly switchable fluorescent proteins, features three distinctly modulated illumination patterns crafted and combined to gain fluorescence ON–OFF switching cycles and image contrast. By maximizing the detected photon flux, MoNaLISA enables prolonged (40–50 frames) and large (50 × 50 µm2) recordings at 0.3–1.3 Hz with enhanced optical sectioning ability. We demonstrate the general use of our approach by 4D imaging of organelles and fine structures in epithelial human cells, colonies of mouse embryonic stem cells, brain cells, and organotypic tissues.

Recent progress of fluorescence lifetime imaging microscopy technology and its application

In the past decade, fluorescence lifetime imaging microscopy (FLIM) has been widely used in biomedical research and other fields. As the fluorescence lifetime is unaffected by probe concentration, excitation intensity and photobleaching, the FLIM has the advantages of high specificity, high sensitivity and capability of quantitative measurement in monitoring microenvironment changes and reflecting the intermolecular interactions. Despite decades of technical development, the FLIM technology still faces some challenges in practical applications. For example, its resolution is still difficult to overcome the diffraction limit and the trade-off among imaging speed, image quality and lifetime accuracy needs to be considered. In recent years, a great advance in FLIM and its application has been made due to the rapid development of hardware and software and their integration with other optical technologies. In this review, we first introduce the principle and characteristics of FLIM technology based on time domain and frequency domain. We then summarize the latest progress of FLIM technology:1) imaging speed enhancement based on hardware improvement such as optimized time-correlated single photon counting module, single photon avalanche diode array detector, and acousto-optic deflector scanner; 2) lifetime measurement accuracy improvement by the proposed algorithms such as maximum likelihood estimate, Bayesian analysis and compressed sensing; 3) imaging quality enhancement and spatial resolution improvement by integrating FLIM with other optical technologies such as adaptive optics for correcting the aberration generated in the optical path, special illumination for equipping wide-field FLIM with optical sectioning ability, and super-resolution techniques for exceeding the resolution limit. We then highlight some recent applications in biomedical studies such as signal transduction or plant cell growth, disease diagnosis and treatment in cancers, Alzheimer's disease and skin diseases, assessment for toxicity and treatment efficiency of nanomaterials developed in the past few years. Finally, we present a short discussion on the current challenges and provide an outlook of the future development of enhanced imaging performance for FLIM technology. We hope that our summary on the state-of-the-art FLIM, our commentary on future challenges, and some proposed avenues for further advances will contribute to the development of FLIM technology and its applications in relevant fields.

Contrast and resolution enhanced optical sectioning in scattering tissue using line-scanning two-photon structured illumination microscopy.

Optical sectioning imaging with high spatial resolution deep inside scattering samples such as mammalian brain is of great interest in biological study. Conventional two-photon microscopy deteriorates in focus when light scattering increases. Here we develop an optical sectioning enhanced two-photon technique which incorporates structured illumination into line-scanning spatial-temporal focusing microscopy (LTSIM), and generate patterned illumination via laser intensity modulation synchronized with scanning. LTSIM brings scattering background elimination and in-focus contrast enhancement, and realizes nearly 2-fold increase in spatial resolution to ∼208 nm laterally and ∼0.94 µm axially. In addition, the intensity modulated line-scanning implementation of LTSIM enables fast and flexible generation of structured illumination, permitting adjustable spatial frequency profiles to optimize image contrast. The highly qualified optical sectioning ability of our system is demonstrated on samples including tissue phantom, C. elegans and mouse brain at depths over hundreds of microns.

Two-photon light-sheet nanoscopy by fluorescence fluctuation correlation analysis

Advances in light-sheet microscopy have enabled the fast three-dimensional (3D) imaging of live cells and bulk specimens with low photodamage and phototoxicity. Combining light-sheet illumination with super-resolution imaging is expected to resolve subcellular structures. Actually, such kind of super-resolution light-sheet microscopy was recently demonstrated using a single-molecule localization algorithm. However, the imaging depth and temporal resolution of this method are limited owing to the requirements of precise single molecule localization and reconstruction. In this work, we present two-photon super-resolution light-sheet imaging via stochastic optical fluctuation imaging (2PLS-SOFI), which acquires high spatiotemporal resolution and excellent optical sectioning ability. 2PLS-SOFI is based on non-linear excitation of fluctuation/blinking probes using our recently developed fast two-photon three-axis digital scanned light-sheet microscope (2P3A-DSLM), which enables both deep penetration and thin sheet of light. Overall, 2PLS-SOFI demonstrates up to 3-fold spatial resolution enhancement compared with conventional two-photon light-sheet (2PLS) microscopy and about 40-fold temporal resolution enhancement compared with individual molecule localization-selective plane illumination microscopy (IML-SPIM). Therefore, 2PLS-SOFI is promising for 3D long-term, deep-tissue imaging with high spatiotemporal resolution.

Rapid imaging of large tissues using high-resolution stage-scanning microscopy.

Rapid and high-resolution imaging of large tissues is essential in biological research, like brain neuron connectivity research and cancer margins imaging. Here a novel stage-scanning confocal microscopy was developed for rapid imaging of large tissues. Line scanning methods and strip imaging strategy were used to increase the imaging speed. The scientific CMOS was used as line detector in sub-array mode and the optical sectioning ability can be easily adjusted by changing the number of line detectors according to different samples. Fluorescent beads imaging showed resolutions of 0.47 μm, 0.56 μm, and 1.56 μm in the X, Y, and Z directions, respectively, with a 40 × objective lens. A 10 × 10 mm(2) coronal plane with enough signal intensity could be imaged in about 88 sec at a sampling resolution of 0.16 μm/pixel. Rapid imaging of mouse brain slices demonstrated the applicability of this system in visualizing neuronal details at high frame rate.

Multimodal optical microscopy methods reveal polyp tissue morphology and structure in Caribbean reef building corals.

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues comprising the Caribbean reef building corals Montastraeaannularis and M. faveolata. These approaches include fluorescence microscopy (FM), serial block face imaging (SBFI), and two-photon confocal laser scanning microscopy (TPLSM). SBFI provides deep tissue imaging after physical sectioning; it details the tissue surface texture and 3D visualization to tissue depths of more than 2 mm. Complementary FM and TPLSM yield ultra-high resolution images of tissue cellular structure. Results have: (1) identified previously unreported lobate tissue morphologies on the outer wall of individual coral polypsand (2) created the first surface maps of the 3D distribution and tissue density of chromatophores and algae-like dinoflagellate zooxanthellae endosymbionts. Spectral absorption peaks of 500 nm and 675 nm, respectively, suggest that M. annularis and M. faveolata contain similar types of chlorophyll and chromatophores. However, M. annularis and M. faveolata exhibit significant differences in the tissue density and 3D distribution of these key cellular components. This study focusing on imaging methods indicates that SBFI is extremely useful for analysis of large mm-scale samples of decalcified coral tissues. Complimentary FM and TPLSM reveal subtle submillimeterscale changes in cellular distribution and density in nondecalcified coral tissue samples. The TPLSM technique affords: (1) minimally invasive sample preparation,(2) superior optical sectioning ability,and (3) minimal light absorption and scattering, while still permitting deep tissue imaging.

Multimodal Optical Microscopy Methods Reveal Polyp Tissue Morphology and Structure in Caribbean Reef Building Corals

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues comprising the Caribbean reef building corals Montastraeaannularis and M. faveolata. These approaches include fluorescence microscopy (FM), serial block face imaging (SBFI), and two-photon confocal laser scanning microscopy (TPLSM). SBFI provides deep tissue imaging after physical sectioning; it details the tissue surface texture and 3D visualization to tissue depths of more than 2 mm. Complementary FM and TPLSM yield ultra-high resolution images of tissue cellular structure. Results have: (1) identified previously unreported lobate tissue morphologies on the outer wall of individual coral polyps and (2) created the first surface maps of the 3D distribution and tissue density of chromatophores and algae-like dinoflagellate zooxanthellae endosymbionts. Spectral absorption peaks of 500 nm and 675 nm, respectively, suggest that M. annularis and M. faveolata contain similar types of chlorophyll and chromatophores. However, M. annularis and M. faveolata exhibit significant differences in the tissue density and 3D distribution of these key cellular components. This study focusing on imaging methods indicates that SBFI is extremely useful for analysis of large mm-scale samples of decalcified coral tissues. Complimentary FM and TPLSM reveal subtle submillimeter scale changes in cellular distribution and density in nondecalcified coral tissue samples. The TPLSM technique affords: (1) minimally invasive sample preparation, (2) superior optical sectioning ability, and (3) minimal light absorption and scattering, while still permitting deep tissue imaging.