Fast photothermal spatial light modulation for quantitative phase imaging at the nanoscale.

Spatial light modulators have become an essential tool for advanced microscopy, enabling breakthroughs in 3D, phase, and super-resolution imaging. However, continuous spatial-light modulation that is capable of capturing sub-millisecond microscopic motion without diffraction artifacts and polarization dependence is challenging. Here we present a photothermal spatial light modulator (PT-SLM) enabling fast phase imaging for nanoscopic 3D reconstruction. The PT-SLM can generate a step-like wavefront change, free of diffraction artifacts, with a high transmittance and a modulation efficiency independent of light polarization. We achieve a phase-shift > π and a response time as short as 70 µs with a theoretical limit in the sub microsecond range. We used the PT-SLM to perform quantitative phase imaging of sub-diffractional species to decipher the 3D nanoscopic displacement of microtubules and study the trajectory of a diffusive microtubule-associated protein, providing insights into the mechanism of protein navigation through a complex microtubule network.

Scratch assay microscopy: A reaction-diffusion equation approach for common instruments and data.

Scratch assay is an easy and widely used "in vitro" technique to study cell migration and proliferation. In this work we focus on its modelling and on the capability to distinguish between these two phenomena that the simpler and common models are not able to disentangle. We adapted a model based on reaction-diffusion equation for being used with common microscopy instruments/data and therefore taking place in the gap between simpler modelling approaches and complex ones. An optimized image analysis pipeline and numerical least-squares fit provide estimates of the scratch proliferation and diffusion coefficients l and D. This work is intended as a first of a series in which the model is tested and its robustness and reproducibility are evaluated. Test samples were NIH3T3 cells scratch assays with proliferation and migration stimulated by varying the foetal bovine serum amount in the culture medium (10%, 7.5%, 5% and 2.5%). Results demonstrate, notwithstanding an expected l-D anticorrelation, the model capability to disentangle them. The 7.5% serum treatment can be identified as the model sensitivity limit. Treat-control l and D variations showed an intra-experiment reproducibility (∼±0.05∕h and ∼±200µm2∕h respectively) consistent with single fit typical uncertainties (∼±0.02∕h and ∼±300µm2∕h respectively).

Green Fluorescent Protein and Phase-Contrast Image Fusion via Generative Adversarial Networks.

In the field of cell and molecular biology, green fluorescent protein (GFP) images provide functional information embodying the molecular distribution of biological cells while phase-contrast images maintain structural information with high resolution. Fusion of GFP and phase-contrast images is of high significance to the study of subcellular localization, protein functional analysis, and genetic expression. This paper proposes a novel algorithm to fuse these two types of biological images via generative adversarial networks (GANs) by carefully taking their own characteristics into account. The fusion problem is modelled as an adversarial game between a generator and a discriminator. The generator aims to create a fused image that well extracts the functional information from the GFP image and the structural information from the phase-contrast image at the same time. The target of the discriminator is to further improve the overall similarity between the fused image and the phase-contrast image. Experimental results demonstrate that the proposed method can outperform several representative and state-of-the-art image fusion methods in terms of both visual quality and objective evaluation.

High-Resolution and Quantitative X-Ray Phase-Contrast Tomography for Mouse Brain Research.

Imaging techniques for visualizing cerebral vasculature and distinguishing functional areas are essential and critical to the study of various brain diseases. In this paper, with the X-ray phase-contrast imaging technique, we proposed an experiment scheme for the ex vivo mouse brain study, achieving both high spatial resolution and improved soft-tissue contrast. This scheme includes two steps: sample preparation and volume reconstruction. In the first step, we use heparinized saline to displace the blood inside cerebral vessels and then replace it with air making air-filled mouse brain. After sample preparation, X-ray phase-contrast tomography is performed to collect the data for volume reconstruction. Here, we adopt a phase-retrieval combined filtered backprojection method to reconstruct its three-dimensional structure and redesigned the reconstruction kernel. To evaluate its performance, we carried out experiments at Shanghai Synchrotron Radiation Facility. The results show that the air-tissue structured cerebral vasculatures are highly visible with propagation-based phase-contrast imaging and can be clearly resolved in reconstructed cross-images. Besides, functional areas, such as the corpus callosum, corpus striatum, and nuclei, are also clearly resolved. The proposed method is comparable with hematoxylin and eosin staining method but represents the studied mouse brain in three dimensions, offering a potential powerful tool for the research of brain disorders.

Segmentation and Tracking of Lymphocytes Based on Modified Active Contour Models in Phase Contrast Microscopy Images.

The paper proposes an improved active contour model for segmenting and tracking accurate boundaries of the single lymphocyte in phase-contrast microscopic images. Active contour models have been widely used in object segmentation and tracking. However, current external-force-inspired methods are weak at handling low-contrast edges and suffer from initialization sensitivity. In order to segment low-contrast boundaries, we combine the region information of the object, extracted by morphology gray-scale reconstruction, and the edge information, extracted by the Laplacian of Gaussian filter, to obtain an improved feature map to compute the external force field for the evolution of active contours. To alleviate initial location sensitivity, we set the initial contour close to the real boundaries by performing morphological image processing. The proposed method was tested on live lymphocyte images acquired through the phase-contrast microscope from the blood samples of mice, and comparative experimental results showed the advantages of the proposed method in terms of the accuracy and the speed. Tracking experiments showed that the proposed method can accurately segment and track lymphocyte boundaries in microscopic images over time even in the presence of low-contrast edges, which will provide a good prerequisite for the quantitative analysis of lymphocyte morphology and motility.

Laplace field microscopy for label-free imaging of dynamic biological structures.

We present Laplace field microscopy as a method for generating intrinsic contrast of transparent specimens. This technique uses a spatial light modulator to perform the Laplacian of the field in the Fourier plane of a microscope image. The resulting image incorporates phase information and thus renders high contrast images from phase objects. We demonstrate the potential of the method by imaging index-matched beads, unlabeled tissue slices, and dynamic live cells.

Beyond the lateral resolution limit by phase imaging.

We present a theory to extend the classical Abbe resolution limit by introducing a spatially varying phase into the illumination beam of a phase imaging system. It allows measuring lateral and axial distance differences between point sources to a higher accuracy than intensity imaging alone. Various proposals for experimental realization are debated. Concretely, the phase of point scatterers' interference is experimentally visualized by high numerical aperture (NA = 0.93) digital holographic microscopy combined with angular scanning. Proof-of-principle measurements are presented by using sub-wavelength nanometric holes on an opaque metallic film. In this manner, Rayleighs classical two-point resolution condition can be rebuilt. With different illumination phases, enhanced bandpass information content is demonstrated, and its spatial resolution is theoretically shown to be potentially signal-to-noise ratio limited.

Restoration of weak phase-contrast images recorded with a high degree of defocus: the "twin image" problem associated with CTF correction.

Relatively large values of objective-lens defocus must normally be used to produce detectable levels of image contrast for unstained biological specimens, which are generally weak phase objects. As a result, a subsequent restoration operation must be used to correct for oscillations in the contrast transfer function (CTF) at higher resolution. Currently used methods of CTF correction assume the ideal case in which Friedel mates in the scattered wave have contributed pairs of Fourier components that overlap with one another in the image plane. This "ideal" situation may be only poorly satisfied, or not satisfied at all, as the particle size gets smaller, the defocus value gets larger, and the resolution gets higher. We have therefore investigated whether currently used methods of CTF correction are also effective in restoring the single-sideband image information that becomes displaced (delocalized) by half (or more) the diameter of a particle of finite size. Computer simulations are used to show that restoration either by "phase flipping" or by multiplying by the CTF recovers only about half of the delocalized information. The other half of the delocalized information goes into a doubly defocused "twin" image of the type produced during optical reconstruction of an in-line hologram. Restoration with a Wiener filter is effective in recovering the delocalized information only when the signal-to-noise ratio (S/N) is orders of magnitude higher than that which exists in low-dose images of biological specimens, in which case the Wiener filter approaches division by the CTF (i.e. the formal inverse). For realistic values of the S/N, however, the "twin image" problem seen with a Wiener filter is very similar to that seen when either phase flipping or multiplying by the CTF is used for restoration. The results of these simulations suggest that CTF correction is a poor alternative to using a Zernike-type phase plate when imaging biological specimens, in which case the images can be recorded in a close-to-focus condition, and delocalization of high-resolution information is thus minimized.

Quantitative amplitude and phase contrast imaging in a scanning transmission X-ray microscope.

Phase contrast in X-ray imaging provides lower radiation dose, and dramatically higher contrast at multi-keV photon energies when compared with absorption contrast. We describe here the use of a segmented detector in a scanning transmission X-ray microscope to collect partially coherent bright field images. We have adapted a Fourier filter reconstruction technique developed by McCallum, Landauer and Rodenburg to retrieve separate, quantitative maps of specimen phase shift and absorption. This is demonstrated in the imaging of a germanium test pattern using 525eV soft X-rays.