Thick Biological Specimens Research Articles

Towards More Efficient Use of Electrons: Demonstrating Cryo-4D-STEM Phase Imaging Techniques on Thick and Thin Biological Specimens, from Organelles to Proteins

Towards More Efficient Use of Electrons: Demonstrating Cryo-4D-STEM Phase Imaging Techniques on Thick and Thin Biological Specimens, from Organelles to Proteins

Pixel-reassigned line-scanning microscopy for fast volumetric super-resolution imaging.

Super-resolution microscopy has revolutionized the field of biophotonics by revealing detailed 3D biological structures. Nonetheless, the technique is still largely limited by the low throughput and hampered by increased background signals for dense or thick biological specimens. In this paper, we present a pixel-reassigned continuous line-scanning microscope for large-scale high-speed 3D super-resolution imaging, which achieves an imaging resolution of 0.41 µm in the lateral direction, i.e., a 2× resolution enhancement from the raw images. Specifically, the recorded line images are first reassigned to the line-excitation center at each scanning position to enhance the resolution. Next, a modified HiLo algorithm is applied to reduce the background signals. Parametric models have been developed to simulate the imaging results of randomly distributed fluorescent beads. Imaging experiments were designed and performed to verify the predicted performance on various biological samples, which demonstrated an imaging speed of 3400 pixels/ms on millimeter-scale specimens. These results confirm the pixel-reassigned line-scanning microscopy is a facile and powerful method to realize large-area super-resolution imaging on thick or dense biological samples.

Enhancing obSTORM imaging performance with cubic spline PSF modeling.

Oblique plane microscopy-based single molecule localization microscopy (obSTORM) has shown great potential for super-resolution imaging of thick biological specimens. Despite its compatibility with tissues and small animals, prior uses of the Gaussian point spread function (PSF) model have resulted in limited imaging resolution and a narrow axial localization range. This is due to the poor fit of the Gaussian PSF model with the actual PSF shapes in obSTORM. To overcome these limitations, we have employed cubic splines for a more accurate modeling of the experimental PSF shapes. This refined PSF model enhances three-dimensional localization precision, leading to significant improvements in obSTORM imaging of mouse retina tissues, such as an approximately 1.2 times increase in imaging resolution, seamless stitching of single molecules between adjacent optical sections, and a doubling of the sectional interval in volumetric obSTORM imaging due to the extended axial range of usable section thickness. The cubic spline PSF model thus offers a path towards more accurate and faster volumetric obSTORM imaging of biological specimens.

Explorations of Fluorescence Modulation of DNA Functionalized Single Wall Carbon Nanotubes by Catechol-Bearing Compounds

This work describes explorations into analyte-mediated modulation of the fluorescence of single-stranded DNA functionalized single wall carbon nanotubes (SWCNT). SWCNTs have several photophysical properties that make them attractive for imaging in biology. They fluoresce in the near-infrared (NIR) region of the spectrum (900 – 1300 nm), which is suitable for imaging in thick biological specimens because of reduced scattering of NIR photons and minimal tissue autofluorescence. SWCNTs exhibit superior photostability and can be considered to be non-photobleaching on time scales of interest to biological imaging. In addition to these advantageous photophysical properties, SWCNT fluorescence can be selectively sensitized to local chemical environments, which has served as a basis for the synthesis and application of SWCNT-based optical biosensors for a variety of biologically relevant compounds including dopamine, norepinephrine, serotonin, and oxytocin, among others. However, the mechanistic basis for fluorescence modulation of functionalized nanotubes by various classes of analytes remains incompletely understood. One class of optical biosensors that have been particularly successful in biological research includes (GT)N oligonucleotide functionalized SWCNTs that exhibit exquisite turn-on response to compounds that contain catechol-like motifs. In this work, we offer new insights into the mechanism of molecular recognition and fluorescence modulation of this sensor class by catechol-like compounds. We first carried out a fragment-based structure activity relationship study to establish how the structural and electronic properties of these compounds correlated with their ability to modulate SWCNT-fluorescence. This helped us establish a spectrum of substrate functionalities that are detectable by this sensor class. Intriguingly however, our experiments revealed that solution phase fluorescence turn-on events are critically influenced by factors that are not intrinsic to the analytes, including solution pH. We will present the pH dependence of fluorescence turn-on events and establish structural and electronic correlates for these dependencies. As a results of these explorations, we have been able to achieve an improved understanding of sensor dynamics and sensing mechanism. We expect that the insights gleaned from this work will contribute to the body of knowledge that underpins SWCNT-based optical sensors.

Mapping nanoscale topographic features in thick tissues with speckle diffraction tomography

Resolving three-dimensional morphological features in thick specimens remains a significant challenge for label-free imaging. We report a new speckle diffraction tomography (SDT) approach that can image thick biological specimens with ~500 nm lateral resolution and ~1 μm axial resolution in a reflection geometry. In SDT, multiple-scattering background is rejected through spatiotemporal gating provided by dynamic speckle-field interferometry, while depth-resolved refractive index maps are reconstructed by developing a comprehensive inverse-scattering model that also considers specimen-induced aberrations. Benefiting from the high-resolution and full-field quantitative imaging capabilities of SDT, we successfully imaged red blood cells and quantified their membrane fluctuations behind a turbid medium with a thickness of 2.8 scattering mean-free paths. Most importantly, we performed volumetric imaging of cornea inside an ex vivo rat eye and quantified its optical properties, including the mapping of nanoscale topographic features of Dua’s and Descemet’s membranes that had not been previously visualized.

Transmission electron microscopy of thick polymer and biological specimens

We explore the properties of elastic and inelastic scattering in a thick organic specimen, together with the mechanisms that provide contrast in a transmission electron microscope (TEM) and scanning-transmission electron microscope (STEM). Experimental data recorded from amorphous carbon are used to predict the bright-field image intensity, mass-thickness contrast and dose-limited resolution as a function of thickness, objective-aperture size, and primary-electron energy E0. Combining this information with estimates of chromatic aberration, objective-aperture diffraction and beam broadening in the specimen, we calculate the achievable TEM and STEM resolution to be around 4 nm at E0 = 300 keV (or below 3 nm at MeV energies) for a 10 µm-diameter objective aperture and 1 – 2 µm thickness of hydrated biological tissue. The 3 MeV resolution for a 10-μm tissue sample is probably closer to 10 nm. We also comment on the error involved in quadrature addition of resolution factors, when one or more of the point-spread functions are non-Gaussian.

Improving two-photon excitation microscopy for sharper and faster biological imaging.

Two-photon excitation laser scanning microscopy (TPLSM) has provided many insights into the life sciences, especially for thick biological specimens, because of its superior penetration depth and less invasiveness owing to the near-infrared wavelength of its excitation laser light. This paper introduces our four kinds of studies to improve TPLSM by utilizing several optical technologies as follows: (1) A high numerical aperture objective lens significantly deteriorates the focal spot size in deeper regions of specimens. Thus, approaches to adaptive optics were proposed to compensate for optical aberrations for deeper and sharper intravital brain imaging. (2) TPLSM spatial resolution has been improved by applying super-resolution microscopic techniques. We also developed a compact stimulated emission depletion (STED) TPLSM that utilizes electrically controllable components, transmissive liquid crystal devices, and laser diode-based light sources. The spatial resolution of the developed system was five times higher than conventional TPLSM. (3) Most TPLSM systems adopt moving mirrors for single-point laser beam scanning, resulting in the temporal resolution caused by the limited physical speed of these mirrors. For high-speed TPLSM imaging, a confocal spinning-disk scanner and newly-developed high-peak-power laser light sources enabled approximately 200 foci scanning. (4) Several researchers have proposed various volumetric imaging technologies. However, most technologies require large-scale and complicated optical setups based on deep expertise for microscopic technologies, resulting in a high threshold for biologists. Recently, an easy-to-use light-needle-creating device was proposed for conventional TPLSM systems to achieve one-touch volumetric imaging.

Airy beam assisted NIR-II light-sheet microscopy

Near-infrared II (1000–1700 nm) three-dimensional (3D) microscopy addresses a significant improvement on the penetration depth for bioimaging, but is challenging in high z-direction spatial resolution, leading to imaging distortion in deep tissue bioimaging. Here, we developed Airy beam-assisted NIR-II light-sheet Microscope (NIR-II Airy LSM) to improve z-direction spatial resolution with large field of view (FOV) on thick biological specimens via self-healing properties of the propagation-invariant field. The axial resolution was measured to be consistently 3.5 µm across the whole FOV (∼ 600 µm), about 4 times higher than traditional Gaussian-beam based microscopy (∼ 15 µm). Besides, the NIR-II Airy LSM demonstrates 2–3 times higher signal background ratio (SBR) than NIR-I imaging, at the penetration depth of approximately 2.5 mm. The increased spatial resolution and SBR allows us to observe pathological conditions and progression of glomeruli and tubules of radiation induced kidney injury and discriminated integral damage degree of intestinal mucosal cells from intestinal tissue accurately via 3D volumetric multiplexed imaging. The improved NIR-II light-sheet microscopy exhibit complete 3D imaging capabilities of whole bio tissues without sacrificing axial resolution and avoid imaging distortion under deep tissue.

Imaging biological macromolecules in thick specimens: The role of inelastic scattering in cryoEM

We investigate potential improvements in using electron cryomicroscopy to image thick specimens with high-resolution phase contrast imaging. In particular, using model experiments, electron scattering theory, Monte Carlo and multislice simulations, we determine the potential for improving electron cryomicrographs of proteins within a cell using chromatic aberration (Cc) correction. We show that inelastically scattered electrons lose a quantifiable amount of spatial coherence as they transit the specimen, yet can be used to enhance the signal from thick biological specimens (in the 1000 to 5000 Å range) provided they are imaged close to focus with an achromatic lens. This loss of information quantified here, which we call “specimen induced decoherence”, is a fundamental limit on imaging biological molecules in situ. We further show that with foreseeable advances in transmission electron microscope technology, it should be possible to directly locate and uniquely identify sub-100 kDa proteins without the need for labels, in a vitrified specimen taken from a cell.

Wavefront engineered light needle microscopy for axially resolved rapid volumetric imaging.

Increasing the acquisition speed of three-dimensional volumetric images is important-particularly in biological imaging-to unveil the structural dynamics and functionalities of specimens in detail. In conventional laser scanning fluorescence microscopy, volumetric images are constructed from optical sectioning images sequentially acquired by changing the observation plane, limiting the acquisition speed. Here, we present a novel method to realize volumetric imaging from two-dimensional raster scanning of a light needle spot without sectioning, even in the traditional framework of laser scanning microscopy. Information from multiple axial planes is simultaneously captured using wavefront engineering for fluorescence signals, allowing us to readily survey the entire depth range while maintaining spatial resolution. This technique is applied to real-time and video-rate three-dimensional tracking of micrometer-sized particles, as well as the prompt visualization of thick fixed biological specimens, offering substantially faster volumetric imaging.

Low-invasive 5D visualization of mitotic progression by two-photon excitation spinning-disk confocal microscopy

Non-linear microscopy, such as multi-photon excitation microscopy, offers spatial localities of excitations, thereby achieving 3D cross-sectional imaging with low phototoxicity even in thick biological specimens. We had developed a multi-point scanning two-photon excitation microscopy system using a spinning-disk confocal scanning unit. However, its severe color cross-talk has precluded multi-color simultaneous imaging. Therefore, in this study, we introduced a mechanical switching system to select either of two NIR laser light pulses and an image-splitting detection system for 3- or 4-color imaging. As a proof of concept, we performed multi-color fluorescent imaging of actively dividing human HeLa cells and tobacco BY-2 cells. We found that the proposed microscopy system enabled time-lapse multi-color 3D imaging of cell divisions while avoiding photodamage. Moreover, the application of a linear unmixing method to the 5D dataset enabled the precise separation of individual intracellular components in multi-color images. We thus demonstrated the versatility of our new microscopy system in capturing the dynamic processes of cellular components that could have multitudes of application.

Variable immersion microscopy with a high numerical aperture.

Three-dimensional (3D) optical microscopy with a high numerical aperture (NA) remains challenging for thick biological specimens owing to aberrations arising from interface refractions. We developed a variable immersion lens (VIL) to passively minimize these aberrations. A VIL is a high-NA concentric meniscus lens and was used in combination with an aberration-corrected high-NA reflecting objective (TORA-FUJI mirror). Wave-optics simulation at a wavelength of 488 nm showed that a VIL microscope enables diffraction-limited 1.2-NA imaging in water (refractive index of 1.34) at a depth of 0.3 mm by minimizing aberrations due to refraction of a sample interface. Another aberration due to the refractive index mismatching between a mounting medium, and an object can also be corrected by the VIL system, because various fluids with different refractive indices can be used as mounting media for the VIL. As a result of correcting the two aberrations at the same time, we experimentally demonstrated that a 6 µm diameter fluorescent bead can be imaged to the true dimensions in 3D.

Two-Photon Phosphorescence Lifetime Microscopy.

Two-photon Phosphorescence Lifetime Microscopy (2PLM) is an emerging nonlinear optical technique that has great potential to improve our understanding of the basic biology underlying human health and disease. Although analogous to 2-photon Fluorescence Lifetime Imaging Microscopy (2P-FLIM), the contrast in 2PLM is fundamentally different from various intensity-based forms of imaging since it is based on the lifetime of an excited state and can be regarded as a "functional imaging" technique. 2PLM signal originates from the deactivation of the excited triplet state (phosphorescence) [1, 2]. Typically, this triplet state is a much longer-lived excited state than the singlet excited state resulting in phosphorescence emission times of microseconds to milliseconds at room temperature as opposed to nanoseconds for fluorescence emission [3]. The long-lived nature of the triplet state makes it highly sensitive to quenching molecules in the surrounding environment such as biomolecular oxygen (O2). Therefore, 2PLM can provide not only information on the distribution pattern of the probe in the sample (via intensity) but also determine the local oxygen tension (via phosphorescence lifetime quenching) [1]. The ability to create three-dimensional optical sections in the plane of focus within a thick biological specimen while maintaining relatively low phototoxicity due to the use of near-infrared wavelengths for two-photon excitation gives 2PLM powerful advantages over other techniques for longitudinal imaging and monitoring of oxygen within living organisms [4]. In this chapter, we will provide background on the development of 2PLM, discuss the most common oxygen sensing measurement methods and concepts, and explain the general principles and optical configuration of a 2PLM system. We also discuss the key characteristics and strategies for improvement of the technique. Finally, we will present an overview of the current primary scientific literature of how 2PLM has been used for oxygen sensing in biological applications and how this technique is improving our understanding of the basic biology underlying several areas of human health.

Deep-learning super-resolution light-sheet add-on microscopy (Deep-SLAM) for easy isotropic volumetric imaging of large biological specimens.

Isotropic 3D histological imaging of large biological specimens is highly desired but remains highly challenging to current fluorescence microscopy technique. Here we present a new method, termed deep-learning super-resolution light-sheet add-on microscopy (Deep-SLAM), to enable fast, isotropic light-sheet fluorescence imaging on a conventional wide-field microscope. After integrating a minimized add-on device that transforms an inverted microscope into a 3D light-sheet microscope, we further integrate a deep neural network (DNN) procedure to quickly restore the ambiguous z-reconstructed planes that suffer from still insufficient axial resolution of light-sheet illumination, thereby achieving isotropic 3D imaging of thick biological specimens at single-cell resolution. We apply this easy and cost-effective Deep-SLAM approach to the anatomical imaging of single neurons in a meso-scale mouse brain, demonstrating its potential for readily converting commonly-used commercialized 2D microscopes to high-throughput 3D imaging, which is previously exclusive for high-end microscopy implementations.

Measuring two-photon microscopy ultrafast laser pulse duration at the sample plane using time-correlated single-photon counting

.Two-photon microscopy (2PM) has revolutionized biomedical imaging by allowing thin optical sectioning in relatively thick biological specimens. Because dispersive microscope components in 2PM, such as objective lens, can alter temporal laser pulse width (typically being broader at the sample plane), for accurate measurements of two-photon absorption properties, it is important to characterize pulse duration at the sample plane. We present a simple modification to a two-photon microscope light path that allows for second-harmonic-generation-based interferometric autocorrelation measurements to characterize ultrafast laser pulse duration at the sample plane using time-correlated single-photon counting (TCSPC). We show that TCSPC can be used as a simple and versatile method to estimate the zero time delay step value between two adjacent ultrafast laser pulses for these measurements. To demonstrate the utility of this modification, we measured the Coherent Chameleon-Ultra II Ti:sapphire laser pulse width at the sample plane using a air, air, or water-immersion objective lens. At 950-nm two-photon excitation, the measured pulse width was , , and (, ), respectively.

Quantification of intraepidermal nerve fiber density using three-dimensional microscopy.

Three-dimensional microscopy provides more extended depth of penetration compared with conventional light microscopy and is known to be useful in clinical evaluation of thick biological specimens. Skin nerve biopsy together with the quantification of intraepidermal nerve fibers in multiple thick sections has been widely adopted for evaluating peripheral neuropathies. The aim of the present study was to evaluate the effectivity of three-dimensional microscopy in reducing the required time and inter-rater discrepancies, especially in the case of personnel not familiar with the quantification methods. A total of six cryo-sectioned specimens were analyzed for the study and the skin samples were collected from one patient with postherpetic neuralgia who voluntarily participated in the study. Two investigators, a physician and non-physician assessed the intraepidermal nerve fiber densities and required analysis time using three different methods including direct visualization of tissue slides, and analysis with two- and three-dimensional images. Three-dimensional microscopy could produce images that enabled reliable evaluation of intraepidermal nerve fibers; the accuracy of analysis was statistically comparable between the physician and non-physician (p > .05). Three-dimensional microscopy also enabled the non-physician to proceed meaningfully faster evaluation compared with the direct visualization method (p = .03). Three-dimensional microscopy could be one of the useful methods to improve accuracy and convenience of the analysis of intraepidermal nerve fibers especially appropriate for unaccustomed physician or non-physician. RESEARCH HIGHLIGHTS: Three-dimensional microscopy is capable of producing images with more extended depth of penetration compared with conventional light microscopy and has been known to be suitable for clinical evaluation of thick biological specimens. Cutaneous nerve biopsy and the quantification of nerve fibers in thick sections has been widely adopted for evaluating peripheral neuropathies. Three-dimensional microscopy could be especially appropriate for unaccustomed physician or non-physician to improve accuracy and convenience of the analysis of intraepidermal nerve fibers.

Aqueous mounting media increasing tissue translucence improve image quality in Structured Illumination Microscopy of thick biological specimen

Structured Illumination Microscopy (SIM) is a super-resolution microscopy method that has significantly advanced studies of cellular structures. It relies on projection of illumination patterns onto a fluorescently labelled biological sample. The information derived from the sample is then shifted to a detectable band, and in the process of image calculation in Fourier space the resolution is doubled. Refractive index homogeneity along the optical path is crucial to maintain a highly modulated illumination pattern necessary for high-quality SIM. This applies in particular to thick samples consisting of large cells and tissues. Surprisingly, sample mounting media for SIM have not undergone a significant evolution for almost a decade. Through identification and systematic evaluation of a number of non-hazardous, water-soluble chemical components of mounting media, we demonstrate an unprecedented improvement in SIM-image quality. Mounting solutions presented in this research are capable of reducing abundant light scattering which constitutes the limiting factor in 3D-SIM imaging of large Hodgkin’s lymphoma and embryonic stem cells as well as 10 µm tissue sections. Moreover, we demonstrate usefulness of some of the media in single molecule localisation microscopy. The results presented here are of importance for standardisation of 3D-SIM data acquisition pipelines for an expanding community of users.

Light-sheet microscopy with attenuation-compensated propagation-invariant beams.

Scattering and absorption limit the penetration of optical fields into tissue. We demonstrate a new approach for increased depth penetration in light-sheet microscopy: attenuation-compensation of the light field. This tailors an exponential intensity increase along the illuminating propagation-invariant field, enabling the redistribution of intensity strategically within a sample to maximize signal and minimize irradiation. A key attribute of this method is that only minimal knowledge of the specimen transmission properties is required. We numerically quantify the imaging capabilities of attenuation-compensated Airy and Bessel light sheets, showing that increased depth penetration is gained without compromising any other beam attributes. This powerful yet straightforward concept, combined with the self-healing properties of the propagation-invariant field, improves the contrast-to-noise ratio of light-sheet microscopy up to eightfold across the entire field of view in thick biological specimens. This improvement can significantly increase the imaging capabilities of light-sheet microscopy techniques using Airy, Bessel, and other propagation-invariant beam types, paving the way for widespread uptake by the biomedical community.

Three-Dimensional Imaging of Biological Tissue by Cryo X-Ray Ptychography

High-throughput three-dimensional cryogenic imaging of thick biological specimens is valuable for identifying biologically- or pathologically-relevant features of interest, especially for subsequent correlative studies. Unfortunately, high-resolution imaging techniques at cryogenic conditions often require sample reduction through sequential physical milling or sectioning for sufficient penetration to generate each image of the 3-D stack. This study represents the first demonstration of using ptychographic hard X-ray tomography at cryogenic temperatures for imaging thick biological tissue in a chemically-fixed, frozen-hydrated state without heavy metal staining and organic solvents. Applied to mammalian brain, this label-free cryogenic imaging method allows visualization of myelinated axons and sub-cellular features such as age-related pigmented cellular inclusions at a spatial resolution of ~100 nanometers and thicknesses approaching 100 microns. Because our approach does not require dehydration, staining or reduction of the sample, we introduce the possibility for subsequent analysis of the same tissue using orthogonal approaches that are expected to yield direct complementary insight to the biological features of interest.

Optical diffraction tomography microscopy with transport of intensity equation using a light-emitting diode array

Optical diffraction tomography (ODT) is an effective label-free technique for quantitatively refractive index imaging, which enables long-term monitoring of the internal three-dimensional (3D) structures and molecular composition of biological cells with minimal perturbation. However, existing optical tomographic methods generally rely on interferometric configuration for phase measurement and sophisticated mechanical systems for sample rotation or beam scanning. Thereby, the measurement is suspect to phase error coming from the coherent speckle, environmental vibrations, and mechanical error during data acquisition process. To overcome these limitations, we present a new ODT technique based on non-interferometric phase retrieval and programmable illumination emitting from a light-emitting diode (LED) array. The experimental system is built based on a traditional bright field microscope, with the light source replaced by a programmable LED array, which provides angle-variable quasi-monochromatic illumination with an angular coverage of ±37 degrees in both x and y directions (corresponding to an illumination numerical aperture of ∼0.6). Transport of intensity equation (TIE) is utilized to recover the phase at different illumination angles, and the refractive index distribution is reconstructed based on the ODT framework under first Rytov approximation. The missing-cone problem in ODT is addressed by using the iterative non-negative constraint algorithm, and the misalignment of the LED array is further numerically corrected to improve the accuracy of refractive index quantification. Experiments on polystyrene beads and thick biological specimens show that the proposed approach allows accurate refractive index reconstruction while greatly reduced the system complexity and environmental sensitivity compared to conventional interferometric ODT approaches.