Imaging Of Biological Cells Research Articles

Nanostructures imaging via numerical solution of a 3-D inverse scattering problem without the phase information

Inverse scattering problems without the phase information arise in imaging of nanostructures, whose sizes are hundreds of nanometers, as well as in imaging of biological cells. The governing equation is the 3-D generalized Helmholtz equation with the unknown coefficient, which represents the spatially distributed dielectric constant. It is assumed in the classical inverse scattering problem that both the modulus and the phase of the complex valued scattered wave field are measured outside of a scatterer. Unlike this, it is assumed here that only the modulus of the complex valued scattered wave field is measured on a certain interval of frequencies. The phase is not measured. In this paper a substantially modified reconstruction procedure of [25] is developed and numerically implemented. Ranges of parameters, which are realistic for imaging of nanostructures, are used in numerical examples. Note that numerical studies were not carried out in [25].

An empirical study of parallel solutions for GLCM calculation of diffraction images.

Feature calculation of large amount of images is time consuming. The GPU based CUDA framework offers an affordable solution for calculating image features in parallel. The research focused on an empirical study of different implementations of a general-purpose GPU-based solution for calculating Gray-Level Co-occurrence Matrices (GLCM) and associated features of diffraction images of biological cells. The GLCM features calculated from the diffraction images are used for rapid cell classification with the machine learning algorithm Support Vector Machine (SVM). CPU and GPU versions of the GLCM and textural feature calculations were implemented and evaluated with different configurations. The results of the optimized GPU implementation showed an average speedup of 7 times for GLCM calculation of diffraction images, and average speedup of 9.83 times for feature calculation over the CPU version.

Implementation of a Coherent Anti-Stokes Raman Scattering (CARS) System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope.

Laser scanning microscopes combining a femtosecond Ti:sapphire laser and an optical parametric oscillator (OPO) to duplicate the laser line have become available for biologists. These systems are primarily designed for multi-channel two-photon fluorescence microscopy. However, without any modification, complementary non-linear optical microscopy such as second-harmonic generation (SHG) or third harmonic generation (THG) can also be performed with this set-up, allowing label-free imaging of structured molecules or aqueous medium-lipid interfaces. These techniques are well suited for in-vivo observation, but are limited in chemical specificity. Chemically selective imaging can be obtained from inherent vibration signals based on Raman scattering. Confocal Raman microscopy provides 3D spatial resolution, but it requires high average power and long acquisition time. To overcome these difficulties, recent advances in laser technology have permitted the development of nonlinear optical vibrational microscopy, in particular coherent anti-Stokes Raman scattering (CARS). CARS microscopy has therefore emerged as a powerful tool for biological and live cell imaging, by chemically mapping lipids (via C-H stretch vibration), water (via O-H stretch vibrations), proteins or DNA. In this work, we describe the implementation of the CARS technique on a standard OPO-coupled multiphoton laser scanning microscope. It is based on the in-time synchronization of the two laser lines by adjusting the length of one of the laser beam path. We present a step-by-step implementation of this technique on an existing multiphoton system. A basic background in experimental optics is helpful and the presented system does not require expensive supplementary equipment. We also illustrate CARS imaging obtained on myelin sheaths of sciatic nerve of rodent, and we show that this imaging can be performed simultaneously with other nonlinear optical imaging, such as standard two-photon fluorescence technique and second-harmonic generation.

Characterization of the temperature rise in a single cell during photoacoustic tomography at the nanoscale.

We are developing a label-free nanoscale photoacoustic tomography (nPAT) for imaging a single living cell. nPAT uses a laser-induced acoustic pulse to generate a nanometer-scale image. The primary motivation behind this imaging technique is the imaging of biological cells in the context of diagnosis without fluorescent tagging. During this procedure, thermal damage due to the laser pulse is a potential risk that may damage the cells. A physical model is built to estimate the temperature rise and thermal relaxation during the imaging procedure. Through simulations using finite element methods, two lasers (532 nm at 5 ps pulse duration and 830 nm at 0.2 ps pulse duration) were simulated for imaging red blood cells (RBCs). We demonstrate that a single 5-ps pulse laser with a 400-Hz repetition rate will generate a steady state temperature rise of less than a Kelvin on the surface of the RBCs. All the simulation results show that there is no significant temperature rise in an RBC in either single pulse or multiple pulse illumination with a 532-nm laser with 219 W fluence. Therefore, our simulation results demonstrate the thermal safety of an nPAT system. The photoacoustic signal generated by this laser is on the order of 2.5 kPa, so it should still be large enough to generate high-resolution images with nPAT. Frequency analysis of this signal shows a peak at 1.47 GHz, with frequencies as high as 3.5 GHz still being present in the spectrum. We believe that nPAT will open an avenue for disease diagnosis and cell biology studies at the nanometer-level.

Simultaneously Synchrotron X-ray Fluorescence and Ptychographic Imaging of Frozen Biological Single Cells

Simultaneously Synchrotron X-ray Fluorescence and Ptychographic Imaging of Frozen Biological Single Cells

Specimen preparation for cryogenic coherent X-ray diffraction imaging of biological cells and cellular organelles by using the X-ray free-electron laser at SACLA.

Coherent X-ray diffraction imaging (CXDI) allows internal structures of biological cells and cellular organelles to be analyzed. CXDI experiments have been conducted at 66 K for frozen-hydrated biological specimens at the SPring-8 Angstrom Compact Free-Electron Laser facility (SACLA). In these cryogenic CXDI experiments using X-ray free-electron laser (XFEL) pulses, specimen particles dispersed on thin membranes of specimen disks are transferred into the vacuum chamber of a diffraction apparatus. Because focused single XFEL pulses destroy specimen particles at the atomic level, diffraction patterns are collected through raster scanning the specimen disks to provide fresh specimen particles in the irradiation area. The efficiency of diffraction data collection in cryogenic experiments depends on the quality of the prepared specimens. Here, detailed procedures for preparing frozen-hydrated biological specimens, particularly thin membranes and devices developed in our laboratory, are reported. In addition, the quality of the frozen-hydrated specimens are evaluated by analyzing the characteristics of the collected diffraction patterns. Based on the experimental results, the internal structures of the frozen-hydrated specimens and the future development for efficient diffraction data collection are discussed.

Quantitative phase imaging of biological cells using spatially low and temporally high coherent light source.

In this Letter, we demonstrate quantitative phase imaging of biological samples, such as human red blood cells (RBCs) and onion cells using narrow temporal frequency and wide angular frequency spectrum light source. This type of light source was synthesized by the combined effect of spatial, angular, and temporal diversity of speckle reduction technique. The importance of using low spatial and high temporal coherence light source over the broad band and narrow band light source is that it does not require any dispersion compensation mechanism for biological samples. Further, it avoids the formation of speckle or spurious fringes which arises while using narrow band light source.

X-rays Reveal the Internal Structure of Keratin Bundles in Whole Cells

In recent years, X-ray imaging of biological cells has emerged as a complementary alternative to fluorescence and electron microscopy. Different techniques were established and successfully applied to macromolecular assemblies and structures in cells. However, while the resolution is reaching the nanometer scale, the dose is increasing. It is essential to develop strategies to overcome or reduce radiation damage. Here we approach this intrinsic problem by combing two different X-ray techniques, namely ptychography and nanodiffraction, in one experiment and on the same sample. We acquire low dose ptychography overview images of whole cells at a resolution of 65 nm. We subsequently record high-resolution nanodiffraction data from regions of interest. By comparing images from the two modalities, we can exclude strong effects of radiation damage on the specimen. From the diffraction data we retrieve quantitative structural information from intracellular bundles of keratin intermediate filaments such as a filament radius of 5 nm, hexagonal geometric arrangement with an interfilament distance of 14 nm and bundle diameters on the order of 70 nm. Thus, we present an appealing combined approach to answer a broad range of questions in soft-matter physics, biophysics and biology.

A novel water-soluble fluorescent polymer based on perylene bisimides dyes: one-pot preparation and its bio-imaging

Perylene bisimides dye-based water-soluble fluorescent polymer P3, N,N’-bis(3-amyl)-1-bromo-7-{4’-[3’’-(S-poly(N-acryloyl ethylene diamine hydrochloride)-2’’’-methyl propionic acid)propionyloxy hexyloxy]phenyl} perylene-3,4:9,10-tetracarboxylic bisimides, was synthesized with polyelectrolyte modification via one-pot reaction (the reduction reaction of trithioester and click reaction between the thiol group and carbon–carbon double bond were simultaneously conducted in one pot with high conversion). One-pot method can overcome the limitation that usual click reaction between thiol and other groups has low conversion because thiol group is subject to rapid oxidation during purification and storage. Chemical, structural, and optical properties of P3 and intermediate products were fully characterized by nuclear magnetic resonance spectroscopy, Fourier transform infrared, gel permeation chromatograph, UV–vis spectra, and fluorescence spectra, respectively. The results revealed that P3 displayed excellent water solubility and not only exhibited red strong fluorescence emission band in water but also had the similar photoluminescent spectra to those of intermediate products (M4 and P2) in chloroform. Allowing for the potential application in biological detection field, cell viability and live cell imaging with the presence of P3 were further investigated with Hela cells. The results showed that P3 had low cytotoxicity with strong intracellular fluorescence entry. Meanwhile, with the augment of concentration of P3 (0–0.500 mg mL−1), the cell uptake and accumulation of P3 increased and thereby result in enhancement of the intracellular fluorescence. These experiment results suggested that P3 had enormous potential as a fluorescence probe to be an important component in biological detection field.

Thin-film transducers for the detection and imaging of Brillouin oscillations in transmission on cultured cells

Mechanical imaging and characterisation of biological cells has been a subject of interest for the last twenty years. Ultrasonic imaging based on the scanning acoustic microscope (SAM) and mechanical probing have been extensively reported. Large acoustic attenuation at high frequencies and the use of conventional piezo-electric transducers limit the operational frequency of a SAM. This limitation results in lower resolution compared to an optical microscope. Direct mechanical probing in the form of applied stress by contacting probes causes stress to cells and exhibits poor depth resolution. More recently, laser ultrasound has been reported to detect ultrasound in the GHz range via Brillouin oscillations on biological cells. This technique offers a promising new high resolution acoustic cell imaging technique. In this work, we propose, design and apply a thin-film based opto-acoustic transducer for the detection in transmission of Brillouin oscillations on cells. The transducer is used to generate acoustic waves, protect the cells from laser radiation and enhance signal-to-noise ratio (SNR). Experimental traces are presented in water films as well as images of the Brillouin frequency of phantom and fixed 3T3 fibroblast cells.

Optical diffraction tomography techniques for the study of cell pathophysiology

Three-dimensional imaging of biological cells is crucial for the investigation of cell biology, providing valuable information to reveal the mechanisms behind pathophysiology of cells and tissues. Recent advances in optical diffraction tomography (ODT) have demonstrated the potential for the study of various cells with its unique advantages of quantitative and label-free imaging capability. To provide insight on this rapidly growing field of research and to discuss its applications in biology and medicine, we present the summary of the ODT principle and highlight recent studies utilizing ODT with the emphasis on the applications to the pathophysiology of cells.

Two reconstruction procedures for a 3D phaseless inverse scattering problem for the generalized Helmholtz equation

The 3D inverse scattering problem of the reconstruction of the unknown dielectric permittivity in the generalized Helmholtz equation is considered. Applications are in imaging of nanostructures and biological cells. The main difference with the conventional inverse scattering problems is that only the modulus of the scattering wave field is measured. The phase is not measured. The initializing wave field is the incident plane wave. On the other hand, in the previous recent works of the authors about the ‘phaseless topic’ the case of the point source was considered (Klibanov and Romanov 2015 J. Inverse Ill-Posed Problem 23 415–28; J. Inverse Ill-Posed Problem 23 187–93). Two reconstruction procedures are developed.

SIMcheck: a Toolbox for Successful Super-resolution Structured Illumination Microscopy

Three-dimensional structured illumination microscopy (3D-SIM) is a versatile and accessible method for super-resolution fluorescence imaging, but generating high-quality data is challenging, particularly for non-specialist users. We present SIMcheck, a suite of ImageJ plugins enabling users to identify and avoid common problems with 3D-SIM data, and assess resolution and data quality through objective control parameters. Additionally, SIMcheck provides advanced calibration tools and utilities for common image processing tasks. This open-source software is applicable to all commercial and custom platforms, and will promote routine application of super-resolution SIM imaging in cell biology.

Thin-film optoacoustic transducers for subcellular Brillouin oscillation imaging of individual biological cells.

At low frequencies ultrasound is a valuable tool to mechanically characterize and image biological tissues. There is much interest in using high-frequency ultrasound to investigate single cells. Mechanical characterization of vegetal and biological cells by measurement of Brillouin oscillations has been demonstrated using ultrasound in the GHz range. This paper presents a method to extend this technique from the previously reported single-point measurements and line scans into a high-resolution acoustic imaging tool. Our technique uses a three-layered metal-dielectric-metal film as a transducer to launch acoustic waves into the cell we want to study. The design of this transducer and measuring system is optimized to overcome the vulnerability of a cell to the exposure of laser light and heat without sacrificing the signal-to-noise ratio. The transducer substrate shields the cell from the laser radiation, efficiently generates acoustic waves, facilitates optical detection in transmission, and aids with heat dissipation away from the cell. This paper discusses the design of the transducers and instrumentation and presents Brillouin frequency images on phantom, fixed, and living cells.

One-Pot Synthesis of Cy5-Encapsulated Photostable Fluorescent Silica Nanoparticles for Bioimaging

A new type of Cy5-encapsulated photostable fluorescent silica nanoparticles (FSNPs) bearing positive charges have been successfully fabricated by a reverse microemulsion synthesis in one-pot. The Cy5 dye containing four primary amines are embedded into silica via covalent bonds through a silane coupling agent (GPTMS), followed by co-condensation with tetraethylorthosilicate. The uniform-sized, spherical and monodispersed FSNPs have high fluorescence intensity and photostability. The FSNPs exhibit high stability, good biocompatibility as well as low cytotoxicity. These FSNPs can be internalized into live cells and thus fluorescently label the cells. This study provides a simple synthesis approach that can be applied to other water-soluble and amino-modified organic dye molecules for biological targeting and fluorescent cell imaging.

High-contrast epi-fluorescence wide-field imaging of biological cells using integrating-bucket method

We present an optical lock-in detection scheme, called the integrating-bucket technique, as a signal-to-background ratio enhancement method for wide-field fluorescence imaging of biological cells. The proposed method uses sinusoidally modulated illumination light and captures four frames of fluorescence images, one per each quarter of the modulation period, by integrating the fluorescence intensity signal. The capability of this technique is demonstrated by imaging fluorescent bead solutions as well as labeled cells. The results show that the method yields a 4–10dB higher signal contrast than conventional fluorescence microscopy, and a background-free fluorescence image can be extracted within a sub-second time scale. Our findings indicate that the proposed method could be advantageous for the long-term study of live cells.

Super-resolution microscopy based on fluorescence emission difference of cylindrical vector beams

We propose a novel fluorescence emission difference microscopy (FED) system based on focusing cylindrical vector beams. In conventional FED, a Gaussian beam and a 0–2π vortex phase plate are used to generate solid and hollow spots. We focus radially polarized and azimuthally polarized cylindrical vector beams to obtain an expanded solid spot and a shrunken hollow spot, taking advantage of the optical properties of cylindrical vector beams to improve the conventional FED performance. Our novel method enhances FED performance because the hollow spot size determines the FED resolution and an expanded solid spot effectively reduces negative side-lobe emergence during image processing. We demonstrate improved performance theoretically and experimentally using an in-house built FED. Our FED achieved resolution of less than λ/4 in test images of 100nm nanoparticles, better than the confocal image resolution by a factor of approximately 1/3. We also discuss detailed simulation analyses and FED imaging of biological cells.

Three-dimensional imaging of biological cells with picosecond ultrasonics

We use picosecond ultrasonics to image animal cells in vitro—a bovine aortic endothelial cell and a mouse adipose cell—fixed to Ti-coated sapphire. Tightly focused ultrashort laser pulses generate and detect GHz acoustic pulses, allowing three-dimensional imaging (x, y, and t) of the ultrasonic propagation in the cells with ∼1 μm lateral and ∼150 nm depth resolutions. Time-frequency representations of the continuous-wavelet-transform amplitude of the optical reflectivity variations inside and outside the cells show GHz Brillouin oscillations, allowing the average sound velocities of the cells and their ultrasonic attenuation to be obtained as well as the average bulk moduli.

Nonlinear fluorescence imaging by photoinduced charge separation

Manipulation of the optical property of fluorescent probes has been a powerful strategy to establish super-resolution microscopy. We describe a new strategy to realize a probe with a nonlinear fluorescence response by using photoinduced charge separation. In this scheme, the first photon is used for the generation of the charge-separated state and the second photon is for fluorescence excitation. This stepwise two-photon absorption was confirmed by detection of a second-order nonlinear fluorescence response. Transient absorption spectra studies and simulation indicate that fluorescence is emitted through the photophysical pathways we proposed. Fluorescence imaging of biological cells showed marked improvements in image contrast and resolution, demonstrating the usefulness of the fluorescent probe in laser scanning confocal microscopy.

Fluorescence axial nanotomography with plasmonics.

We present a novel imaging technique with super-resolution axial sensitivity, exploiting the changes in fluorescence lifetime above a plasmonic substrate. Using conventional confocal fluorescence lifetime imaging, we show that it is possible to deliver down to 6 nm axial position sensitivity of fluorophores in whole biological cell imaging. We employ this technique to map the topography of the cellular membrane, and demonstrate its application in an investigation of receptor-mediated endocytosis in carcinoma cells.