Imaging Of Biological Systems Research Articles

High-fidelity 3D live-cell nanoscopy through data-driven enhanced super-resolution radial fluctuation

Live-cell super-resolution microscopy enables the imaging of biological structure dynamics below the diffraction limit. Here we present enhanced super-resolution radial fluctuations (eSRRF), substantially improving image fidelity and resolution compared to the original SRRF method. eSRRF incorporates automated parameter optimization based on the data itself, giving insight into the trade-off between resolution and fidelity. We demonstrate eSRRF across a range of imaging modalities and biological systems. Notably, we extend eSRRF to three dimensions by combining it with multifocus microscopy. This realizes live-cell volumetric super-resolution imaging with an acquisition speed of ~1 volume per second. eSRRF provides an accessible super-resolution approach, maximizing information extraction across varied experimental conditions while minimizing artifacts. Its optimal parameter prediction strategy is generalizable, moving toward unbiased and optimized analyses in super-resolution microscopy.

Visualization Tools for Detecting Microcystin-LR in the Biological System via Near-Infrared Fluorescent Probes.

Numerous toxicological and epidemiological studies have shown that microcystin-LR (MC-LR) could cause a variety of toxicity to humans and animals. However, the absence of effective methods to trace MC-LR in biological systems has hindered the in-depth understanding of the mechanism of MC-LR toxicity. Near-infrared (NIR) fluorescent probes are crucial tools for accurate visualization and in-depth study of specific molecules in biological systems. Due to the lack of effective design strategies, NIR fluorescent probes for imaging MC-LR specifically in biological systems have not been reported yet. In order to address this pressing issue, herein, we have introduced a new and facile strategy to improve MC-LR detection and imaging in biological systems, and based on this design strategy, three NIR fluorescence probes (MC-RdTPA1, MC-RdTPA2, and MC-RdTPE1) have been constructed. These probes have several advantages: (i) have long emission wavelength and large Stokes shifts, which have great potential in vivo imaging applications; (ii) could selectively visualize MC-LR in cells; and (iii) showed stable fluorescence intensity in the pH range of 5.0-7.0. This work may provide a new avenue for the detection of MC-LR in biological systems and new tool to advance our knowledge of the mechanism of MC-LR toxicity.

Flexible infrared photodetector based on polyethylene terephthalate (PET) supported lead sulfide thin film

Flexible photodetectors are thought to have uses in optoelectronics, biological imaging, and smart wearable systems. The goal of this work was to develop a simple and low-cost method for fabricating a flexible infrared photodetector based on a lead sulfide (PbS) thin film produced on a PET substrate utilizing a simple low-temperature spray pyrolysis technique. The device has a highest sensitivity to infrared light (562%), a high responsivity (1.06 mA/W), and a short response time (1 ms) at an intensity of 1.72 mW/cm2. During 100 bending tests at a 40° angle, the performance of this flexible devices showed no signs of significant deterioration. The device exhibits a good, reproducible, and steady photoresponse even after bending (under tensile strain). This implies that this Ag/PbS-PET/Ag photodetector will find uses in wearable technology that require the detection of infrared radiation.

Orthogonal activation of a DNA molecular reaction network for proteinase-free and highly specific mRNA imaging in live cells

Construction of artificial DNA molecular reaction networks (DMRNs) represents an effective approach in biosensing. Here we developed a reaction network consisting of a dual catalytic hairpin assembly-mediated tandem molecular circuit combined with strand displacement reaction (DCHA-SDR) for proteinase-free and highly specific mRNA imaging in living cells. The strand displacement reaction (SDR) was designed to be orthogonally activated in exact order by two fuel strands that separately split into inactive species and grafted into the dual catalytic hairpin assembly-mediated molecular tandem circuit (DCHA). The fuel strands can be activated only when the specific mRNA is introduced, and DCHA is initiated by using two different pieces of mRNA sequences as target. In this way, the target mRNA can be automatically matched twice to obtain an accurate and amplified recognition. Comparative analysis indicates that this orthogonal strategy for target identification is superior to the strategy consisting of single CHA-mediated molecular circuit and strand displacement reaction (SCHA-SDR). Moreover, we demonstrate that the DCHA-SDR system comes with the benefit of proteinase-free progress, which makes the approach suitable for operation in living cells. Final application in various cancer cells and normal cells reveals its enormous potential for highly specific mRNA imaging in biological systems.

1,2‐Dioxetane‐based Chemiluminescence Probes for Tumor Optical Molecular Imaging

Abstract1,2‐Dioxetane‐based chemiluminescent probes have recently emerged as a promising tool for imaging biological systems, including disease diagnosis, staging, therapeutic response monitoring and surgical guidance. The advantages of these probes, such as high sensitivity and specificity and real‐time imaging capabilities, are based on their ability to generate chemiluminescent signals without the need for additional excitation sources, enzymes or oxidants after being triggered by target biomarkers under physiological conditions. Over the past decade, numerous 1,2‐dioxetane‐based chemiluminescent probes have been developed and tested for tumor imaging. In this review, we first introduce the trigger mechanism and structural improvements of these probes, followed by an overview of the design principles and applications of 1,2‐dioxetane‐based chemiluminescent probes in in vrtio and in vivo tumour imaging. In addition, the challenges and potential prospects of this technology in the field of bioanalysis and diagnostics are discussed, with the aim of stimulating further progress in the development of new chemiluminescence methods.

Recent Advances in Nanomaterial-Based Chemiluminescence Probes for Biosensing and Imaging of Reactive Oxygen Species.

Reactive oxygen species (ROS) play important roles in organisms and are closely related to various physiological and pathological processes. Due to the short lifetime and easy transformation of ROS, the determination of ROS content in biosystem has always been a challenging task. Chemiluminescence (CL) analysis has been widely used in the detection of ROS due to its advantages of high sensitivity, good selectivity and no background signal, among which nanomaterial-related CL probes are rapidly developing. In this review, the roles of nanomaterials in CL systems are summarized, mainly including their roles as catalysts, emitters, and carriers. The nanomaterial-based CL probes for biosensing and bioimaging of ROS developed in the past five years are reviewed. We expect that this review will provide guidance for the design and development of nanomaterial-based CL probes and facilitate the wider application of CL analysis in ROS sensing and imaging in biological systems.

White Light and Color‐Tuning Long Persistent Luminescence from Metal Halide Based Metal‐Organic Frameworks

AbstractRoom temperature phosphorescence (RTP) materials with long persistent luminescence (LPL) have raised particular attention among researchers for their potential uses in chemical sensing, optical recording devices, biological imaging, and security systems applications, due to the long‐lived triplet states. In this study, one‐pot method of reaction of appropriate ratio of crown ether ligand (15‐Crown‐5) and metallic salts (CdX2) is utilized to yield three metal halide‐based complexes (namely 15‐5‐CdCl2, 15‐5‐CdBr2, and 15‐5‐CdI2). In solid state, three complexes exhibit nearly standard white light emission with CIE coordinates of (0.28, 0.32), (0.31, 0.39), (0.30, 0.34) at room temperature under UV radiation of 340 nm. Meanwhile, they display the excitation‐dependent room temperature phosphorescence from blue/cyan to green/yellow, especially, 15‐5‐CdCl2 and 15‐5‐CdBr2 exhibit color‐tunable and visible LPL with time‐resolved luminescence lifetime as high as 1–2 s. Single‐crystal X‐ray diffraction analysis and theoretical calculations reveal that the bright LPL of 15‐5‐CdCl2 and 15‐5‐CdBr2 arise from the crown ether ligand and aggregation state induced by halogen‐bond. The thus obtained LPL provides potentials in lighting and displaying devices, optical recording devices, security systems and so on.

SEM3De: image restoration for FIB-SEM.

FIB-SEM (Focused Ion Beam-Scanning Electron Microscopy) is a technique to generate 3D images of samples up to several microns in depth. The principle is based on the alternate use of SEM to image the surface of the sample (a few nanometers thickness) and of FIB to mill the surface of the sample a few nanometers at the time. In this way, huge stacks of images can thus be acquired.Although this technique has proven useful in imaging biological systems, the presence of some visual artifacts (stripes due to sample milling, detector saturation, charge effects, focus or sample drift, etc.) still raises some challenges for image interpretation and analyses. With the aim of meeting these challenges, we developed a freeware (SEM3De) that either corrects artifacts with state-of-the-art approaches or, when artifacts are impossible to correct, enables the replacement of artifactual slices by an in-painted image created from adjacent non-artifactual slices. Thus, SEM3De improves the overall usability of FIB-SEM acquisitions. SEM3De can be downloaded from https://sourceforge.net/projects/sem3de/ as a plugin for ImageJ.

A large Stokes shift NIR fluorescent probe for visual monitoring of mitochondrial peroxynitrite during inflammation and ferroptosis and in an Alzheimer's disease model.

The excessive formation of peroxynitrite (ONOO-) in mitochondria has been implicated in various pathophysiological processes and diseases. However, owing to short emission wavelengths and small Stokes shifts, previously reported fluorescent probes pose significant challenges for mitochondrial ONOO- imaging in biological systems. In this study, a near-infrared (NIR) fluorescent probe, denoted as DCO-POT, is designed for the visual monitoring of mitochondrial ONOO-, displaying a remarkable Stokes shift of 170 nm. The NIR fluorophore of DCO-CHO is released by DCO-POT upon the addition of ONOO-, resulting in off-on NIR fluorescence at 670 nm. This phenomenon facilitates the high-resolution confocal laser scanning imaging of ONOO- generated in biological systems. The practical applications of DCO-POT as an efficient fluorescence imaging tool are verified in this study. DCO-POT enables the fluorometric visualization of ONOO- in organelles, cells, and organisms. In particular, ONOO- generation is analyzed during cellular and organism-level (zebrafish) inflammation during ferroptosis and in an Alzheimer's disease mouse model. The excellent visual monitoring performance of DCO-POT in vivo makes it a promising tool for exploring the pathophysiological effects of ONOO-.

Generation of low-loss single TM-mode terahertz using plasma fiber

Using the transfer matrix method, this article proposes a novel single -mode plasma fiber in the applicable terahertz frequency range. Accordingly, by examining the two theoretical models, an applied model is designed that has a very loss-discrimination rate, even up to 500 between the desired mode and unwanted modes, and imposes a very low loss on the transmitted modes. The theoretical results of the present paper indicate that the proposed fiber can examine empirically to find the low-loss single -mode terahertz fibers which are especially utilizable in biological and security imaging systems where the short waveguiding distance is required.

Duo Emission of CVD Nanodiamonds Doped by SiV and GeV Color Centers: Effects of Growth Conditions.

The investigation of the hot filament chemical vapor deposition nanodiamonds with simultaneously embedded luminescent GeV− and SiV− color centers from solid sources showed that both the absolute and relative intensities of their zero-phonon lines (at 602 and 738 nm) depend on nanodiamond growth conditions (a methane concentration in the CH4/H2 gas mixture, growth temperature, and time). It is shown that a controlled choice of parameters of hot filament chemical vapor deposition synthesis makes it possible to select the optimal synthesis conditions for tailoring bicolor fluorescence nanodiamond labels for imaging biological systems.

Toxicity and Surface Modification of Dendrimers: A Critical Review.

Compared to other nano polymers, dendrimers have novel three-dimensional, synthetic hyperbranched, nano-polymeric structures. These supramolecular dendritic structures have a high degree of significant surface and core functionality in the transportation of drugs for targeted therapy, specifically in host-guest response, gene transfer therapy, and imaging of biological systems. However, there are conflicting shreds of evidence regarding biological safety and dendrimers toxicity due to their positive charge at the surface. It includes cytotoxicity, hemolytic toxicity, haematological toxicity, immunogenicity, and in vivo toxicity. Surface modification of the dendrimer group is one of the methods to resolve these issues. This review aimed at investigating different strategies that can reduce toxicity and improve the biocompatibility of different dendrimers. From that viewpoint, we broaden the structural and safe characteristics of the dendrimers in the biomedical and pharmaceutical fields.

Bio-Inspired Multimodal Imaging in Reduced Visibility

The visual systems found in nature rely on capturing light under different modalities, in terms of spectral sensitivities and polarization sensitivities. Numerous imaging techniques are inspired by this variety, among which, the most famous is color imaging inspired by the trichromacy theory of the human visual system. We investigate the spectral and polarimetric properties of biological imaging systems that will lead to the best performance on scene imaging through haze, i.e., dehazing. We design a benchmark experiment based on modalities inspired by several visual systems, and adapt state-of-the-art image reconstruction algorithms to those modalities. We show the difference in performance of each studied systems and discuss it in front of our methodology and the statistical relevance of our data.

Recent progress in small-molecule fluorescent probes for endoplasmic reticulum imaging in biological systems.

Endoplasmic reticulum (ER) is an indispensable organelle in eukaryotic cells involved in protein synthesis and processing, as well as calcium storage and release. Therefore, maintaining the quality of ER is of great importance for cellular homeostasis. Aberrant fluctuations of bioactive species in the ER will result in homeostasis disequilibrium and further cause ER stress, which has evolved to contribute to the pathogenesis of various diseases. Therefore, the real-time monitoring of various bioactive species in the ER is of high priority to ascertain the mysterious roles of ER, which will contribute to unveiling the corresponding mechanism of organism disturbances. Recently, fluorescence imaging has emerged as a robust technique for the direct visualization of molecular events due to its outstanding sensitivity, high temporal-spatial resolution and noninvasive nature. In this review, we comprehensively summarize the recent progress in design strategies, bioimaging applications, potential directions and challenges of ER-targetable small-molecular fluorescent probes.

A workflow for rapid unbiased quantification of fibrillar feature alignment in biological images.

Measuring the organisation of the cellular cytoskeleton and the surrounding extracellular matrix (ECM) is currently of wide interest as changes in both local and global alignment can highlight alterations in cellular functions and material properties of the extracellular environment. Different approaches have been developed to quantify these structures, typically based on fibre segmentation or on matrix representation and transformation of the image, each with its own advantages and disadvantages. Here we present AFT-Alignment by Fourier Transform, a workflow to quantify the alignment of fibrillar features in microscopy images exploiting 2D Fast Fourier Transforms (FFT). Using pre-existing datasets of cell and ECM images, we demonstrate our approach and compare and contrast this workflow with two other well-known ImageJ algorithms to quantify image feature alignment. These comparisons reveal that AFT has a number of advantages due to its grid-based FFT approach. 1) Flexibility in defining the window and neighbourhood sizes allows for performing a parameter search to determine an optimal length scale to carry out alignment metrics. This approach can thus easily accommodate different image resolutions and biological systems. 2) The length scale of decay in alignment can be extracted by comparing neighbourhood sizes, revealing the overall distance that features remain anisotropic. 3) The approach is ambivalent to the signal source, thus making it applicable for a wide range of imaging modalities and is dependent on fewer input parameters than segmentation methods. 4) Finally, compared to segmentation methods, this algorithm is computationally inexpensive, as high-resolution images can be evaluated in less than a second on a standard desktop computer. This makes it feasible to screen numerous experimental perturbations or examine large images over long length scales. Implementation is made available in both MATLAB and Python for wider accessibility, with example datasets for single images and batch processing. Additionally, we include an approach to automatically search parameters for optimum window and neighbourhood sizes, as well as to measure the decay in alignment over progressively increasing length scales.

Recent Advancement in Fluorescent Probes for Sensing and Imaging Tyrosinase Activity in Living Cells

Tyrosinase, a multi-copper oxidase enzyme, is widely distributed in different organisms and plays a vital role in the melanogenesis and enzymatic browning in fruits and vegetables. An abnormal level of tyrosinase in the living system is often associated with different kinds of skin diseases including albinism, vitiligo, skin hyperpigmentation etc. Moreover, overexpressed tyrosinase has become a prognostic biomarker for melanoma. Therefore early detection of tyrosinase activity both in vivo and in vitro has a potential diagnostic and therapeutic application. Small-molecule fluorescent probes have become a powerful device over the traditional biochemical method for the detection and imaging of enzymatic activities in biological systems by virtue of their superior sensitivity, nondestructive fast analysis, spatiotemporal resolution and real-time detection abilities. Moreover, due to their structural tunability, several small-molecule fluorescent probes have been developed to meet various aspects such as enhancing sensitivity, selectivity, cell permeability, real-time monitoring and easy imaging in biological systems. This review article sums up the recent progress of small-molecules fluorescent probes for tyrosinase activity, including their synthesis strategies, mechanistic paths and potential applications based on reports mainly in the past five years. The rapid advancement in this field suggests that fluorescence detection and imaging is a promising technology and widen up new horizons for early diagnosis of melanoma. 

Shear wave generation by mode conversion in picosecond ultrasonics: Impact of grain orientation and material properties

AbstractPicosecond ultrasonics has seen wide application in the investigation of interfaces in semiconductors and 3D imaging in biological systems. Shear ultrasonic waves are important for elastic constant and grain orientation measurement in ceramic materials. In this study, we investigate the impact of grain orientation and material's elastic properties on the generation efficiency of shear waves by mode conversion at a transducer thin film/substrate interface. The solution of acoustic wave equations suggests that crystal grain orientation has a strong impact on the generated shear wave amplitude. This dependence is found to be closely related to the magnitude of longitudinal component of wave displacements. The applicability of analytical model is validated by the experimental results from time‐domain Brillouin scattering. Moreover, material properties determine acoustic wave amplitudes based on the acoustic mismatch model and, particularly, large elastic anisotropy defined by Zener ratio favors strong shear waves. In the light of this analysis, several recommendations on suitable grain orientations and film materials are made to facilitate shear wave detection.

Refractive Index Imaging of Cellular Structures

Label free microscopic approaches have become a necessity to explore, image, and understand, complex biological systems with minimal sample modifications [1]. These innovative microscopes exploit the native photophysical processes present when light interacts with matter, presenting a plethora of options not only for introducing image contrast, but in providing information unobtainable with traditional approaches [2]. When paired with the recent and significant advances in computational approaches, label free imaging is a profoundly powerful tool to image biological systems in innovative ways. In particular, the way in which light is slowed when passing through a sample, known as it's phase shift, is a label-free paradigm particularly useful for biological samples. This is because the refractive index is essentially a relation of the different chemical or physical properties of the sample, and if obtained quantitatively can be used to characterize the contents and distribution of cellular contents [3]. In this work the phase information from our custom ptychographic microscope is compared and combined with traditional fluorescent images, as well as advanced label-free techniques [4], to extract refractive index information of healthy and unhealthy cells. Our results showcase the ability of ptychography to not only provide high quality images of samples, but to also deliver complimentary information about the processes within a cell, currently not able to be captured with standard high-resolution approaches.

Highly Efficient Aggregation-Induced Red-Emissive Organic Thermally Activated Delayed Fluorescence Materials with Prolonged Fluorescence Lifetime for Time-Resolved Luminescence Bioimaging

Organic thermally activated delayed fluorescence (TADF) materials are emerging as potential candidates for time-resolved fluorescence imaging in biological systems. However, the development of purely organic TADF materials with bright aggregated-state emissions in the red/near-infrared (NIR) region remains challenging. Here, we report three donor-acceptor-type TADF molecules as promising candidates for time-resolved fluorescence imaging, which are engineered by direct connection of electron-donating moieties (phenoxazine or phenothiazine) and an electron-acceptor 1,8-naphthalimide (NI). Theoretically and experimentally, we elucidate that three TADF materials possessed remarkably small ΔEST to promote the occurrence of reverse intersystem crossing (RISC). Moreover, they all exhibit aggregation-induced red emissions and long delayed fluorescence lifetimes without the influence of molecular oxygen. More importantly, these long-lived and biocompatible TADF materials, especially the phenoxazine-substituted NI fluorophores, show great potential for high-contrast fluorescence lifetime imaging in living cells. This study provides further a molecular design strategy for purely organic TADF materials and expands the versatile biological application of long-lived fluorescence research in time-resolved luminescence imaging.

Design, development, and performance comparison of wide field lensless and lens-based optical systems for point-of-care biological applications

Lensless biological imaging systems are an emerging alternative to conventional microscopic systems because they enable a wide field of view imaging. While most microscopic systems sacrifice the field of view for magnification, lensless systems have taken advantage of small imaging pixel size, projection, digital magnification, and post-processing to compensate for diffracted images. A new lens-based system is designed to have the exact same wide field of view as that of a basic lensless setup. A new compound lens system design is utilized to achieve an explicit aim to have the same fields of view as the lensless setup. Then the characteristics of these two optical imaging setups (lensless and lens-based setups) are compared at this level of complexity to see what the minimal systems principles are needed to achieve the biological imaging goals for simplified and less expensive future designs. For both imaging systems, images of biological entities are recorded with the help of the same CMOS imaging device and computer software. The main contribution of this work is an exhaustive comparison between the performance characteristics of both systems using optical standards and biological images.