Multi-wavelength multi-direction laser light scattering for cell characterization using machine learning-based methods.

Cell identification and analysis play a crucial role in many biology- and health-related applications. The internal and surface structures of a cell are complex and many of the features are sub-micron in scale. Well-resolved images of these features cannot be obtained using optical microscopy. Previous studies have reported that the single-cell angular laser-light scattering patterns (ALSP) can be used for label-free cell identification and analysis. The ALSP can be affected by cell properties and the wavelength of the probing laser. Two cell properties, cell surface roughness and the number of mitochondria, are investigated in this study. The effects of probing laser wavelengths (blue, green, and red) and the directions of scattered light collection (forward, side, and backward) are studied to determine the optimum conditions for distinguishing the two cell properties. Machine learning (ML) analysis has been applied to ALSP obtained from numerical simulations. The results of ML analysis show that the backward scattering is the best direction for characterizing the surface roughness, while the forward scattering is the best direction for differentiating the number of mitochondria. The laser light having red or green wavelength is found to perform better than that having the blue wavelength in differentiating the surface roughness and the number of mitochondria. This study provides important insights into the effects of probing laser wavelength on gaining information about cells from their ALSP.

Integration of light scattering with machine learning for label free cell detection.

Light scattering has been used for label-free cell detection. The angular light scattering patterns from the cells are unique to them based on the cell size, nucleus size, number of mitochondria, and cell surface roughness. The patterns collected from the cells can then be classified based on different image characteristics. We have also developed a machine learning (ML) method to classify these cell light scattering patterns. As a case study we have used this light scattering technique integrated with the machine learning to analyze staurosporine-treated SH-SY5Y neuroblastoma cells and compare them to non-treated control cells. Experimental results show that the ML technique can provide a classification accuracy (treated versus non-treated) of over 90%. The predicted percentage of the treated cells in a mixed solution is within 5% of the reference (ground-truth) value and the technique has the potential to be a viable method for real-time detection and diagnosis.

Crossed beam energy transfer between optically smoothed laser beams in inhomogeneous plasmas.

Crossed beam energy transfer, CBET, in high-intensity laser-plasma interaction is investigated for the case of optically smoothed laser beams. In the two approaches to laser-driven inertial confinement fusion experiments, the direct-drive and the indirect-drive, CBET is of great importance because it governs the coupling of laser energy to the plasma. We use the two-dimensional wave-coupling code Harmony to simulate the transfer between two laser beams with speckle structure that overlap in a plasma with an inhomogeneous flow profile. We compare the CBET dynamics for laser beams with spatial incoherence and with spatio-temporal incoherence; in particular we apply the smoothing techniques using random phase plates (RPPs) and smoothing by spectral dispersion (SSD), respectively. It is found that for laser beams (wavelength λ0) with intensities (IL) above IL ∼ 2 × 1015 W cm-2(λ0/0.35 µm)-2(Te/keV), both the so-called plasma-induced smoothing as well as self-focusing in intense laser speckles induce temporal incoherence; the latter affects the CBET and the angular distribution of the light transmitted behind the zone of beam overlap. For RPP-smoothed incident beams, the resulting band width of the transmitted light can already be of the same order as the effective band width of the SSD available at major laser facilities. We examine the conditions when spatio-temporal smoothing techniques become efficient for CBET. This article is part of a discussion meeting issue 'Prospects for high gain inertial fusion energy (part 1)'.

Physical characterization of hematopoietic stem cells using multidirectional label-free light scatterings.

An experimental setup capable of measuring simultaneous 2D scattered light angular distribution from two directions to study cell morphology without the use of bio-labels was developed. Experiments with hematopoietic stem cells (CD34+ cells) show good agreement with detailed numerical simulations of light scattering. Numerical simulations and computer models of cells are used to identify physical features of cells with the largest scattering cross sections. This allows for determination of size, geometry of the nucleus and distribution of mitochondria in hematopoietic stem cells by means of our label-free method.

Label-free and noninvasive optical detection of the distribution of nanometer-size mitochondria in single cells.

A microfluidic flow cytometric technique capable of obtaining information on nanometer-sized organelles in single cells in a label-free, noninvasive optical manner was developed. Experimental two-dimensional (2D) light scattering patterns from malignant lymphoid cells (Jurkat cell line) and normal hematopoietic stem cells (cord blood CD34+ cells) were compared with those obtained from finite-difference time-domain simulations. In the simulations, we assumed that the mitochondria were randomly distributed throughout a Jurkat cell, and aggregated in a CD34+ cell. Comparison of the experimental and simulated light scattering patterns led us to conclude that distinction from these two types of cells may be due to different mitochondrial distributions. This observation was confirmed by conventional confocal fluorescence microscopy. A method for potential cell discrimination was developed based on analysis of the 2D light scattering patterns. Potential clinical applications using mitochondria as intrinsic biological markers in single cells were discussed in terms of normal cells (CD34+ cell and lymphocytes) versus malignant cells (THP-1 and Jurkat cell lines).

Microscope-based label-free microfluidic cytometry.

A microscope-based label-free microfluidic cytometer capable of acquiring two dimensional light scatter patterns from single cells, pattern analysis of which determines cellular information such as cell size, orientation and inner nanostructure, was developed. Finite-difference time-domain numerical simulations compared favorably with experimental scatter patterns from micrometer-sized beads and cells. The device was capable of obtaining light scattering patterns from the smallest mature blood cells (platelets) and cord blood hematopoietic stem/progenitor cells (CD34 + cells) and myeloid precursor cells. The potential for evaluation of cells using this label-free microfluidic cytometric technique was discussed.

Wide-angle light-scattering differentiation of organelle-size particle distributions in whole cells.

A finite-difference time-domain (FDTD) method is used to study the multiple scattering from many organelle-size particles distributed in a biological cell. Conventional flow cytometry, where the small-angle forward scatter (FSC) intensity and side scatter (SSC) intensity are used for cell characterizations, may have difficulties to differentiate the organelle distributions in biological cells. Based on the FDTD simulations, a light-scattering methodology is proposed here to overcome such a problem. This method differentiates the dense and sparse distributions of organelle-size particles in a cell, by counting the peak numbers in both large-angle FSC and wide-angle SSC, with the multiple scattering effects being considered. Implemented with a wide-angle microfluidic cytometer, the approach demonstrated in this theoretical study may find potential applications in clinics for label-free cell physiological study.

Light scattering characterization of mitochondrial aggregation in single cells.

Three dimensional finite-difference time-domain (FDTD) simulations are employed to show that light scattering techniques may be used to infer the mitochondrial distributions that exist within single biological cells. Two-parameter light scattering plots of the FDTD light scattering spectra show that the small angle forward scatter can be used to differentiate the case of a random distribution of mitochondria within a cell model from that in which the mitochondria are aggregated to the nuclear periphery. Fourier transforms of the wide angle side scatter spectra show a consistent highest dominant frequency, which may be used for size differentiation of biological cells with distributed mitochondria.

Quasimonoenergetic electron beams with relativistic energies and ultrashort duration from laser-solid interactions at 0.5 kHz.

We investigate the production of electron beams from the interaction of relativistically-intense laser pulses with a solid-density SiO(2) target in a regime where the laser pulse energy is approximately mJ and the repetition rate approximately kHz. The electron beam spatial distribution and spectrum were investigated as a function of the plasma scale length, which was varied by deliberately introducing a moderate-intensity prepulse. At the optimum scale length of lambda/2, the electrons are emitted in a collimated beam having a quasimonoenergetic distribution that peaked at approximately 0.8 MeV. A highly reproducible structure in the spatial distribution exhibits an evacuation of electrons along the laser specular direction and suggests that the electron beam duration is comparable to that of the laser pulse. Particle-in-cell simulations which are in good agreement with the experimental results offer insights on the acceleration mechanism by the laser field.

Measurements of light scattering in an integrated microfluidic waveguide cytometer.

An integrated microfluidic planar optical waveguide system for measuring light scattered from a single scatterer is described. This system is used to obtain 2D side-scatter patterns from single polystyrene microbeads in a fluidic flow. Vertical fringes in the 2D scatter patterns are used to infer the location of the 90-deg scatter (polar angle). The 2D scatter patterns are shown to be symmetrical about the azimuth angle at 90 deg. Wide-angle comparisons between the experimental scatter patterns and Mie theory simulations are shown to be in good agreement. A method based on the Fourier transform analysis of the experimental and Mie simulation scatter patterns is developed for size differentiation.

2D light scattering patterns of mitochondria in single cells.

The ability to characterize the mitochondria in single living cells may provide a powerful tool in clinical applications. We have recently developed a 2D (both polar angle and azimuth angle dependences) light scattering cytometric technique which we apply here to assess experimental 2D light scattering patterns from single biological cells (yeast and human). We compare these patterns to those obtained from simulations using a 3D Finite-Difference Time-Domain (FDTD) method and demonstrate that microstructure (e.g., the cytoplasm and/or nucleus) of cells generates fringes of scattered light, while in the larger human cells the light scattered by the mitochondria dominates the scatter pattern, forming compact regions of high intensity that we term 'blobs'. These blobs provide information on the mitochondria within the cell and their analysis may ultimately be useful as a diagnostic technique.

A miniaturized wide-angle 2D cytometer.

BACKGROUND: We present an optical waveguide based cytometer that is capable of simultaneously collecting the light scattered by cells over a wide range of solid angles. Such comprehensive scattering data are a prerequisite for the microstructural characterization of cells. METHODS: We use latex beads as cell mimics, and demonstrate the ability of this new cytometer to collect back-scattered light in two dimensions (2D). This cytometer is based on a liquid-core optical waveguide, excited by prism coupling, that also serves as the microfluidic channel. In principle, our use of a hemispherical lens allows the collection of scattered light from 0 to 180 degrees in 2D. RESULTS: The experimentally observed positions of the intensity peaks of the back-scattered light agree well with theoretical prediction of scattering from both 4.0- and 9.6-mum diameter latex beads. The position of the bead, relative to the axes of the hemispherical lens and the microchannel, strongly affects the scattering pattern. We discuss a computational method for determining these offsets. CONCLUSIONS: We show that wide-angle 2D light scattering patterns of cell-sized latex beads can be observed in a microfluidic-based optical cytometer that uses leaky waveguide mode excitation. This chip-based system is compatible with emerging chip-based technologies.