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.

Radial profile detection of multiple spherical particles in contact with interacting surfaces.

Adhesive interactions of soft materials play an important role in nature and technology. Interaction energies can be quantified by determining contact areas of deformable microparticles with the help of reflection interference contrast microscopy (RICM). For high throughput screening of adhesive interactions, a method to automatically evaluate large amounts of interacting microparticles was developed. An image is taken which contains circular interference patterns with visual characteristics that depend on the probe's shape due to its surface interaction. We propose to automatically detect radial profiles in images, and to measure the contact radius and size of the spherical probe, allowing the determination of particle-surface interaction energy in a simple and fast imaging and image analysis setup. To achieve this, we analyze the image gradient and we perform template matching that utilizes the physical foundations of reflection interference contrast microscopy.

Embedded pupil function recovery for Fourier ptychographic microscopy.

We develop and test a pupil function determination algorithm, termed embedded pupil function recovery (EPRY), which can be incorporated into the Fourier ptychographic microscopy (FPM) algorithm and recover both the Fourier spectrum of sample and the pupil function of imaging system simultaneously. This EPRY-FPM algorithm eliminates the requirement of the previous FPM algorithm for a priori knowledge of the aberration in the imaging system to reconstruct a high quality image. We experimentally demonstrate the effectiveness of this algorithm by reconstructing high resolution, large field-of-view images of biological samples. We also illustrate that the pupil function we retrieve can be used to study the spatially varying aberration of a large field-of-view imaging system. We believe that this algorithm adds more flexibility to FPM and can be a powerful tool for the characterization of an imaging system's aberration.

Automatic classification of the interferential tear film lipid layer using colour texture analysis.

The tear film lipid layer is heterogeneous among the population. Its classification depends on its thickness and can be done using the interference pattern categories proposed by Guillon. This papers presents an exhaustive study about the characterisation of the interference phenomena as a texture pattern, using different feature extraction methods in different colour spaces. These methods are first analysed individually and then combined to achieve the best results possible. The principal component analysis (PCA) technique has also been tested to reduce the dimensionality of the feature vectors. The proposed methodologies have been tested on a dataset composed of 105 images from healthy subjects, with a classification rate of over 95% in some cases.

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.

Using ultrahigh sensitive optical microangiography to achieve comprehensive depth resolved microvasculature mapping for human retina.

This paper presents comprehensive and depth-resolved retinal microvasculature images within human retina achieved by a newly developed ultrahigh sensitive optical microangiography (UHS-OMAG) system. Due to its high flow sensitivity, UHS-OMAG is much more sensitive to tissue motion due to the involuntary movement of the human eye and head compared to the traditional OMAG system. To mitigate these motion artifacts on final imaging results, we propose a new phase compensation algorithm in which the traditional phase-compensation algorithm is repeatedly used to efficiently minimize the motion artifacts. Comparatively, this new algorithm demonstrates at least 8 to 25 times higher motion tolerability, critical for the UHS-OMAG system to achieve retinal microvasculature images with high quality. Furthermore, the new UHS-OMAG system employs a high speed line scan CMOS camera (240 kHz A-line scan rate) to capture 500 A-lines for one B-frame at a 400 Hz frame rate. With this system, we performed a series of in vivo experiments to visualize the retinal microvasculature in humans. Two featured imaging protocols are utilized. The first is of the low lateral resolution (16 µm) and a wide field of view (4 × 3 mm(2) with single scan and 7 × 8 mm(2) for multiple scans), while the second is of the high lateral resolution (5 µm) and a narrow field of view (1.5 × 1.2 mm(2) with single scan). The great imaging performance delivered by our system suggests that UHS-OMAG can be a promising noninvasive alternative to the current clinical retinal microvasculature imaging techniques for the diagnosis of eye diseases with significant vascular involvement, such as diabetic retinopathy and age-related macular degeneration.

Detrended fluctuation analysis of membrane flickering in discocyte and spherocyte red blood cells using quantitative phase microscopy.

Dynamic analyses of vibrational motion in cell membranes provide a lot of information on the complex dynamic motilities of a red blood cell (RBC). Here, we present the correlation properties of membrane fluctuation in discocyte and spherocyte RBCs by using quantitative phase microscopy (QPM). Since QPM can provide nanometer sensitivity in thickness measurement within a millisecond time scale, we were able to observe the membrane flicking of an RBC in nanometer resolution up to the bandwidth of 50 Hz. The correlation properties of the vibrational motion were analyzed with the detrended fluctuation analysis (DFA) method. Fractal scaling exponent α in the DFA method was calculated for the vibrational motion of a cell surface at various surface points for normal discocyte and abnormal spherocyte RBCs. Measured α values for normal RBCs are distributed between 0.7 and 1.0, whereas those for abnormal spherocyte RBCs are within a range from 0.85 to 1.2. We have also verified that the vibrational motion of background fluid outside of a cell has an α value close to 0.5, which is a typical property of an uncorrelated white noise.

Combination of Raman tweezers and quantitative differential interference contrast microscopy for measurement of dynamics and heterogeneity during the germination of individual bacterial spores.

Raman tweezers and quantitative differential interference contrast (DIC) microscopy are combined to monitor the dynamic germination of individual bacterial spores of Bacillus species, as well as the heterogeneity in this process. The DIC bias phase is set properly such that the brightness of DIC images of individual spores is proportional to the dipicolinic acid (DPA) level of the spores, and an algorithm is developed to retrieve the phase image of an individual spore from its DIC image. We find that during germination, the rapid drop in both the intensity of the original DIC image and the intensity of the reconstructed phase image precisely corresponds to the release of all DPA from that spore. The summed pixel intensity of the DIC image of individual spores adhered on a microscope coverslip is not sensitive to the drift of the slide in both horizontal and vertical directions, which facilitates observation of the germination of thousands of individual spores for long periods of time. A motorized stage and synchronized image acquisition system is further developed to effectively expand the field of view of the DIC imaging. This quantitative DIC technique is used to track the germination of hundreds or thousands of individual spores simultaneously.

Transmission digital holographic microscopy based on a beam-splitter cube interferometer.

A new optical configuration for digital holographic microscopy is presented. Digital off-axis holograms are recorded by use of a single cube beam splitter in a nonconventional configuration to both split and combine a diverging spherical wavefront as it emerges from a single point source. Both the amplitude and the phase can then be reconstructed, yielding intensity and phase images with improved resolution. The novelty of the proposed configuration is its simplicity, minimal number of optical elements, insensitivity to vibration, and its inherent capability to compensate for the phase curvature that results from the illuminating wavefront in the case of microscopic samples.

Simultaneous dynamic optical and electrical properties of endothelial cell attachment on indium tin oxide bioelectrodes.

This study quantifies the dynamic attachment and spreading of porcine pulmonary artery endothelial cells (PPAECs) on optically thin, indium tin oxide (ITO) biosensors using simultaneous differential interference contrast microscopy (DICM) and electrical microimpedance spectroscopy. A lock-in amplifier circuit monitored the impedance of PPAECs cultivated on the transparent ITO bioelectrodes as a function of frequency between 10 Hz and 100 kHz and as a function of time, while DICM images were simultaneously acquired. A digital image processing algorithm quantified the cell-covered electrode area as a function of time. The results of this study show that the fraction of the cell-covered electrode area is in qualitative agreement with the electrical impedance during the attachment phase following the cell settling on the electrode surface. The possibility of several distinctly different states of electrode coverage and cellular attachment giving rise to similar impedance signals is discussed.