Live Biological Cells Research Articles

Investigation of an Improved Angular Spectrum Method Based on Holography

Digital holography (DH) is a novel, real-time, non-destructive, and quantitative phase-contrast imaging method that is particularly suitable for label-free live biological cell imaging and real-time dynamic monitoring. It is currently a research hotspot in the interdisciplinary field of optics and biomedical sciences, both domestically and internationally. This article proposes an improved angle spectrum algorithm based on holographic technology, which reconstructs a cellular hologram based on phase information. Optical images and chromosome cell images, reconstructed using holographic technology at different diffraction distances under the improved angle spectrum algorithm, were analyzed and compared. The optimal diffraction distance for reconstructing chromosome cell images was selected, and chromosome cell images reproduced using traditional angle spectrum algorithms, angle spectrum algorithms combined with GS, and improved angle spectrum algorithms were compared. Comparative experiments with the different models show that the proposed algorithm is superior to traditional angle spectrum algorithms in reconstructing cell images based on phase information. Furthermore, experiments have shown that images reconstructed using the improved algorithm can resolve high signal-to-noise ratio information. This algorithmic improvement provides new applications for cellular detection in clinical diagnostics and is more suitable for cell phase reconstruction in practical applications.

Dynamic signal of live biological cells under interferometric scattering (iSCAT) microscopy and its impacts on single-particle tracking

Interferometric scattering (iSCAT) microscopy has been demonstrated as a powerful tool for tracking single nanoparticles at ultrahigh spatial and temporal resolutions. The unmatched performance of iSCAT single-particle tracking (SPT) relies on the fact that the scattering signal is steady and can be linearly increased under strong illumination, circumventing the problems of photobleaching and saturation of fluorescence-based approaches. However, the scattering-based imaging is complicated by the presence of nonspecific scattering background, e.g. in the complex samples of biological cells. To distinguish the signal of interest from the nonspecific scattering background, metallic nanoparticles are often used as the efficient scattering probes. In many applications, reduction of the particle size is favorable because the loading of particle may introduce labeling artifacts. To work with smaller particles of weaker signal, suppression of the heterogeneous background is necessary. In this work, we start with the characterization of dynamic iSCAT signal of living cells by calculating its spatial and temporal Fourier spectra. To reduce the influence of cell background to SPT, a common strategy of background estimation and correction by temporal average filtering is considered. The effect of residual cell background to SPT is evaluated systematically with simulated image data of various signal-to-background ratios. This work benchmarks the localization errors caused by the cell background in SPT, providing a guideline for the interpretation of single-particle diffusion data of iSCAT microscopy.

Deep learning-based cell identification and disease diagnosis using spatio-temporal cellular dynamics in compact digital holographic microscopy

We demonstrate a successful deep learning strategy for cell identification and disease diagnosis using spatio-temporal cell information recorded by a digital holographic microscopy system. Shearing digital holographic microscopy is employed using a low-cost, compact, field-portable and 3D-printed microscopy system to record video-rate data of live biological cells with nanometer sensitivity in terms of axial membrane fluctuations, then features are extracted from the reconstructed phase profiles of segmented cells at each time instance for classification. The time-varying data of each extracted feature is input into a recurrent bi-directional long short-term memory (Bi-LSTM) network which learns to classify cells based on their time-varying behavior. Our approach is presented for cell identification between the morphologically similar cases of cow and horse red blood cells. Furthermore, the proposed deep learning strategy is demonstrated as having improved performance over conventional machine learning approaches on a clinically relevant dataset of human red blood cells from healthy individuals and those with sickle cell disease. The results are presented at both the cell and patient levels. To the best of our knowledge, this is the first report of deep learning for spatio-temporal-based cell identification and disease detection using a digital holographic microscopy system.

The Talbot effect in self-assembled red blood cells investigated by digital holography

The Talbot effect, also known as self-imaging, is a well-established phenomenon observed when a beam of light is transmitted through a periodic pattern and the image of the pattern is reproduced at a regular interval along the optical axis, namely the Talbot length. This effect has been widely investigated and exploited for several applications in different fields. Here we discuss for the first time, to the best of our knowledge, the self-imaging effect due to a self-assembled and quasi-ordered array of live biological cells under illumination by a coherent light beam. In particular, self-assembly of red blood cells (RBCs) provides a monolayer of cells that appear to be quasi-ordered in a trigonal array geometry. Thanks to the recent proof that RBCs can be modeled as a microlens array, the Talbot length can be predicted and the corresponding self-imaging can be observed experimentally. In particular, we investigate the Talbot effect of self-assembled RBC arrays for two different RBC body shapes, i.e. discocytes and spherocytes, by using digital holography as tool for imaging and quantifying this phenomenon. This research could open up a new way to investigate biological material by exploiting its photonics properties.

Quantitative Scanning Microwave Microscopy of the Evolution of a Live Biological Cell in a Physiological Buffer

This article reports the first quantitative characterization by scanning microwave microscopy (SMM) of a live cell in its physiological buffer. The challenges for quantitative SMM of soft matter in liquid were overcome mainly by characterizing the probe–sample interaction through multiscale finite-element full-wave electromagnetic simulation of not only the probe tip but also the probe body and holder. Using quantitative SMM, the relative permittivity of the center of a live L6 cell on the top of its nucleus was determined to be ( $32\pm 6)-j(20 \pm 4)$ , which was in general agreement with the literature and simple estimation. Moreover, taking advantage of the noninvasiveness and subsurface sensitivity of SMM, it was used to monitor the physiological condition of the cell for hours. The results showed that the gradual shrinking of the cell footprint did not impact cell vitality significantly. These results implied that SMM could be a valuable technique for label-free noninvasive characterization of subcellular structures in a live cell, as well as its physio-pathological conditions.

Inverted scanning microwave microscope for in vitro imaging and characterization of biological cells

This paper presents an instrument called an inverted scanning microwave microscope (iSMM), which is capable of performing noninvasive and label-free imaging and characterization of intracellular structures of a live cell on the nanometer scale. In particular, the iSMM is sensitive to not only surface structures but also electromagnetic properties up to one micrometer below the surface. Conveniently, the iSMM can be constructed through straightforward conversion of any scanning probe microscope, such as an atomic force microscope and a scanning tunneling microscope, with a simple metal probe to outperform a traditional SMM in terms of ruggedness, bandwidth, sensitivity, and dynamic range. By contrast, the application of the traditional SMM to date has been limited to mainly surface physics and semiconductor technology because the traditional SMM requires a fragile and expensive probe and is incompatible with saline solution or live cells.

Simultaneous off-axis multiplexed holography and regular fluorescence microscopy of biological cells.

We present a new technique for obtaining simultaneous multimodal quantitative phase and fluorescence microscopy of biological cells, providing both quantitative phase imaging and molecular specificity using a single camera. Our system is based on an interferometric multiplexing module, externally positioned at the exit of an optical microscope. In contrast to previous approaches, the presented technique allows conventional fluorescence imaging, rather than interferometric off-axis fluorescence imaging. We demonstrate the presented technique for imaging fluorescent beads and live biological cells.

Integral refractive index imaging of flowing cell nuclei using quantitative phase microscopy combined with fluorescence microscopy.

We suggest a new multimodal imaging technique for quantitatively measuring the integral (thickness-average) refractive index of the nuclei of live biological cells in suspension. For this aim, we combined quantitative phase microscopy with simultaneous 2-D fluorescence microscopy. We used 2-D fluorescence microscopy to localize the nucleus inside the quantitative phase map of the cell, as well as for measuring the nucleus radii. As verified offline by both 3-D confocal fluorescence microscopy and 2-D fluorescence microscopy while rotating the cells during flow, the nucleus of cells in suspension that are not during division can be assumed to be an ellipsoid. The entire shape of a cell in suspension can be assumed to be a sphere. Then, the cell and nucleus 3-D shapes can be evaluated based on their in-plain radii available from the 2-D phase and fluorescent measurements, respectively. Finally, the nucleus integral refractive index profile is calculated. We demonstrate the new technique on cancer cells, obtaining nucleus refractive index values that are lower than those of the cytoplasm, coinciding with recent findings. We believe that the proposed technique has the potential to be used for flow cytometry, where full 3-D refractive index tomography is too slow to be implemented during flow.

Rapid-scan Fourier-transform coherent anti-Stokes Raman scattering spectroscopy with heterodyne detection.

High-speed Raman spectroscopy has become increasingly important for analyzing chemical dynamics in real time. To address the need, rapid-scan Fourier-transform coherent anti-Stokes Raman scattering (FT-CARS) spectroscopy has been developed to realize broadband CARS measurements at a scan rate of more than 20,000scans/s. However, the detection sensitivity of FT-CARS spectroscopy is inherently low due to the limited number of photons detected during each scan. In this Letter, we show our experimental demonstration of enhanced sensitivity in rapid-scan FT-CARS spectroscopy by heterodyne detection. Specifically, we implemented heterodyne detection by superposing the CARS electric field with an external local oscillator (LO) for their interference. The CARS signal was amplified by simply increasing the power of the LO without the need for increasing the incident power onto the sample. Consequently, we achieved enhancement in signal intensity and the signal-to-noise ratio by factors of 39 and 5, respectively, compared to FT-CARS spectroscopy with homodyne detection. The sensitivity-improved rapid-scan FT-CARS spectroscopy is expected to enable the sensitive real-time observation of chemical dynamics in a broad range of settings, such as combustion engines and live biological cells.

A review of impedance measurements of whole cells.

Impedance measurement of live biological cells is widely accepted as a label free, non-invasive and quantitative analytical method to assess cell status. This method is easy-to-use and flexible for device design and fabrication. In this review, three typical techniques for impedance measurement, i.e., electric cell-substrate impedance sensing, Impedance flow cytometry and electric impedance spectroscopy, are reviewed from the aspects of theory, to electrode design and fabrication, and applications. Benefiting from the integration of microelectronic and microfluidic techniques, impedance sensing methods have expanded their applications to nearly all aspects of biology, including living cell counting and analysis, cell biology research, cancer research, drug screening, and food and environmental safety monitoring. The integration with other techniques, the fabrication of devices for certain biological assays, and the development of point-of-need diagnosis devices is predicted to be future trend for impedance sensing techniques.

Common-path diffraction optical tomography for investigation of three-dimensional structures and dynamics of biological cells

We present an optical holographic micro-tomographic technique for imaging both the three-dimensional structures and dynamics of biological cells. Optical light field images of a sample, illuminated by a plane wave with various illumination angles, are measured in a common-path interferometry, and thus both the three-dimensional refractive index tomogram and two-dimensional dynamics of live biological cells are measured with extremely high sensitivity. The applicability of the technique is demonstrated through quantitative and measurements of morphological, chemical, and mechanical parameters at the individual cell level.

Quantitative phase imaging with single shot digital holography

We demonstrate quantitative phase imaging using single shot digital holography for a calibrated spiral phase object. A single frame of near on-axis digital hologram of a spiral phase plate is recorded and the complex object field in the hologram plane is retrieved using a constrained optimization approach. Experimental results show the feasibility of a quantitative phase imaging technique which has superior performance to conventional Fourier filtering methods. Single shot capability suggests that this method is suitable for holographic imaging of dynamic objects such as live biological cells.

In situ observation of cellular organelles with a contact x-ray microscope

A contact x-ray microscope coupled with a highly intense laser plasma soft x-ray source has been developed and in situ observations of cellular organelles have been conducted. The soft x-rays were generated by irradiating a high power laser pulse onto a thin-foiled gold target and about 1.3×1015 photons/sr were obtained, which allowed the inner structures of live wet biological cells to be imaged. Single shot flash imaging is a key technique to image cellular organelles of live biological cells avoiding degradation of the spatial resolution of the images resulting from motion blur and radiation damage. The use of a fluorescence microscope to identify cellular organelles in conjunction with the soft x-ray microscope has allowed several cellular organelles to be identified precisely in the soft x-ray images. Combining the fluorescence microscope with the soft x-ray microscope will be very useful and will establish the technique as a powerful tool to analyze living function of biological cells.

Transport of NaYF4:Er3+, Yb3+ up-converting nanoparticles into HeLa cells

An effective, simple and practically useful method to incorporate fluorescent nanoparticles inside live biological cells was developed. The internalization time and concentration dependence of a frequently used liposomal transfection factor (Lipofectamine 2000) was studied. A user friendly, one-step technique to obtain water and organic solvent soluble Er3+ and Yb3+ doped NaYF4 nanoparticles coated with polyvinylpyrrolidone was obtained. Structural analysis of the nanoparticles confirmed the formation of nanocrystals of the desired sizes and spectral properties. The internalization of NaYF4 nanoparticles in HeLa cervical cancer cells was determined at different nanoparticle concentrations and for incubation periods from 3 to 24 h. The images revealed a redistribution of nanoparticles inside the cell, which increases with incubation time and concentration levels, and depends on the presence of the transfection factor. The study identifies, for the first time, factors responsible for an effective endocytosis of the up-converting nanoparticles to HeLa cells. Thus, the method could be applied to investigate a wide range of future ‘smart’ theranostic agents. Nanoparticles incorporated into the liposomes appear to be very promising fluorescent probes for imaging real-time cellular dynamics.

Simultaneous immersion Mirau interferometry

A novel technique for label-free imaging of live biological cells in aqueous medium that is insensitive to ambient vibrations is presented. This technique is a spin-off from previously developed immersion Mirau interferometry. Both approaches utilize a modified Mirau interferometric attachment for a microscope objective that can be used both in air and in immersion mode, when the device is submerged in cell medium and has its internal space filled with liquid. While immersion Mirau interferometry involves first capturing a series of images, the resulting images are potentially distorted by ambient vibrations. Overcoming these serial-acquisition challenges, simultaneous immersion Mirau interferometry incorporates polarizing elements into the optics to allow simultaneous acquisition of two interferograms. The system design and production are described and images produced with the developed techniques are presented.

Aberration-free FTIR spectroscopic imaging of live cells in microfluidic devices

The label-free, non-destructive chemical analysis offered by FTIR spectroscopic imaging is a very attractive and potentially powerful tool for studies of live biological cells. FTIR imaging of live cells is a challenging task, due to the fact that cells are cultured in an aqueous environment. While the synchrotron facility has proven to be a valuable tool for FTIR microspectroscopic studies of single live cells, we have demonstrated that high quality infrared spectra of single live cells using an ordinary Globar source can also be obtained by adding a pair of lenses to a common transmission liquid cell. The lenses, when placed on the transmission cell window, form pseudo hemispheres which removes the refraction of light and hence improve the imaging and spectral quality of the obtained data. This study demonstrates that infrared spectra of single live cells can be obtained without the focus shifting effect at different wavenumbers, caused by the chromatic aberration. Spectra of the single cells have confirmed that the measured spectral region remains in focus across the whole range, while spectra of the single cells measured without the lenses have shown some erroneous features as a result of the shift of focus. It has also been demonstrated that the addition of lenses can be applied to the imaging of cells in microfabricated devices. We have shown that it was not possible to obtain a focused image of an isolated cell in a droplet of DPBS in oil unless the lenses are applied. The use of the approach described herein allows for well focused images of single cells in DPBS droplets to be obtained.

Three-dimensional differential interference contrast microscopy using synthetic aperture imaging

We implement differential interference contrast (DIC) microscopy using high-speed synthetic aperture imaging that expands the passband of coherent imaging by a factor of 2.2. For an aperture synthesized coherent image, we apply for the numerical post-processing and obtain a high-contrast DIC image for arbitrary shearing direction and bias retardation. In addition, we obtain images at different depths without a scanning objective lens by numerically propagating the acquired coherent images. Our method achieves high-resolution and high-contrast 3-D DIC imaging of live biological cells. The proposed method will be useful for monitoring 3-D dynamics of intracellular particles.

Full-field and single-shot quantitative phase microscopy using dynamic speckle illumination

We developed an off-axis quantitative phase microscopy that works for a light source with an extremely short spatial coherence length in order to reduce the diffraction noise and enhance the spatial resolution. A dynamic speckle wave whose coherence length is 440 nm was used as an illumination source. To implement an off-axis interferometry for a source of low spatial coherence, a diffraction grating was inserted in the reference beam path. In doing so, an oblique illumination was generated without rotation of the wavefront, which leads to a full-field and single-shot phase recording with improved phase sensitivity of more than a factor of 10 in comparison with coherent illumination. The spatial resolution, both laterally and axially, and the depth selectivity are significantly enhanced due to the wide angular spectrum of the speckle wave. We applied our method to image the dynamics of small intracellular particles in live biological cells. With enhanced phase sensitivity and speed, the proposed method will serve as a useful tool to study the dynamics of biological specimens.

High-speed synthetic aperture microscopy for live cell imaging

We present a high-speed synthetic aperture microscopy for quantitative phase imaging of live biological cells. We measure 361 complex amplitude images of an object with various directions of illumination covering an NA of 0.8 in less than one-thirteenth of a second and then combine the images with a phase-referencing method to create a synthesized phase image. Because of the increased depth selectivity, artifacts from diffraction that are typically present in coherent imaging are significantly suppressed, and lateral resolution of phase imaging is improved. We use the instrument to demonstrate high-quality phase imaging of live cells, both static and dynamic, and thickness measurements of a nanoscale cholesterol helical ribbon.

Parallel on-axis holographic phase microscopy of biological cells and unicellular microorganism dynamics

We apply a wide-field quantitative phase microscopy technique based on parallel two-step phase-shifting on-axis interferometry to visualize live biological cells and microorganism dynamics. The parallel on-axis holographic approach is more efficient with camera spatial bandwidth consumption compared to previous off-axis approaches and thus can capture finer sample spatial details, given a limited spatial bandwidth of a specific digital camera. Additionally, due to the parallel acquisition mechanism, the approach is suitable for visualizing rapid dynamic processes, permitting an interferometric acquisition rate equal to the camera frame rate. The method is demonstrated experimentally through phase microscopy of neurons and unicellular microorganisms.