Four-dimensional motility tracking of biological cells by digital holographic microscopy.
ABSTRACT: Three-dimensional profiling and tracking by digital holography microscopy (DHM) provide label-free and quantitative analysis of the characteristics and dynamic processes of objects, since DHM can record real-time data for microscale objects and produce a single hologram containing all the information about their three-dimensional structures. Here, we have utilized DHM to visualize suspended microspheres and microfibers in three dimensions, and record the four-dimensional trajectories of free-swimming cells in the absence of mechanical focus adjustment. The displacement of microfibers due to interactions with cells in three spatial dimensions has been measured as a function of time at subsecond and micrometer levels in a direct and straightforward manner. It has thus been shown that DHM is a highly efficient and versatile means for quantitative tracking and analysis of cell motility.
Project description:Significance: Over the past decade, laser-based digital holographic microscopy (DHM), an important approach in the field of quantitative-phase imaging techniques, has become a significant label-free modality for live-cell imaging and used particularly in cellular neuroscience. However, coherent noise remains a major drawback for DHM, significantly limiting the possibility to visualize neuronal processes and precluding important studies on neuronal connectivity. Aim: The goal is to develop a DHM technique able to sharply visualize thin neuronal processes. Approach: By combining a wavelength-tunable light source with the advantages of hologram numerical reconstruction of DHM, an approach called polychromatic DHM (P-DHM), providing OPD images with drastically decreased coherent noise, was developed. Results: When applied to cultured neuronal networks with an air microscope objective ( 20× , 0.8 NA), P-DHM shows a coherent noise level typically corresponding to 1 nm at the single-pixel scale, in agreement with the 1/N -law, allowing to readily visualize the 1-?m -wide thin neuronal processes with a signal-to-noise ratio of ?5 . Conclusions: Therefore, P-DHM represents a very promising label-free technique to study neuronal connectivity and its development, including neurite outgrowth, elongation, and branching.
Project description:Digital holographic microscopy (DHM) has its intrinsic ability to refocusing a sample by numerically propagating an object wave from its hologram plane to its image plane. In this paper opposite-view digital holographic microscopy (OV-DHM) is demonstrated for autofocusing, namely, digitally determining the location of the image plane, and refocusing the object wave without human intervention. In OV-DHM, a specimen is illuminated from two sides in a 4?-alike configuration, and two holograms are generated and recorded by a CCD camera along two orthogonal polarization orientations. The image plane of the sample is determined by finding the minimal variation between the two object waves, and consequently refocusing is performed by propagating the waves to the image plane. Furthermore, the field of view (FOV) of OV-DHM can be extended by combining the two object waves which have an angle in-between. The proposed technique also has the potential to reduce speckle noise and out-of-focus background.
Project description:We describe an optimized digital holographic microscopy system (DHM) suitable for high-resolution visualization of living cells under conditions of altered macroscopic mechanical forces such as those that arise from changes in gravitational force. Experiments were performed on both a ground-based microgravity simulation platform known as the random positioning machine (RPM) as well as during a parabolic flight campaign (PFC). Under these conditions the DHM system proved to be robust and reliable. In addition, the stability of the system during disturbances in gravitational force was further enhanced by implementing post-processing algorithms that best exploit the intrinsic advantages of DHM for hologram autofocusing and subsequent image registration. Preliminary results obtained in the form of series of phase images point towards sensible changes of cytoarchitecture under states of altered gravity.
Project description:Many daily situations require us to track multiple objects and people. This ability has traditionally been investigated in observers tracking objects in a plane. This simplification of reality does not address how observers track objects when targets move in three dimensions. Here, we study how observers track multiple objects in 2D and 3D while manipulating the average speed of the objects and the average distance between them. We show that performance declines as speed increases and distance decreases and that overall tracking accuracy is always higher in 3D than in 2D. The effects of distance and dimensionality interact to produce a more than additive improvement in performance during tracking in 3D compared to 2D. We propose an ideal observer model that uses the object dynamics and noisy observations to track the objects. This model provides a good fit to the data and explains the key findings of our experiment as originating from improved inference of object identity by adding the depth dimension.
Project description:<h4>Significance</h4>Digital holographic microscopy (DHM) is a promising technique for the study of semitransparent biological specimen such as red blood cells (RBCs). It is important and meaningful to detect and count biological cells at the single cell level in biomedical images for biomarker discovery and disease diagnostics. However, the biological cell analysis based on phase information of images is inefficient due to the complexity of numerical phase reconstruction algorithm applied to raw hologram images. New cell study methods based on diffraction pattern directly are desirable.<h4>Aim</h4>Deep fully convolutional networks (FCNs) were developed on raw hologram images directly for high-throughput label-free cell detection and counting to assist the biological cell analysis in the future.<h4>Approach</h4>The raw diffraction patterns of RBCs were recorded by use of DHM. Ground-truth mask images were labeled based on phase images reconstructed from RBC holograms using numerical reconstruction algorithm. A deep FCN, which is UNet, was trained on the diffraction pattern images to achieve the label-free cell detection and counting.<h4>Results</h4>The implemented deep FCNs provide a promising way to high-throughput and label-free counting of RBCs with a counting accuracy of 99% at a throughput rate of greater than 288 cells per second and 200???m??×??200???m field of view at the single cell level. Compared to convolutional neural networks, the FCNs can get much better results in terms of accuracy and throughput rate.<h4>Conclusions</h4>High-throughput label-free cell detection and counting were successfully achieved from diffraction patterns with deep FCNs. It is a promising approach for biological specimen analysis based on raw hologram directly.
Project description:We demonstrate the use of two-color digital holographic microscopy (DHM) for imaging microbiological subjects. The use of two wavelengths significantly reduces artifacts present in the reconstructed data, allowing us to image weakly-scattering objects in close proximity to strongly-scattering objects. We demonstrate this by reconstructing the shape of the flagellum of a unicellular eukaryotic parasite Leishmania mexicana in close proximity to a more strongly-scattering cell body. Our approach also yields a reduction of approximately one third in the axial position uncertainty when tracking the motion of swimming cells at low magnification, which we demonstrate with a sample of Escherichia coli bacteria mixed with polystyrene beads. The two-wavelength system that we describe introduces minimal additional complexity into the optical system, and provides significant benefits.
Project description:Single-shot, two-frame, ?-shifted spatially multiplexed interference microscopy (?-SMIM) is presented as an improvement to previous SMIM implementations, introducing a versatile, robust, fast, and accurate method for cumbersome, noisy, and low-contrast phase object analysis. The proposed ?-SMIM equips a commercially available nonholographic microscope with a high-speed (video frame rate) enhanced quantitative phase imaging (QPI) capability by properly placing a beam-splitter in the microscope embodiment to simultaneously (in a single shot) record two holograms mutually phase shifted by ? radians at the expense of reducing the field of view. Upon subsequent subtractive superimposition of holograms, a ?-hologram is generated with reduced background and improved modulation of interference fringes. These features determine superior phase retrieval quality, obtained by employing the Hilbert spiral transform on the ?-hologram, as compared with a single low-quality (low signal-to-noise ratio) hologram analysis. In addition, ?-SMIM enables accurate in-vivo analysis of high dynamic range phase objects, otherwise measurable only in static regime using time-consuming phase-shifting. The technique has been validated utilizing a 20??×????/??0.46 NA objective in a regular Olympus BX-60 upright microscope for QPI of different lines of prostate cancer cells and flowing microbeads.
Project description:We propose and demonstrate a novel technique for efficient local fixing of photorefractive polymer hologram using a laser beam. In the technique, a CO(2) laser beam is used to heat the sample and a local hologram can be fixed easier than previous methods. By using thinner glass substrates for the photorefractive device, the hologram can be fixed efficiently and at much faster speed. The fixation efficiency can be greater than 80% and the hologram can be fixed in the order of a second. This technique is critical for dynamic holographic three-dimensional display and holographic data storage.
Project description:Neurons and neural networks often extend hundreds of micrometers in three dimensions. Capturing the calcium transients associated with their activity requires volume imaging methods with subsecond temporal resolution. Such speed is a challenge for conventional two-photon laser-scanning microscopy, because it depends on serial focal scanning in 3D and indicators with limited brightness. Here we present an optical module that is easily integrated into standard two-photon laser-scanning microscopes to generate an axially elongated Bessel focus, which when scanned in 2D turns frame rate into volume rate. We demonstrated the power of this approach in enabling discoveries for neurobiology by imaging the calcium dynamics of volumes of neurons and synapses in fruit flies, zebrafish larvae, mice and ferrets in vivo. Calcium signals in objects as small as dendritic spines could be resolved at video rates, provided that the samples were sparsely labeled to limit overlap in their axially projected images.
Project description:Molecular trafficking within cells, tissues and engineered three-dimensional multicellular models is critical to the understanding of the development and treatment of various diseases including cancer. However, current tracking methods are either confined to two dimensions or limited to an interrogation depth of ?15??m. Here we present a three-dimensional tracking method capable of quantifying rapid molecular transport dynamics in highly scattering environments at depths up to 200??m. The system has a response time of 1?ms with a temporal resolution down to 50??s in high signal-to-noise conditions, and a spatial localization precision as good as 35?nm. Built on spatiotemporally multiplexed two-photon excitation, this approach requires only one detector for three-dimensional particle tracking and allows for two-photon, multicolour imaging. Here we demonstrate three-dimensional tracking of epidermal growth factor receptor complexes at a depth of ?100??m in tumour spheroids.