Imaging Of Biological Cells Research Articles

Label-free superior contrast with c-band ultra-violet extinction microscopy

In 1934, Frits Zernike demonstrated that it is possible to exploit the sample’s refractive index to obtain superior contrast images of biological cells. The refractive index contrast of a cell surrounded by media yields a change in the phase and intensity of the transmitted light wave. This change can be due to either scattering or absorption caused by the sample. Most cells are transparent at visible wavelengths, which means the imaginary component of their complex refractive index, also known as extinction coefficient k, is close to zero. Here, we explore the use of c-band ultra-violet (UVC) light for high-contrast high-resolution label-free microscopy, as k is naturally substantially higher in the UVC than at visible wavelengths. Using differential phase contrast illumination and associated processing, we achieve a 7- to 300-fold improvement in contrast compared to visible-wavelength and UVA differential interference contrast microscopy or holotomography, and quantify the extinction coefficient distribution within liver sinusoidal endothelial cells. With a resolution down to 215 nm, we are, for the first time in a far-field label-free method, able to image individual fenestrations within their sieve plates which normally requires electron or fluorescence superresolution microscopy. UVC illumination also matches the excitation peak of intrinsically fluorescent proteins and amino acids and thus allows us to utilize autofluorescence as an independent imaging modality on the same setup.

Aza-phenol Based Macrocyclic Probes Design for "CHEF-on" Multi Analytes Sensor: Crystal Structure Elucidation and Application in Biological Cell Imaging.

Metal bound macrocyclic compounds found in biological systems inspired us to design and synthesize two Robson-type macrocyclic Schiff-base chemosensors, H 2 L1 (H 2 L1=1,11-dimethyl-6,16-dithia-3,9,13,19-tetraaza-1,11(1,3)-dibenzenacycloicosaphane-2,9,12,19-tetraene-1,11-diol) and H 2 L2 (H 2 L2=1,11-dimethyl-6,16-dioxa-3,9,13,19-tetraaza-1,11(1,3)-dibenzenacycloicosaphane-2,9,12,19-tetraene-1,11-diol). Both the chemosensors have been characterized with different spectroscopic techniques. They act as multianalyte sensor and exhibit "turn-on" fluorescence toward different metal ions in 1X PBS (Phosphate Buffered Saline) solution. In presence of Zn2+, Al3+, Cr3+ and Fe3+ ions, H 2 L1 exhibits ∼6-fold enhancement of emission intensity, while H 2 L2 shows ∼6-fold enhancement of emission intensity in the presence of Zn2+, Al3+ and Cr3+ ions. The interaction between the different metal ion and chemosensor have been examined by absorption, emission, and 1H NMR spectroscopy as well as by ESI-MS+ analysis. We have successfully isolated and solved the crystal structure of the complex [Zn(H 2 L1)(NO3)]NO3 (1) by X-ray crystallography. The crystal structure of 1 shows 1:1 metal:ligand stoichiometry and helps to understand the observed PET-Off-CHEF-On sensing mechanism. LOD values of H 2 L1 and H 2 L2 toward metal ions are found to be ∼10-8 and ∼10-7 M, respectively. Large Stokes shifts of the probes against analytes (∼100 nm) make them a suitable candidate for biological cell imaging studies. Robson type phenol based macrocyclic fluorescence sensors are very scarce in the literature. Therefore, the tuning of structural parameters as the number and nature of donor atoms, their relative locations and presence of rigid aromatic groups can lead to the design of new chemosensors, which can accommodate different charged/neutral guest(s) inside its cavity. The study of the spectroscopic properties of this type of macrocyclic ligands and their complexes might open a new avenue of chemosensors.

Sound velocity mapping from GHz Brillouin oscillations in transparent materials by optical incidence from the side of the sample

Time-domain Brillouin scattering (TDBS) is an all-optical experimental technique for investigating transparent materials based on laser picosecond ultrasonics. Its application ranges from imaging thin-films, polycrystalline materials and biological cells to physical properties such as residual stress, temperature gradients and nonlinear coherent nano-acoustic pulses. When the sample refractive index is spatially uniform and known in TDBS, analysis by windowed Fourier transforms allows one to depth-profile the sound velocity. Here, we present a new method in TDBS for extracting sound velocity without a knowledge of the refractive index, by use of probe light obliquely incident on a side face—as opposed to the usual top face—of the sample. We demonstrate this method using a fused silica sample with a titanium transducer film and map the sound velocity in the depth direction. In future, it should be possible to map the sound velocity distribution in three dimensions in inhomogeneous samples, with applications to the imaging of biological cells.

Phase retrieval without phase unwrapping for white blood cells in deep-learning phase-shifting digital holography

Phase retrieval and phase unwrapping are the two important problems for enabling quantitative phase imaging of cells in phase-shifting digital holography. To simultaneously cope with these two problems, a deep-learning phase-shifting digital holography method is proposed in this paper. The proposed method can establish the continuous mapping function of the interferogram to the ground-truth phase using the end-to-end convolutional neural network. With a well-trained deep convolutional neural network, this method can retrieve the phase from one-frame blindly phase-shifted interferogram, without phase unwrapping. The feasibility and applicability of the proposed method are verified by the simulation experiments of the microsphere and white blood cells, respectively. This method will pave the way to the quantitative phase imaging of biological cells with complex substructures.

Quantitative phase imaging of biological cells using lensless inline holographic microscopy through sparsity-assisted iterative phase retrieval algorithm

The twin image-free phase reconstruction is still a challenge with single-shot inline holographic systems. Existing solutions mostly are based on the inverse problem approaches or alternating projections. However, there exists a trade-off between phase retrieval and twin image elimination. Recent studies have introduced a hybrid method involving both the approaches to mitigate this trade-off. Following these works, we propose a single-shot sparsity-assisted iterative phase retrieval approach that applies a sparsity constraint in the object domain and formulates phase retrieval as a minimization problem. We demonstrate lensless digital inline holographic microscopy for imaging transparent and weakly scattering biological samples over a large field-of-view of ∼29mm2. The proposed method achieves high fidelity phase reconstruction with faster convergence compared to the existing single-shot phase retrieval methods. We further demonstrate the phase quantification of label-free biological samples, such as cervical cells and RBCs, to highlight the potential of our technique in clinical applications.

Bond-selective intensity diffraction tomography

Recovering molecular information remains a grand challenge in the widely used holographic and computational imaging technologies. To address this challenge, we developed a computational mid-infrared photothermal microscope, termed Bond-selective Intensity Diffraction Tomography (BS-IDT). Based on a low-cost brightfield microscope with an add-on pulsed light source, BS-IDT recovers both infrared spectra and bond-selective 3D refractive index maps from intensity-only measurements. High-fidelity infrared fingerprint spectra extraction is validated. Volumetric chemical imaging of biological cells is demonstrated at a speed of ~20 s per volume, with a lateral and axial resolution of ~350 nm and ~1.1 µm, respectively. BS-IDT’s application potential is investigated by chemically quantifying lipids stored in cancer cells and volumetric chemical imaging on Caenorhabditis elegans with a large field of view (~100 µm x 100 µm).

Fluorescent property of carbon dots extracted from cigarette smoke and the application in bio-imaging.

Cigarette smoke is one of the six major pollution sources in the room air. It contains large number of particles with size less than 10 nm. There exist carbon dots (CDs) in cigarette smoke which have strong fluorescence and with good bio-compatibility and low toxicity. CDs in cigarette smoke can be applied in bio-imaging which has great potential applications in the integration of cancer diagnosis and treatment. In this paper, CDs were extracted from cigarette smoke. Then, sodium borohydride was added to CDs aqueous solution for reduction and the reduced CDs (R-CDs) were used for biological cell imaging. The results indicate that the CDs with the particle size <10 nm in cigarette smoke are self-assembled by the polymerizated polycyclic aromatic hydrocarbons (PAHs) and ammonium nitrite which are disk nano-structure composed of sp2/sp3 carbon and oxygen/nitrogen groups or polymers. Sodium borohydride can reduce the carbonyl group on the surface of CDs to hydroxyl group and increase the ratio of the Na 1s ratio of the CDs from 1.86 to 7.42. The CDs can emit blue fluorescence under ultraviolet irradiation. After reduction, the R-CDS have the intensity of fluorescence 7.2 times than before and the fluorescence quantum yield increase from 6.13% to 8.86%. The photoluminescence (PL) wavelength of R-CDS have red-shift of 7 nm which was due to the increasing of Na element ratio. The onion epidermal cells labeled with R-CDs show that the CDs could pass through the cell wall into the cell and reach the nucleus. The cell wall and the nucleus could be clearly visualized. CDs also shows low toxicity to human bronchial epithelial cells (BEAS-2B) with good biological activity. The obtained results indicate that the CDs and R-CDs have good fluorescent property which could be used as bio-imaging agent.

Brewster differential microscopy

Imaging of transparent samples such as cells is important in the biomedicine field; however, insignificant absorption and weakly scattering limit the imaging contrast of phase objects. Here, we propose and demonstrate Brewster differential microscopy based on simple optical reflection at the glass interface. The combination of spin–orbit interaction of light and the Brewster effect can perform two-dimensional differentiation to the incident light distribution and, thus, achieves isotropic edge-enhanced imaging of pure phase objects, which overcomes the limitation of traditional one-dimensional imaging. Furthermore, by introducing bias retardation, we also reconstruct the original phase distribution. The proposed microscopic imaging mechanism does not involve any complex modulation devices and takes advantages of simple and low-cost structure. The results indicate that our research shows promising applications for nondestructive imaging of biological cells.

Ring stage classification of Babesia microti and Plasmodium falciparum using optical diffraction 3D tomographic technique

BackgroundBabesia is an intraerythrocytic parasite often misdiagnosed as a malaria parasite, leading to inappropriate treatment of the disease especially in co-endemic areas. In recent years, optical diffraction tomography (ODT) has shown great potential in the field of pathogen detection by quantification of three-dimensional (3D) imaging tomograms. The 3D imaging of biological cells is crucial to investigate and provide valuable information about the mechanisms behind the pathophysiology of cells and tissues.MethodsThe early ring stage of P. falciparum were obtained from stored stock of infected RBCs and of B. microti were obtained from infected patients during diagnosis. The ODT technique was applied to analyze and characterize detailed differences between P. falciparum and B. microti ring stage at the single cell level. Based on 3D quantitative information, accurate measurement was performed of morphological, biochemical, and biophysical parameters.ResultsAccurate measurements of morphological parameters indicated that the host cell surface area at the ring stage in B. microti was significantly smaller (140.2 ± 17.1 µm2) than that in P. falciparum (159.0 ± 15.2 µm2), and sphericities showed higher levels in B. microti-parasitized cells (0.66 ± 0.05) than in P. falciparum (0.60 ± 0.04). Based on biochemical parameters, host cell hemoglobin level was significantly higher and membrane fluctuations were respectively more active in P. falciparum-infected cells (30.25 ± 2.96 pg; 141.3 ± 24.68 nm) than in B. microti (27.28 ± 3.52 pg; 110.1 ± 38.83 nm). The result indicates that P. falciparum more actively altered host RBCs than B. microti.ConclusionAlthough P. falciparum and B. microti often show confusable characteristics under the microscope, and the actual three-dimensional properties are different. These differences could be used in differential clinical diagnosis of erythrocytes infected with B. microti and P. falciparum.Graphical

Three-dimensional imaging of biological cells using surface plasmon coupled emission.

.SignificanceBiological cell imaging has become one of the most crucial research interests because of its applications in biomedical and microbiology studies. However, three-dimensional (3D) imaging of biological cells is critically challenging and often involves prohibitively expensive and complex equipment. Therefore, a low-cost imaging technique with a simpler optical arrangement is immensely needed.AimThe proposed approach will provide an accurate cell image at a low cost without needing any microscope or extensive processing of the collected data, often used in conventional imaging techniques.ApproachWe propose that patterns of surface plasmon coupled emission (SPCE) features from a fluorescently labeled biological cell can be used to image the cell. An imaging methodology has been developed and theoretically demonstrated to create 3D images of cells from the detected SPCE patterns. The 3D images created from the different SPCE properties at the far-field closely match the actual cell structures.ResultsThe developed technique has been applied to different regular and irregular cell shapes. In each case, the calculated root-mean-square error (RMSE) of the created images from the cell structures remains within a few percentages. Our work recreates the base of a circular-shaped cell with an RMSE of . In addition, the images of irregular-shaped cell bases have an RMSE of . Finally, we obtained a 3D image with an RMSE of for a random cellular structure.ConclusionsDespite being in its initial stage of development, the proposed technique shows promising results considering its simplicity and the nominal cost it would require.

A series of silane-modified rhodamine dyes for near-IR biological fluorescence imaging of Hela cells and A549 lung cancer cells: Synthesis and sensing behavior towards ions, amino acids and hypocholorous acid

HClO is recognized as an indicator for phagocytosis and inflammatory response. Excess HClO ruptures lysosomes and leads to cell apoptosis, which has been proposed as a cure for cancer treatment. As a consequence, the real-time and in situ cell imaging for HClO is highly appealed in the field of anticancer pharmacy. In this work, a series of silane-modified rhodamine fluorescers with various functional groups were designed, hoping to find a fluorescer with near IR emission for bio-imaging in live cells, along with unique sensing response towards ClO− over competing species. These silane-modified rhodamine fluorescers were carefully characterized, their photophysical parameters and responses towards ClO− and competing species were compared systematically. An optimal probe showed emission quantum yield of 0.26 with emission wavelength at 681 nm. Emission “turn-on” effect was observed, with calibration equation of F = 0.720*[ClO−]2+40.047*[ClO−]+54.982, R2 = 0.999, and the limit of detection (LOD) was calculated as 0.03 μM. Corresponding sensing mechanism was revealed as an HClO-mediated cyclization reaction. Fluorescence imaging for Hela cells and A549 lung cancer cells was performed. The lysosome-targetable feature was confirmed.

Methods of Preparing Nanoscale Vitreous Ice Needles for High-Resolution Cryogenic Characterization.

New high-resolution imaging methods for biological samples such as atom probe tomography (APT), facilitated by the invention of laser-pulsed atom probes and cryo-transfer procedures, have recently emerged. However, ensuring the vitreous state of the fabricated aqueous needle-shaped APT samples remains a challenge despite it being crucial for characterizing biomolecules such as proteins and cellular architectures in their near-native state. Our work investigated three potential approaches: (1) open microcapillary (OMC) method, (2) high-pressure freezing method (HPF), and (3) graphene encapsulation method. Diffraction patterns of the needle specimens acquired by cryo-TEM have demonstrated the vitreous state of the ice needles, although limited to the tip regions, has been achieved with the three proposed approaches. With the capability to prepare vitreous ice needles from hydrated samples of up to ∼200 μm thickness (HPF), combined use of the three approaches opens new avenues for future near-atomic imaging of biological cells in their near-native state.

Integrated optical device for Structured Illumination Microscopy.

Structured Illumination Microscopy (SIM) is a key technology for high resolution and super-resolution imaging of biological cells and molecules. The spread of portable and easy-to-align SIM systems requires the development of novel methods to generate a light pattern and to shift it across the field of view of the microscope. Here we show a miniaturized chip that incorporates optical waveguides, splitters, and phase shifters, to generate a 2D structured illumination pattern suitable for SIM microscopy. The chip creates three point-sources, coherent and controlled in phase, without the need for further alignment. Placed in the pupil of a microscope's objective, the three sources generate a hexagonal illumination pattern on the sample, which is spatially translated thanks to thermal phase shifters. We validate and use the chip, upgrading a commercial inverted fluorescence microscope to a SIM setup and we image biological sample slides, extending the resolution of the microscope.

Imaging cell biology.

Imaging cell biology.

Recent Trends in AFM Imaging Speed and Improvement Methods

: Atomic force microscope (AFM) has become the main tool for observation and manipulation in nanotechnology research due to its nano-meter high resolution, but the slow imaging speed is one of the important reasons hindering the further development of AFM. This article first introduces the applications of AFM in cell biology in recent years, expresses the importance of rapid imaging in cell biology, and then summarizes the reasons affecting the imaging speed of AFM from three aspects: the limited bandwidth of system mechanical components, obvious inherent characteristics of piezoelectric scanners, and complex image processing algorithms. The improvement and optimization methods of mechanical parts or structure, control algorithm and image processing are reviewed for different influence reasons. Then, the advantages and of different improvement methods, as well as the improved imaging speed and imaging quality improvement effects, are compared. Imaging speed and resolution are both several to dozens of times higher than before, while ensuring image quality and without damaging the samples. The aim of this review is to enable students, the public and even experts of different knowledge backgrounds to learn directly, and select realizable improvement methods according to realistic conditions. Finally, the future development trend and further prospects of high-speed AFM are discussed.

Real-Time Phase Imaging with an Asymmetric Transfer Function Metasurface

The conversion of phase variations in an optical wavefield into intensity information is of fundamental importance for optical imaging technology including microscopy of biological cells. While conventional approaches to phase-imaging commonly rely on bulky optical components or computational post processing, meta-optical devices have recently demonstrated all-optical, ultracompact image processing methods. Here we describe a metasurface that exploits photonic spin-orbit coupling to create an asymmetric optical transfer function for real time phase-imaging. The effect of the asymmetry on transmission through the device is demonstrated experimentally with the generation of high contrast pseudo-3D intensity images of phase variations in an optical wavefield without the need for post-processing. This non-interferometric method has potential applications in biological live cell imaging and real-time wavefront sensing.

Dynamic Tomographic Phase Microscopy by Double Six-Pack Holography.

Three-dimensional (3D) optical imaging of rapidly moving biological cells is difficult to achieve as such samples cannot be scanned over time. Here, we present a dynamic scan-free optical tomography approach for stain-free 3D imaging of biological cells using our new double six-pack tomography technique, whereby 12 off-axis holograms are captured in a single camera exposure without sacrificing resolution or field of view. The proposed system illuminates the sample from 12 angles simultaneously, and 3D refractive index (RI) tomograms are reconstructed from each recorded video frame of the dynamic sample. The technique is verified experimentally by recording flowing silica beads, 3 μm in diameter, with the resulting tomogram RI accuracy being 98.5%. A live swimming sperm cell is also imaged, and dynamic 3D imaging results for both beads and sperm cell are presented. The proposed technique represents a 12-fold increase in dynamic holographic data for tomography.

Switchable two-color graphene quantum dot as a promising fluorescence probe to highly sensitive pH detection and bioimaging

Graphene quantum dots have been widely applied in biosensing, fluorescence imaging, biomedicine, energy storage and conversion and catalysis, but design and synthesis of polychromatic graphene quantum dot with high luminous efficiency still faces great challenges. The study reports synthesis of histidine, serine and pentaethylenehexamine-functionalized and boron-doped graphene quantum dot (HSPB-GQD) via one-step pyrolysis. The resulting HSPB-GQD consists of graphene sheets of 2–5 nm with carboxyl, hydroxyl, amino, imino and imidazole. Synergy of histidine, serine, pentaethylenehexamine and boron atoms improves the luminescence behavior. This realizes unique switchable two-color luminescence. UV excitation of 370 nm produces one strong blue fluorescence with the maximum emission wavelength of 455 nm and quantum yield of 72.34%. Vis. excitation of 480 nm produces one strong yellow fluorescence with the maximum emission wavelength of 560 nm and quantum yield of 72.59%. The multiple proton dissociation system constructed by nitrogen-containing and oxygen-containing groups makes yellow fluorescence sensitive to environmental pH value. The intensity linearly increases with increasing pH in the range of 4.5–10.0. Organic molecules and inorganic ions do not interfere pH detection. HSPB-GQD as a promising fluorescence probe with negligible effect on cell viability was successfully applied to pH detection in biological and environmental water samples and cell imaging.

Metal-free Lewis pairs catalysed synthesis of fluorescently labelled polyester-based amphiphilic polymers for biological imaging

In recent years, the use of fluorescent amphiphilic polymers as the bioimaging agents has achieved significant progress, and promoted the development of the early stage cancer diagnosis and treatment. In this study, a serials of novel nitrobenzoxadiazole (NBD) fluorophore-functionalized polyester-based amphiphilic polymers were designed and synthesized via a two-step process: a hydrophobic polyester backbone was synthesized first by ring-opening alternating copolymerization (ROAC) of phthalic anhydride (PA) and allyl glycidyl ether (AGE) monomers using NBD-OH fluorophore as the initiator and metal-free Lewis pairs as the catalyst; then hydrophilic modified by three different hydrophilic pendants, PEG-SH, mercaptoacetic acid and mono-6-thio-β-cyclodextrin, through thiol-ene click reaction. Notably, directly using fluorophore as the initiator to initiate the ROAC of cyclic anhydrides and epoxides has never been reported. The influence of the feed ratio, type of Lewis pair and reaction temperature upon the polymerization performance were presented and the quantitative introduction of NBD and three kinds of hydrophilic pendants were described in detail as well. Due to the amphiphilic properties, these polymers could facilely self-assemble into fluorescent polymeric nanoparticles, which were characterized by a series of characterization techniques including surface tensiometer, dynamic light scattering (DLS), transmission electronic microscopy (TEM), and fluorescence spectroscopy. These fluorescent polymeric nanoparticles with diameters of tens or hundreds of nanometers showed good particle stability and good fluorescence performance in aqueous solution. The biocompatibility of these fluorescent polymeric nanoparticles was also excellent. In fact, the biological imaging of A549 cells could be realized successfully in the presence of the fluorescent polymeric nanoparticles. This work provides a new strategy and may inspire new approaches to the design and preparation of the biocompatible fluorescent amphiphilic polymers which could be used as the potential biological imaging agents in basic life science research and clinical applications.

Aza-Crown-Based Macrocyclic Probe Design for "PET-off" Multi-Cu2+ Responsive and "CHEF-on" Multi-Zn2+ Sensor: Application in Biological Cell Imaging and Theoretical Studies.

The work represents a rare example of an aza-crown-based macrocyclic chemosensor, H2DTC (H2DTC = 1,16-dihydroxy-tetraaza-30-crown-8) for the selective detection of both Zn2+ and Cu2+ in HEPES buffer medium (pH 7.4). H2DTC exhibits a fluorescence response for both Zn2+ and Cu2+ ions. The reversibility of the chemosensor in its binding with Zn2+ and Cu2+ ions is also examined using a Na2EDTA solution. H2DTC exhibits a chelation-enhanced fluorescence (CHEF) effect in the presence of Zn2+ ions and a quenching effect (CHEQ) in the presence of paramagnetic Cu2+ ions. Furthermore, the geometry and spectral properties of H2DTC and the chemosensor bound to Zn2+ have been studied by DFT and TDDFT calculations. The limit of detection (LOD) values are 0.11 × 10-9 and 0.27 × 10-9 M for Cu2+ and Zn2+, respectively. The formation constants for the Zn2+ and Cu2+ complexes have been measured by pH-potentiometry in 0.15 M NaCl in 70:30 (v:v) water:ethanol at 298.1 K. UV-vis absorption and fluorometric spectral data and pH-potentiometric titrations indicate 1:1 and 2:1 metal:chemosensor species. In the solid state H2DTC is able to accommodate up to four metal ions, as proved by the crystal structures of the complexes [Zn4(DTC)(OH)2(NO3)4] (1) and {[Cu4(DTC)(OCH3)2(NO3)4]·H2O}n (2). H2DTC can be used as a potential chemosensor for monitoring Zn2+ and Cu2+ ions in biological and environmental media with outstanding accuracy and precision. The propensity of H2DTC to detect intracellular Cu2+ and Zn2+ ions in the triple negative human breast cancer cell line MDA-MB-468 and in HeLa cells has been determined by fluorescence cell imaging.