Live Cell Samples Research Articles

An aldehyde-crosslinking mitochondrial probe for STED imaging in fixed cells

Fluorescence labeling of chemically fixed specimens, especially immunolabeling, plays a vital role in super-resolution imaging as it offers a convenient way to visualize cellular structures like mitochondria or the distribution of biomolecules with high detail. Despite the development of various distinct probes that enable super-resolved stimulated emission depletion (STED) imaging of mitochondria in live cells, most of these membrane-potential-dependent fluorophores cannot be retained well in mitochondria after chemical fixation. This lack of suitable mitochondrial probes has limited STED imaging of mitochondria to live cell samples. In this study, we introduce a mitochondria-specific probe, PK Mito Orange FX (PKMO FX), which features a fixation-driven cross-linking motif and accumulates in the mitochondrial inner membrane. It exhibits high fluorescence retention after chemical fixation and efficient depletion at 775 nm, enabling nanoscopic imaging both before and after aldehyde fixation. We demonstrate the compatibility of this probe with conventional immunolabeling and other strategies commonly used for fluorescence labeling of fixed samples. Moreover, we show that PKMO FX facilitates correlative super-resolution light and electron microscopy, enabling the correlation of multicolor fluorescence images and transmission EM images via the characteristic mitochondrial pattern. Our probe further expands the mitochondrial toolkit for multimodal microscopy at nanometer resolutions.

Single-shot differential phase contrast microscopy using ring-shaped polarisation multiplexing illumination.

We propose a differential phase contrast microscopy that enables single-shot phase imaging for unstained biological samples. The proposed approach employs a ring-shaped LED array for polarisation multiplexing illumination and a polarisation camera for image acquisition. As such, multiple images of different polarisation angles can be simultaneously captured with a single shot. Through polarisation demultiplexing, the sample phase can therefore be recovered from the single-shot measurement. Both simulations and experiments demonstrate the effectiveness of the approach. We also demonstrate that ring-shaped illumination enables higher contrast and lower-distortion imaging results than disk-shaped illumination does. The proposed single-shot approach potentially enables phase contrast imaging for live cell samples in vitro. Lay Description: We propose a microscopy that enables imaging of transparent samples, unstained cells, etc. We demonstrate that the proposed method enables higher contrast and lower-distortion imaging results than conventional methods, and significantly improves imaging efficiency. The proposed method potentially enables dynamic imaging for live cell samples in vitro.

Development of an activity-based ratiometric electrochemical probe of the tumor biomarker γ-glutamyl transpeptidase: Rapid and convenient sensing in whole blood, urine and live-cell samples

γ-Glutamyl transpeptidase (GGT) is a key biomarker for cancer diagnosis and post-treatment surveillance. Currently available methods for sensing GGT show high potential, but face certain challenges including an inability to be used to directly sense analytes in turbid biofluid samples such as whole blood without tedious sample pretreatment. To overcome this issue, activity-based electrochemical probes (GTLP and GTLPOH) were herein developed for a convenient and specific direct targeting of GGT activity in turbid biosamples. Both probes were designed to have GGT catalyze the hydrolysis of the gamma-glutamyl amide moiety of the probe, and result in a self-immolative reaction and concomitant ejection of the masked amino ferrocene reporter. The GTLPOH probe, delivered distinctive key results including high sensitivity, high affinity, a wide detection range of 2–100 U/L, and low LOD of 0.38 U/L against GGT. This probe delivered a precise target for sensing GGT and was free of interference from other electroactive biological species. Furthermore, the GTLPOH probe was employed to monitor and quantify the activity of GGT on the surfaces of tumor cells. The designed sensing method was also validated by the direct quantitative measurement of GGT activity in whole blood and urine samples, and the results were found to be consistent with those of the standard fluorometric assay kit. Thus, GTLPOH is of great significance for its promise as a point-of-care tool for early-stage cancer diagnosis as well as a new drug screening method.

Discrimination of Pd-species by an ultra-fast, long wavelength and biocompatible fluorescent sensor: Its on-site kit assay, drug, and cancer/epithelial cell studies

Extensive use of palladium in many catalysts and catalytic converters causes a high degree of pollution of water and soil resources. Therefore, there is an urgent need to develop rapid and sensitive palladium probes. Herein, a novel “turn-on” near-infrared (NIR) fluorescence and colorimetric probe for Pd has been designed on the basis of the deallylation of the probe, followed by the release of NIR emissive fluorophore through the Tsuji-Trost reaction. The probe can selectively discriminate between the oxidation states of Pd0 and Pd2+. Sensing results demonstrates that the probe has excellent selectivity, sensitivity, fast response time, NIR fluorescence, high biocompatibility, and low detection limit for the Pd detection over a series metal ion. The probe has been successfully applied in visualization of residual Pd content from water, soil, drug and living cell samples by fluorescence observation with the naked eye.

High-throughput artifact-free slightly off-axis holographic imaging based on Fourier ptychographic reconstruction

Slightly off-axis digital holographic microscopy (DHM) has recently gained considerable attention due to its unique ability to improve the space-bandwidth product (SBP) of the imaging system while separating the object information from the background intensity to a certain extent. In order to obtain a decent image reconstruction, the spectral aliasing problem still needs to be addressed, which, however, is difficult to be achieved by the conventional linear Fourier domain filtering. To this end, in this paper, we propose a high-throughput artifact-free slightly off-axis holographic reconstruction method based on Fourier ptychographic microscopy (FPM). Inspired by the nonlinear optimized phase reconstruction algorithm of FPM, we perform constrained updates between the real and Fourier domains in an iterative manner to reconstruct the complex amplitude by the hologram intensity. Experimental results on live HeLa cell samples show that the proposed method can provide higher reconstruction accuracy and better image quality compared with the conventional Fourier method and the Kramers–Kronig (KK) relation-based method.

A Carbon Nanotube Sensor Array for the Label-Free Discrimination of Live and Dead Cells with Machine Learning

Developing robust cell recognition strategies is important in biochemical research, but the lack of well-defined target molecules creates a bottleneck in some applications. In this paper, a carbon nanotube sensor array was constructed for the label-free discrimination of live and dead mammalian cells. Three types of carbon nanotube field-effect transistors were fabricated, and different features were extracted from the transfer characteristic curves for model training with linear discriminant analysis (LDA) and support-vector machines (SVM). Live and dead cells were accurately classified in more than 90% of samples in each sensor group using LDA as the algorithm. The recursive feature elimination with cross-validation (RFECV) method was applied to handle the overfitting and optimize the model, and cells could be successfully classified with as few as four features and a higher validation accuracy (up to 97.9%) after model optimization. The RFECV method also revealed the crucial features in the classification, indicating the participation of different sensing mechanisms in the classification. Finally, the optimized LDA model was applied for the prediction of unknown samples with an accuracy of 87.5-93.8%, indicating that live and dead cell samples could be well-recognized with the constructed model.

Improved ClickTags enable live-cell barcoding for highly multiplexed single cell sequencing.

Click chemistry-enabled DNA barcoding of cells provides a universal strategy for sample multiplexing in single-cell RNA-seq (scRNA-seq). However, current ClickTags are limited to fixed samples as they only label cells efficiently in methanol. Herein, we report the development of a new protocol for barcoding live cells with improved ClickTags. The optimized reactions barcoded live cells without perturbing their physiological states, which allowed sample multiplexing of live cells in scRNA-seq. The general applicability of this protocol is demonstrated in diversified types of samples, including murine and human primary samples. Up to 16 samples across these two species are successfully multiplexed and demultiplexed with high consistency. The wide applications of this method could help to increase throughput, reduce cost and remove the batch effect in scRNA-seq, which is especially valuable for studying clinical samples from a large cohort.

Monitoring the Dynamics of Proteome Aggregation in Live Cells Using a Solubilized and Noncovalent Analogue of Fluorescent Protein Chromophores

Stress-induced intracellular proteome aggregation is a hallmark and a biomarker of various human diseases. Current sensors requiring either cellular fixation or covalent modification of the entire proteome are not suitable for live-cell applications and dynamics study. Herein, we report a noncovalent, cell-permeable, and fluorogenic sensor that can reversibly bind to proteome amorphous aggregates and monitor their formation, transition, and clearance in live cells. This sensor was structurally optimized from previously reported fluorescent protein chromophores to enable noncovalent and reversible binding to aggregated proteins. Unlike all previous sensors, the noncovalent and reversible nature of this probe allows for dynamic detection of both the formation and clearance of aggregated proteome in one live-cell sample. Under different cellular stresses, this sensor reveals drastic differences in the morphology and location of aggregated proteome. Furthermore, we have shown that this sensor can detect the transition from proteome liquid-to-liquid phase separation to liquid-to-solid phase separation in a two-color imaging experiment. Overall, the sensor reported here can serve as a facile tool to screen therapeutic drugs and identify cellular pathways that ameliorate pathogenic proteome aggregation in live-cell models.

Using Ligand-Accelerated Catalysis to Repurpose Fluorogenic Reactions for Platinum or Copper.

The development of a fluorescent probe for a specific metal has required exquisite design, synthesis, and optimization of fluorogenic molecules endowed with chelating moieties with heteroatoms. These probes are generally chelation- or reactivity-based. Catalysis-based fluorescent probes have the potential to be more sensitive; however, catalytic methods with a biocompatible fluorescence turn-on switch are rare. Here, we have exploited ligand-accelerated metal catalysis to repurpose known fluorescent probes for different metals, a new approach in probe development. We used the cleavage of allylic and propargylic ethers as platforms that were previously designed for palladium. After a single experiment that combinatorially examined >800 reactions with two variables (metal and ligand) for each ether, we discovered a platinum- or copper-selective method with the ligand effect of specific phosphines. Both metal–ligand systems were previously unknown and afforded strong signals owing to catalytic turnover. The fluorometric technologies were applied to geological, pharmaceutical, serum, and live cell samples and were used to discover that platinum accumulates in lysosomes in cisplatin-resistant cells in a manner that appears to be independent of copper distribution. The use of ligand-accelerated catalysis may present a new blueprint for engineering metal selectivity in probe development.

Abstract 1655: Hyperspectral metabolic imaging with a camera

Abstract New methods to quantify metabolism in cells, organoids, and tissues are highly demanded for cancer diagnosis and treatment. We developed a light sheet-based volumetric imaging platform to rapidly quantify metabolism via fluorescence spectral signatures of intrinsically fluorescent biomolecules. The ability to quantify metabolism in live cells and tissues can be very powerful as the metabolic activity in rapidly proliferating cells, such as cancer cells, is fundamentally different from non-proliferating cells. Most cancer cells rely on aerobic glycolysis to ferment glucose into lactate independent of the presence of oxygen whereas healthy, differentiated cells primarily rely on oxidative phosphorylation. The rapid generation of cytosolic adenosine triphosphate (ATP) during proliferation results in elevated ratios of the reduced form of nicotinamide adenine dinucleotide (NADH) to NAD+ in cancerous cells that can be detected with fluorescence microscopy of intrinsically (auto)fluorescent molecules such as NADH and flavin adenine dinucleotide (FAD) and its variants. This opens a window to quantify ongoing redox reactions as well as important enzymatic reactions via changes of their fluorescence spectral signatures reporting on the metabolic activity of cells. We developed a light sheet-based hyperspectral imaging platform leveraging custom designed sine/cosine filters and spectral phasor data analysis to directly obtain spectral information with a regular camera. The approach was verified by spectrally resolving 3D reconstructions of fluorescence images of fragments of mouse intestinal crypts. The acquisition speed of light sheet-based hyperspectral imaging is on the order of seconds for one 3D stack allowing for sequential, repeated imaging of several samples over the course of hours to days. In summary, the overall goal of this project is to develop a modular imaging platform to overcome the limitations of current, laser scanning-based spectral imaging and to make quantification of metabolism viable in live three-dimensional cell, organoid and tissue samples, that can then be used to, for example, to study the effects of cancer chemotherapy reagents on the metabolism of patient-derived organoids. Citation Format: Per Niklas Hedde, Enrico Gratton. Hyperspectral metabolic imaging with a camera [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1655.

A tri-functional probe mediated exponential amplification strategy for highly sensitive detection of Dnmt1 and UDG activities at single-cell level

Multiplex DNA methylation and glycosylation are ubiquitous in the human body to ensure the normal function and stability of the genome. The methyltransferases and glycosylases rely on varied enzymes with different action mechanism, which still remain challenges for multiple detection. Herein, we developed a tri-functional dsDNA probe mediated exponential amplification strategy for sensitive detection of human DNA (cytosine-5) methyltransferase 1 (Dnmt1) and uracil-DNA glycosylase (UDG) activities. The tri-functional dsDNA probe was rationally designed with M-DNA and U-DNA. M-DNA contains the 5′-GCmGCGC-3′ site for Dnmt1 recognition. U-DNA possesses one uracil as the substrate of UDG and a primer sequence for initiating the amplification reaction. M-DNA was complementary to partial sequence of U-DNA. In the presence of Dnmt1 and UDG, BssHⅡ and Endo Ⅳ were used to nick the 5′-GCGCGC-3′ and AP sites respectively, resulting in the release of single-stranded DNA sequence (primer sequence), respectively. After magnetic separation, the released primer sequence hybridizes with padlock DNA (P-DNA), initiating exponential rolling circle amplification to produce numerous G-quadruplexes for recordable signals. The strategy exhibited the limit of detection as low as 0.009 U mL−1 and 0.003 U mL−1 for Dnmt1 and UDG, respectively. Meanwhile, this strategy was successfully applied to detect Dnmt1 and UDG activities in living cell samples at single-cell level and assay the inhibitors of Dnmt1 and UDG. Therefore, the strategy provided a potential method to detect Dnmt1 and UDG activities in biological samples for early clinic diagnosis and therapeutics.

Structural Effect on the Cellular Selectivity of an NIR-Emitting Cyanine Probe: From Lysosome to Simultaneous Nucleus and Mitochondria Selectivity with Potential for Monitoring Mitochondria Dysfunction in Cells.

Bright red to NIR emitting cyanine probes 2-3 were synthesized in very good yields. Probes 2-3 exhibited excellent fluorescent quantum yields (ϕfl ≈ 0.1-0.4) and large Stokes shift (Δλ > 150 nm) due to efficient intramolecular charge transfer (ICT) in the conjugated π system. Organelle specificity of these probes was investigated by live cell fluorescence confocal microscopy studies. Probe 3 exhibited the ability to visualize the cell nucleus and mitochondria simultaneously in live cell samples during imaging experiments. However, in structurally modified probe 2 with different substituents (i.e., benzothiazolium vs benzothiazole), the selectivity of the probe switched entirely toward cellular lysosomes. Spectrometric DNA titration experiments were conducted to confirm the DNA/nucleus selectivity of probe 3. The study further evaluates the role of the substituent toward DNA selectivity. Probe 3 was identified as a valuable fluorescent marker to visually identify and study mitochondrial dysfunction in live cells via fluorescent confocal microscopy.

Developing a microscopic image dataset in support of intelligent phytoplankton detection using deep learning

AbstractPhytoplankton plays an important role in marine ecological environment and aquaculture. However, the recognition and detection of phytoplankton rely on manual operations. As the foundation of achieving intelligence and releasing human labour, a phytoplankton microscopic image dataset PMID2019 for phytoplankton automated detection is presented. The PMID2019 dataset contains 10 819 phytoplankton microscopic images of 24 different categories. We leverage microscopes to collect images of phytoplankton in the laboratory environment. Each object in the images is manually labelled with a bounding box and category of ground-truth. In addition, living cells move quickly making it difficult to capture images of them. In order to generalize the dataset for in situ applications, we further utilize Cycle-GAN to achieve the domain migration between dead and living cell samples. We built a synthetic dataset to generate the corresponding living cell samples from the original dead ones. The PMID2019 dataset will not only benefit the development of phytoplankton microscopic vision technology in the future, but also can be widely used to assess the performance of the state-of-the-art object detection algorithms for phytoplankton recognition. Finally, we illustrate the performances of some state-of-the-art object detection algorithms, which may provide new ideas for monitoring marine ecosystems.

Simple, one step and sensitive fluorescent nanoprobe for simultaneous detection of Pb2+ and Cu2+ based on pincer enzyme strand and signal amplification strategy

A simple, one step and sensitive fluorescent nanoprobe was developed for simultaneous detection of Pb2+ and Cu2+ based on pincer enzyme strand (E-DNA) and circular catalytic cleavage reaction. A pincer E-DNA can be formed by two split E-DNA. The formed pincer E-DNA can form Pb2+ and Cu2+ dependent DNAzyme by combining with the substrate strand (S-DNA) on the gold nanoparticles (AuNPs). Then, the fluorophore labeled S-DNA is cleaved and released from the surface of AuNPs with the assistance of Pb2+ and Cu2+. Meanwhile, the pincer E-DNA is also released for circular catalytic cleavage reaction, causing significant recovery of the fluorescent intensities for both Pb2+ and Cu2+. This method offers high sensitivity with detection limit of 80 pM for Pb2+ and 30 pM for Cu2+. It also shows high specificity for simultaneously detection of Pb2+ and Cu2+. This strategy has great potential to be used for simultaneous detection and imaging of metal ions in environmental and living cell samples.

Metal-induced energy transfer

Abstract Since about a decade, metal-induced energy transfer (MIET) has become a tool to measure the distance of fluorophores to a metal-coated surface with nanometer accuracy. The energy transfer from a fluorescent molecule to surface plasmons within a metal film results in the acceleration of its radiative decay rate. This can be observed as a reduction of the molecule’s fluorescence lifetime which can be easily measured with standard microscopy equipment. The achievable distance resolution is in the nanometer range, over a total range of about 200 nm. The method is perfectly compatible with biological and even live cell samples. In this review, we will summarize the theoretical and technical details of the method and present the most important results that have been obtained using MIET. We will also show how the latest technical developments can contribute to improving MIET, and we sketch some interesting directions for its future applications in the life sciences.

Quantitative differential phase contrast imaging at high resolution with radially asymmetric illumination.

Half-circle illumination-based differential phase contrast (DPC) microscopy has been utilized to recover phase images through a pair of images along multiple axes. Recently, the half-circle based DPC using 12-axis measurements significantly provides a circularly symmetric phase transfer function to improve accuracy for more stable phase recovery. Instead of using half-circle-based DPC, we propose a new scheme of DPC under radially asymmetric illumination to achieve circularly symmetric phase transfer function and enhance the accuracy of phase recovery in a more stable and efficient fashion. We present the design, implementation, and experimental image data demonstrating the ability of our method to obtain quantitative phase images of microspheres, as well as live fibroblast cell samples.

Isotropic differential phase contrast microscopy for quantitative phase bio-imaging.

Quantitative phase imaging (QPI) has been investigated to retrieve optical phase information of an object and applied to biological microscopy and related medical studies. In recent examples, differential phase contrast (DPC) microscopy can recover phase image of thin sample under multi-axis intensity measurements in wide-field scheme. Unlike conventional DPC, based on theoretical approach under partially coherent condition, we propose a new method to achieve isotropic differential phase contrast (iDPC) with high accuracy and stability for phase recovery in simple and high-speed fashion. The iDPC is simply implemented with a partially coherent microscopy and a programmable thin-film transistor (TFT) shield to digitally modulate structured illumination patterns for QPI. In this article, simulation results show consistency of our theoretical approach for iDPC under partial coherence. In addition, we further demonstrate experiments of quantitative phase images of a standard micro-lens array, as well as label-free live human cell samples.

Airyscan super-resolution microscopy of mitochondrial morphology and dynamics in living tumor cells.

Mitochondrial morphology is regulated by continuous fusion-and-fission events that are essential for maintaining normal function. Despite the prominence of mitochondrial function in energy generation and cell signaling, understanding of processes of fusion and fission dynamics has been hampered by the lack of high-resolution optical systems that accommodate live-cell imaging. We have examined different confocal modalities in terms of resolution and signal-to-noise ratio (SNR) in a point scanning confocal microscope with Airyscan super-resolution (AS-SR). Results indicated that Airyscan (AS) provided speed, super-resolution, and high SNR. This modality was then used for monitoring mitochondrial dynamics in live tumor cells modified to harbor green-fluorescent protein localized to mitochondria. We then compared regular AS and fast-Airyscan modalities in terms of gentleness on the live-cell samples. The fast mode provided unprecedented imaging speed that permits monitoring dynamics both in 2D and also in three-dimensional dataset with time lapses (4D). Alterations to the mitochondrial network in U87 glioblastoma cells occurred within seconds and the cells were not affected by modest inhibition of fission. The super-resolution permitted quantitative measurements of mitochondrial diameter with a precision that enabled detection of significant differences in mitochondrial morphology between cell lines. We have observed swelling of mitochondrial tubules in A549 lung cancer cells after 2hr treatment with deoxynyboquinone, an ROS-generating pharmacologic drug. We also tested different 3D analytical parameters and how they can affect morphometric quantitation. The AS-SR imaging enabled high-speed imaging of mitochondrial dynamics without the compromise to cell morphology or viability that is common with conventional fluorescence imaging due to photo-oxidation.

In Situ Electrochemical Sensing and Real-Time Monitoring Live Cells Based on Freestanding Nanohybrid Paper Electrode Assembled from 3D Functionalized Graphene Framework.

In this work, we develop a new type of freestanding nanohybrid paper electrode assembled from 3D ionic liquid (IL) functionalized graphene framework (GF) decorated by gold nanoflowers (AuNFs), and explore its practical application in in situ electrochemical sensing of live breast cell samples by real-time tracking biomarker H2O2 released from cells. The AuNFs modified IL functionalized GF (AuNFs/IL-GF) was synthesized via a facile and efficient dopamine-assisted one-pot self-assembly strategy. The as-obtained nanohybrid assembly exhibits a typical 3D hierarchical porous structure, where the highly active electrocatalyst AuNFs are well dispersed on IL-GF scaffold. And the graft of hydrophilic IL molecules (i.e., 1-butyl-3-methylimidazolium tetrafluoroborate, BMIMBF4) on graphene nanosheets not only avoids their agglomeration and disorder stacking during the self-assembly but also endows the integrated IL-GF monolithic material with unique hydrophilic properties, which enables it to be readily dispersed in aqueous solution and processed into freestanding paperlike material. Because of the unique structural properties and the combinational advantages of different components in the AuNFs/IL-GF composite, the resultant nanohybrid paper electrode exhibits good nonenzymatic electrochemical sensing performance toward H2O2. When used in real-time tracking H2O2 secreted from different breast cells attached to the paper electrode without or with radiotherapy treatment, the proposed electrochemical sensor based on freestanding AuNFs/IL-GF paper electrode can distinguish the normal breast cell HBL-100 from the cancer breast cells MDA-MB-231 and MCF-7 cells, and assess the radiotherapy effects to different breast cancer cells, which opens a new horizon in real-time monitoring cancer cells by electrochemical sensing platform.

Layered double hydroxide-enhanced luminescence in a Fenton-like system for selective sensing of cobalt in Hela cells

This work presented a facile and eco-friendly method for the determination of cobalt ions (Co(II)) in living cells based on layered double hydroxides (Mg-Al CO3-LDHs) enhanced chemiluminescence (CL) emission of a Co(II)-hydrogen peroxide-sodium hydroxide system. The enhanced CL emission was attributed to the large specific surface area of Mg-Al CO3-LDHs, which facilitates the generation of an excited-stated intermediate. The proposed method displayed high selectivity toward Co(II) over other metal ions. Under the optimal conditions, the increased CL intensity showed a linear response versus Co(II) concentration in the range of 5.0–1000 nM with a detection limit of 3.7 nM (S/N = 3). The relative standard deviation for nine repeated measurements of 100 nM Co(II) was 3.2%. Furthermore, the proposed method was successfully applied to detect Co(II) in living cell samples, and the results were agreed with those obtained by the standard ICP-MS method.