Imaging Of Thick Samples Research Articles

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A reflectional quantitative differential phase microscopy (RQDPM) based on polarized wavefront phase modulation and partially coherent full‐aperture illumination is introduced for imaging thick and opaque samples. RQDPM utilizes the polarization‐dependent characteristic of a phase‐type spatial light odulator to divide the object wave into two identical copies with a certain shear distance. When loading different modulation images to the spatial light modulator, phase derivative images with different shearing directions and phase shifts can be obtained. Finally, the phase distribution of the sample is obtained.For further details please visit the article by Ying Ma, Taiqiang Dai, Lan Yu, Lin Ma, Sha An, Yang Wang, Min Liu, Juanjuan Zheng, Liang Kong, Chao Zuo, and Peng Gao (e202200325).image

Focus image scanning microscopy for sharp and gentle super-resolved microscopy

To date, the feasibility of super-resolution microscopy for imaging live and thick samples is still limited. Stimulated emission depletion (STED) microscopy requires high-intensity illumination to achieve sub-diffraction resolution, potentially introducing photodamage to live specimens. Moreover, the out-of-focus background may degrade the signal stemming from the focal plane. Here, we propose a new method to mitigate these limitations without drawbacks. First, we enhance a STED microscope with a detector array, enabling image scanning microscopy (ISM). Therefore, we implement STED-ISM, a method that exploits the working principle of ISM to reduce the depletion intensity and achieve a target resolution. Later, we develop Focus-ISM, a strategy to improve the optical sectioning and remove the background of any ISM-based imaging technique, with or without a STED beam. The proposed approach requires minimal architectural changes to a conventional microscope but provides substantial advantages for live and thick sample imaging.

Label free deep penetration single photon microscopic imaging with ultralong anti-diffracting beam

Label free single photon microscopic imaging has natural advantages in noninvasive in vivo tissue imaging such as high resolution and rapid imaging speed. Although label free multi-photon microscopy can be used for imaging thick tissue samples, it requires high excitation light power and is phototoxic to the samples. Conventional label free single photon microscopy requires lower excitation light power, but it has limited imaging depth. Observing some highly scattering thick tissue samples with single photon microscopy is a great challenge. To solve the problem, we developed a label free deep penetration single photon microscopic imaging technique with an ultralong anti-diffracting (UAD) beam. The penetrating ability of the UAD beam was verified by passing through turbid media and performed with autofluorescence of chloroplasts in fresh Epipremnum aureum leaves. Benefiting from the anti-diffracting properties and the elongated focal depth of the UAD beam, single photon UAD microscopy has deeper penetration depth and better anti-scattering ability and is one of the ideal methods to observe the deep structure of biological samples.

Integrated Differential Phase Contrast (IDPC)-STEM Utilizing a Multi-Sector Detector for Imaging Thick Samples.

The integrated differential phase contrast (IDPC) method is useful for generating the potential map of a thin sample. We evaluate theoretically the potential of IDPC imaging for thick samples by varying the focus at different sample thicknesses. Our calculations show that high defocus values result in enhanced anisotropy of the contrast transfer function (CTF) and uninterpretable images, if a quadrant detector is applied. We further show that applying a multi-sector detector can result in an almost isotropic CTF. By sector number-dependent calculations for both Cc/C3-corrected and C3-corrected scanning transmission electron microscopy (STEM), we show that the increase of detector sectors not only removes the anisotropy of the CTF, but also improves image contrast and resolution. For a proof-of-principle IDPC-STEM (uncorrected) experiment, we realize the functionality of a 12-sector detector from a physical quadrant detector and demonstrate the improvement in contrast and resolution on the example of InGaN/GaN quantum well structure.

Biological applications of the scanning transmission electron microscope

The STEM has the capability to acquire spatial and chemical information from almost all scattered electrons. Dedicated STEMs do not have any post-specimen lenses but many different detectors instead. The STEM axial bright field mode is quite insensitive to multiple scattering occurring while imaging thick samples such as vitrified whole cells. The blur of small gold sphere has been simulated by Monte Carlo calculations. Direct electron detectors record the rich information of microdiffraction patterns from every irradiated spot while the STEM beam raster-scans over the sample. • The STEM produces distinct images of negatively stained macromolecules (GroEL-ES, top left; antibodies, bottom left). • The STEM produces mass maps of biomolecular assemblies (ClyA; center). • The STEM simultaneously acquires bright (top right) and dark field electron tomograms (bottom right) of thick vitrified samples, allowing heavier elements like phosphorus or calcium to be discriminated from lighter elements like carbon.

Extended field-of-view light-sheet microscopy

Light-sheet fluorescence microscopy enables rapid 3D imaging of biological samples. Unlike confocal and two-photon microscopes, a light-sheet microscope illuminates the focal plane with an objective orthogonal to the detection axis and images it in a single snapshot. Its combination of high contrast and minimal sample exposure make it ideal to image thick samples with sub-cellular resolution. To uniformly illuminate a wide field-of-view without compromising axial resolution, propagation-invariant light-fields such as Bessel and Airy beams have been put forward. These beams do however irradiate the sample with a relatively broad transversal structure. The fluorescence excited by the side lobes of Bessel beams can be blocked physically during recording; though at the cost of increased sample exposure. In contrast, the Airy beam has a fine transversal structure that is both curved and asymmetric. Its fine structure captures all the high-frequency components that enable high axial resolution without the need to discard useful fluorescence. This advantage does not carry over naturally to two-photon excitation where the fine transversal structure is suppressed. We demonstrate a symmetric and planar Airy light-sheet that can be used with two-photon excitation and that does not rely on deconvolution.

Adaptive optics for structured illumination microscopy based on deep learning.

Structured illumination microscopy (SIM) is widely used in biological imaging for its high resolution, fast imaging speed, and simple optical setup. However, when imaging thick samples, the structured illumination patterns in SIM will suffer from optical aberrations, leading to a serious deterioration in resolution. Therefore, it is necessary to reconstruct structured illumination patterns with high quality and efficiency in deep tissue imaging. Here we demonstrate an adaptive optics (AO) correction method based on deep learning in wide-field SIM imaging system. The mapping between the coefficients of the first 15 Zernike modes and their corresponding distorted patterns is established to train the convolution neural network (CNN). The results show that the optimized CNN can predict the aberration phase within ~10.1 ms with a personal computer. The correlation index between the aberration phases and their corresponding predicted aberration phase is up to 0.9986. This method is highly robust and effective for patterns with various spatial densities and illumination conditions and able to effectively correct the imaging distortion caused by optical aberration in SIM system.

Two-photon multifocal structured light microscopy based on high-speed phase-type spatial light modulator

Multifocal structured illumination microscopy (MSIM) can achieve a doubled improvement in the resolution of the diffraction limit within an imaging depth of 50 μm. But when imaging thick samples, scattered light and defocused light limit its optical sectioning capability and image contrast. Two-photon MSIM (2P-MSIM) overcomes the influence of sample tissue scattering and further improves the imaging depth and imaging characteristics. However, the existing 2P-MSIM usually adopts galvanometer based scanning mirrors for precisely scanning imaging, which is a complicated and poor flexibility system. Here we propose a simpler 2P-MSIM. Two-photon multifocal scanning imaging can be realized by a spatial light modulator (SLM) with a high frame rate (< 845 Hz). The phase map of generating multi-focus array and linear phase grating loaded on the SLM simultaneously, high-precision parallel digital random address scanning and excitation imaging on the sample surface can be realized. The mechanical inertia problem of the galvanometer scanner in multifocal imaging can be solved by the proposed method while reducing the complexity of the system and improving flexibility. We finally realize two-photon multifocal imaging of mouse kidney tissue slices and lily of the valley rhizome by this system, which verifies the three-dimensional super-resolution imaging capability of this method. It is of great significance in developing the 2P-MSIM.

Super-resolution and optical sectioning integrated structured illumination microscopy

Super-resolution structured illumination microscopy (SR-SIM) has attracted a great deal of attention in the past few decades. As a wide-field imaging technique, SR-SIM usually suffers from issues relating to out-of-focus background, particularly when imaging thick samples. In this study, we develop an integrated SIM with simultaneous SR and optical sectioning (OS) capabilities, facilitating SR imaging of stacked optical sections, with the out-of-focus background suppressed. The combination of the merits of SR and OS is realized by means of a new image reconstruction algorithm. We confirm the validity of the integrated SIM, both experimentally and in simulation. We anticipate that this integrated SIM will assist biologists in obtaining much clearer SR images in relation to thick specimens.

Label-free imaging of lipid-rich biological tissues by mid-infrared photoacoustic microscopy.

.Significance: Mid-infrared (IR) imaging based on the vibrational transition of biomolecules provides good chemical-specific contrast in label-free imaging of biology tissues, making it a popular tool in both biomedical studies and clinical applications. However, the current technology typically requires thin and dried or extremely flat samples, whose complicated processing limits this technology’s broader translation.Aim: To address this issue, we report mid-IR photoacoustic microscopy (PAM), which can readily work with fresh and thick tissue samples, even when they have rough surfaces.Approach: We developed a transmission-mode mid-IR PAM system employing an optical parametric oscillation laser operating in the wavelength range from 2.5 to . Due to its high sensitivity to optical absorption and the low ultrasonic attenuation of tissue, our PAM achieved greater probing depth than Fourier transform IR spectroscopy, thus enabling imaging fresh and thick tissue samples with rough surfaces.Results: In our spectroscopy study, the symmetric stretching at (3508 nm) was found to be an excellent source of endogenous contrast for lipids. At this wavenumber, we demonstrated label-free imaging of the lipid composition in fresh, manually cut, and unprocessed tissue sections of up to 3-mm thickness.Conclusions: Our technology requires no time-consuming sample preparation procedure and has great potential in both fast clinical histological analysis and fundamental biological studies.

Three-dimensional space optimization for near-field ptychography.

A multiple slices approach in ptychography, termed 3ePIE algorithm, solves the multiple scattering problems of thick samples, which is crucial to biomedical imaging and in situ studies. However, it is unclear how many slices need to be separated and what the distance is between each layer respectively, while these two parameters are sensitive and crucial to the recovery of thick samples. The traditional method is to separate the sample with the same interval for convenience and to test the number of sections for the best reconstructions, which is reasonable for continuous samples and has achieved great results. But this kind of segmentation approach may not be scientific enough for those discrete samples with an uneven spatial distribution. The two inaccurate parameters may yield the algorithm to diverge or generate artifacts, and the empty slices may decrease the reconstruction quality and increase computation time. In addition, repeatedly testing the number of slices is tedious work even for a continuous sample. To this end, a genetic algorithm-based 3ePIE approach, termed the GA-3ePIE method, is proposed to retrieve both the interval between each layer and the number of slices. The performance is verified by both simulations and experiments. The maximum number of sections that can be resolved is also investigated in numerical analysis, which is associated with the sampling and overlap rate in a spatial domain. Our method can be also promoted to image thick samples with coherent X-rays and in the electron regime. The limitations of our method are also discussed.

Considerations for imaging thick, low contrast, and beam sensitive samples with liquid cell transmission electron microscopy

Transmission electron microscopy of whole cells is hindered by the inherently large thickness and low atomic contrast intrinsic of cellular material. Liquid cell transmission electron microscopy allows samples to remain in their native hydrated state and may permit visualizing cellular dynamics in-situ. However, imaging biological cells with this approach remains challenging and identifying an optimal imaging regime using empirical data would help foster new advancements in the field. Recent questions about the role of the electron beam inducing morphological changes or damaging cellular structure and function necessitates further investigation of electron beam-cell interactions, but such comparisons are complicated by variability in imaging techniques used across various studies currently present in literature. The necessity for using low electron fluxes while imaging biological samples requires finding an imaging strategy which produces the strongest contrast and signal to noise ratio for the electron flux used. Here, we experimentally measure and evaluate signal to noise ratios and damage mechanisms between liquid and cryogenic samples of intact cells using multiple electron imaging modalities all on the same instrument and with equivalent beam parameters to standardize the comparison. We also discuss considerations for optimal electron microscopy imaging conditions for future studies on whole cells within liquid environments.

Quantifying Mesoscale Neuroanatomy Using X-Ray Microtomography.

Methods for resolving the three-dimensional (3D) microstructure of the brain typically start by thinly slicing and staining the brain, followed by imaging numerous individual sections with visible light photons or electrons. In contrast, X-rays can be used to image thick samples, providing a rapid approach for producing large 3D brain maps without sectioning. Here we demonstrate the use of synchrotron X-ray microtomography (µCT) for producing mesoscale (∼1 µm 3 resolution) brain maps from millimeter-scale volumes of mouse brain. We introduce a pipeline for µCT-based brain mapping that develops and integrates methods for sample preparation, imaging, and automated segmentation of cells, blood vessels, and myelinated axons, in addition to statistical analyses of these brain structures. Our results demonstrate that X-ray tomography achieves rapid quantification of large brain volumes, complementing other brain mapping and connectomics efforts.

Measurement of oxygen concentrations in bacterial biofilms using transient state monitoring by single plane illumination microscopy

Oxygen is required for respiration in aerobic bacteria and hence plays an essential role in the generation of energy necessary for cell maintenance and growth. The distribution of oxygen within a biofilm can be heterogeneous and may result in bacterial cells exhibiting distinct physiological states depending on their local oxygen availability. Here, we have employed transient state (TRAST) monitoring, a non-invasive method that can measure the relative populations of triplet states by time-modulated illumination, to determine the distribution of oxygen concentrations in biofilm colonies. The method was implemented on a single plane illumination microscope that can perform optical slicing, making it a suitable approach for imaging thick samples. We present a map of triplet relaxation times measured inside a Pseudomonas aeruginosa biofilm at micrometre resolution. We also provide a calibration method that can evaluate oxygen concentration based on TRAST contrast. Our results indicate that anoxic zones exist within the colonies, and oxygen consumption extends outside the areas of high cell densities, establishing a gradient until levels return to air saturation.

Coherent optical adaptive technique improves the spatial resolution of STED microscopy in thick samples.

Stimulated emission depletion microscopy (STED) is one of far-field optical microscopy techniques that can provide sub-diffraction spatial resolution. The spatial resolution of the STED microscopy is determined by the specially engineered beam profile of the depletion beam and its power. However, the beam profile of the depletion beam may be distorted due to aberrations of optical systems and inhomogeneity of specimens' optical properties, resulting in a compromised spatial resolution. The situation gets deteriorated when thick samples are imaged. In the worst case, the sever distortion of the depletion beam profile may cause complete loss of the super resolution effect no matter how much depletion power is applied to specimens. Previously several adaptive optics approaches have been explored to compensate aberrations of systems and specimens. However, it is hard to correct the complicated high-order optical aberrations of specimens. In this report, we demonstrate that the complicated distorted wavefront from a thick phantom sample can be measured by using the coherent optical adaptive technique (COAT). The full correction can effectively maintain and improve the spatial resolution in imaging thick samples.

Adaptive super-resolution microscopy for imaging thick samples

Adaptive super-resolution microscopy for imaging thick samples

Parallel-mode scanning optical sectioning using digital Fresnel holography with three-wave interference phase-shifting.

The Fresnel incoherent correlation holography (FINCH) method is applicable to various techniques of imaging, including fluorescence microscopy. Recently, a FINCH configuration capable of optical sectioning, using a scanning phase pinhole, has been suggested [Optica 1, 70 (2014)]. This capability is highly important in situations that demand the suppression of out-of-focus information from the hologram reconstruction of a specific plane of interest, such as the imaging of thick samples in biology. In this study, parallel-mode scanning using multiple phase pinholes is suggested as a means to shorten the acquisition time in an optical sectioning FINCH configuration. The parallel-mode scanning is enabled through a phase-shifting procedure that extracts the mixed term of two out of three interfering beams.

Functional Imaging of Intact Pancreatic Islets by Inverted Selective Plane Illumination Microscopy

The islet of Langerhans is a complex aggregate of cells ∼100 μm in diameter, which plays a central role in glucose homeostasis through the regulated secretion of glycolytic hormones. However, the mechanisms underlying these secretions are not yet fully understood. The behaviors of individual islet cells completely change when they are dislocated from the islet environment, so it is essential to choose experimental approaches that permit investigations in the whole islet. Fluorescence microscopy is well-suited to functional imaging in biological samples, thanks to its specificity and non-invasive quality. Selective Plane Illumination Microscopy (SPIM) has been proven to be a useful technique for imaging thick samples. It provides intrinsic optical sectioning by specifically illuminating the sample with a thin sheet of light, yielding the imaging speed of a conventional widefield microscope coupled with less photobleaching compared to confocal imaging techniques. We have developed a version of the inverted SPIM (iSPIM) [Wu et al., 2011. PNAS 108:17708-13] on a widefield inverted microscope, to study the functional behavior of intact mice islets of Langerhans. By scanning the light sheet through the static sample, it is possible to obtain a volume reconstruction of the whole islet in less than 3 seconds, enabling the study of fast dynamics within the islet. Moreover, the resolution provided by a 0.8 NA objective allows for definitive measurements of individual cell's functional details within the islet milieu. Together, the results demonstrate the multi-purpose capability of this technique.

TFS: Combined Tilt- and Focal Series Scanning Transmission Electron Microscopy

The primary method currently used for obtaining insight into the three-dimensional (3D) structure of unique samples from biology and materials science at the nanometer scale is tilt-series transmission electron microscopy (TEM) [1]. A 3D volume is reconstructed from images recorded at many projections obtained by mechanically tilting the sample stage. The tilt range strongly influences the resolution and characteristics of the 3D reconstructions. Therefore, one would ideally acquire tilted images covering the entire angular range of ±90°. However, in practice the maximum tilt range is usually only about ±60 -78° due to mechanical limitations of specimen holders and because the effective thickness of the specimen as seen by the electron beam increases as the section is tilted. The tomographic reconstruction then suffers from missing information and a limited resolution on account of this so-called “missing wedge”. The reconstruction of conventional tilt-series tomography data is particularly difficult because its data acquisition involves a large amount of mechanical movements of the stage. The quality of the tomogram is mostly determined by the precision of the alignment of the individual images. The alternative of recording focal-series scanning TEM (STEM) data avoids the problem caused by the mechanical movements but it lacks axial resolution [2]. We have introduced a new recording scheme for 3D STEM that significantly reduces the aforementioned two limitations of tilt series tomography. In the combined tilt- and focal series (TFS) method, the specimen is rotated in relatively large tilt increments over the possible tilt range, and for every tilt direction, a through-focal series is recorded (Fig. 1). The reconstruction method differs from existing methods such as sequential algebraic reconstruction technique (SART). Both the tilt-series and focal-series data are reconstructed into a 3D tomogram in the same software algorithm. The conical shape of the STEM probe is taken into account via forward- and backward projection operators. The TFS method was demonstrated to exhibit reduced “missing wedge” artifacts and a higher axial resolution than obtainable using STEM tilt series [3]. Fig. 2 shows that the missing wedge is still present in the TFS but in the central vertical region, low spatial frequency signal components are now present (arrow). Moreover, the streaks corresponding to the tilt directions are less pronounced in the TFS data. Amongst the most promising applications for STEM tomography with TFS are specimens that require a high precision in the representation of the 3D shapes, whereby TFS reconstruction results in a superior shape reconstruction. The combined tilt- and focal series method could potentially present a better method for atomic 3D resolution while it tolerates a much smaller number of tilt angles than tomography. We expect that a further advantage of TFS STEM can be found for the imaging of micrometers-thick samples. The largest problem when imaging thick samples occurs at the high tilt

Studies of Materials at the Nanometer Scale Using Coherent X-Ray Diffraction Imaging

For many years, x-ray microscopy has been attractive for materials studies with its ability to image thick samples and provide nanometer-scale resolution. However, the ability to manufacture high-resolution x-ray optics has been a hurdle to achieving the full potential of diffraction limited x-ray imaging. Recently, the advent of bright and coherent x-ray sources at synchrotrons and x-ray free electron lasers has enabled a lensless imaging technique called coherent diffractive imaging (CDI). Since it was first demonstrated in 1999, CDI has been rapidly developing into a materials imaging technique with resolutions approaching a few nanometers. This review provides an overview of the development of CDI and several applications to nanometer-scale imaging in two and three dimensions of biological and condensed mater materials. Also, we review the development of tabletop, coherent, soft x-ray sources that provide a complimentary and potentially more accessible source for nanometer-scale coherent imaging of materials.