High space-time bandwidth product imaging in low coherence quantitative phase microscopy.

Current low coherence quantitative phase microscopy (LC-QPM) systems suffer from either reduced field of view (FoV) or reduced temporal resolution due to the short temporal coherence (TC) length of the light source. Here, we propose a hybrid, experimental and numerical approach to address this core problem associated with LC-QPM. We demonstrate high spatial resolution and high phase sensitivity in LC-QPM at high temporal resolution. High space-time bandwidth product is achieved by employing incoherent light source for sample illumination in QPM to increase the spatial resolution and single-shot Hilbert spiral transform (HST) based phase recovery algorithm to enhance the temporal resolution without sacrificing spatial resolution during the reconstruction steps. The high spatial phase sensitivity comes by default due to the use of incoherent light source in QPM which has low temporal coherence length and does not generate speckle noise and coherent noise. The spatial resolution achieved by the HST is slightly inferior to the temporal phase-shifting (TPS) method when tested on a specimen but surpasses that of the single-shot Fourier transform (FT) based phase recovery method. Contrary to HST method, FT method requires high density fringes for lossless phase recovery, which is difficult to achieve in LC-QPM over entire FoV. Consequently, integration of HST algorithm with LC-QPM system makes an attractive route. Here, we demonstrate scalable FoV and resolution in single-shot LC-QPM and experimentally corroborate it on a test object and on both live and fixed biological specimen such as MEF, U2OS and human red blood cells (RBCs). LC-QPM system with HST reconstruction offer high-speed single-shot QPM imaging at high phase sensitivity and high spatial resolution enabling us to study sub-cellular dynamic inside U2OS for extended duration (3 h) and observe high-speed (50 fps) dynamics of human RBCs. The experimental results validate the effectiveness of the present approach and will open new avenues in the domain of biomedical imaging in the future.

Towards ultrafast quantitative phase imaging via differentiable microscopy [Invited].

With applications ranging from metabolomics to histopathology, quantitative phase microscopy (QPM) is a powerful label-free imaging modality. Despite significant advances in fast multiplexed imaging sensors and deep-learning-based inverse solvers, the throughput of QPM is currently limited by the pixel-rate of the image sensors. Complementarily, to improve throughput further, here we propose to acquire images in a compressed form so that more information can be transferred beyond the existing hardware bottleneck of the image sensor. To this end, we present a numerical simulation of a learnable optical compression-decompression framework that learns content-specific features. The proposed differentiable quantitative phase microscopy (∂-QPM) first uses learnable optical processors as image compressors. The intensity representations produced by these optical processors are then captured by the imaging sensor. Finally, a reconstruction network running on a computer decompresses the QPM images post aquisition. In numerical experiments, the proposed system achieves compression of × 64 while maintaining the SSIM of ∼0.90 and PSNR of ∼30 dB on cells. The results demonstrated by our experiments open up a new pathway to QPM systems that may provide unprecedented throughput improvements.

Label-free superior contrast with c-band ultra-violet extinction microscopy.

In 1934, Frits Zernike demonstrated that it is possible to exploit the sample's refractive index to obtain superior contrast images of biological cells. The refractive index contrast of a cell surrounded by media yields a change in the phase and intensity of the transmitted light wave. This change can be due to either scattering or absorption caused by the sample. Most cells are transparent at visible wavelengths, which means the imaginary component of their complex refractive index, also known as extinction coefficient k, is close to zero. Here, we explore the use of c-band ultra-violet (UVC) light for high-contrast high-resolution label-free microscopy, as k is naturally substantially higher in the UVC than at visible wavelengths. Using differential phase contrast illumination and associated processing, we achieve a 7- to 300-fold improvement in contrast compared to visible-wavelength and UVA differential interference contrast microscopy or holotomography, and quantify the extinction coefficient distribution within liver sinusoidal endothelial cells. With a resolution down to 215 nm, we are, for the first time in a far-field label-free method, able to image individual fenestrations within their sieve plates which normally requires electron or fluorescence superresolution microscopy. UVC illumination also matches the excitation peak of intrinsically fluorescent proteins and amino acids and thus allows us to utilize autofluorescence as an independent imaging modality on the same setup.

Quantification of the NA dependent change of shape in the image formation of a z-polarized fluorescent molecule using vectorial diffraction simulations.

The point spread function of a fixed fluorophore with its dipole axis colinear to the optical axis appears donut-shaped when seen through a microscope, and its light distribution in the pupil plane is radially polarized. Yet other techniques, such as photolithography, report that this same light distribution in the pupil plane appears as a solid spot. How can this same distribution lead to a spot in one case but a donut in the other? Here, we show how the tube lens of the system plays a critical role in determining this shape. Using a vectorial treatment of image formation, we simulate the relative contributions of both longitudinal and radial components to the image of a dipole emitter and thus show how the donut (typically reported for z-polarized single molecule fluorescence microscopy) transforms into a solid spot (as commonly reported for photolithography) as the numerical aperture of the tube lens increases. We find that the transition point occurs around 0.7 NA, which is significantly higher than used for most microscopy systems and lower than for common photolithography systems, thus resolving the seeming paradox of dipole shape.

Hilbert phase microscopy based on pseudo thermal illumination in the Linnik configuration.

Quantitative phase microscopy (QPM) is often based on recording an object-reference interference pattern and its further phase demodulation. We propose pseudo Hilbert phase microscopy (PHPM) where we combine pseudo thermal light source illumination and Hilbert spiral transform (HST) phase demodulation to achieve hybrid hardware-software-driven noise robustness and an increase in resolution of single-shot coherent QPM. Those advantageous features stem from physically altering the laser spatial coherence and numerically restoring spectrally overlapped object spatial frequencies. The capabilities of PHPM are demonstrated by analyzing calibrated phase targets and live HeLa cells in comparison with laser illumination and phase demodulation via temporal phase shifting (TPS) and Fourier transform (FT) techniques. The performed studies verified the unique ability of PHPM to combine single-shot imaging, noise minimization, and preservation of phase details.

Fluorescence fluctuation-based super-resolution microscopy using multimodal waveguided illumination.

Photonic chip-based total internal reflection fluorescence microscopy (c-TIRFM) is an emerging technology enabling a large TIRF excitation area decoupled from the detection objective. Additionally, due to the inherent multimodal nature of wide waveguides, it is a convenient platform for introducing temporal fluctuations in the illumination pattern. The fluorescence fluctuation-based nanoscopy technique multiple signal classification algorithm (MUSICAL) does not assume stochastic independence of the emitter emission and can therefore exploit fluctuations arising from other sources, as such multimodal illumination patterns. In this work, we demonstrate and verify the utilization of fluctuations in the illumination for super-resolution imaging using MUSICAL on actin in salmon keratocytes. The resolution improvement was measured to be 2.2-3.6-fold compared to the corresponding conventional images.

Study of waveguide background at visible wavelengths for on-chip nanoscopy.

On-chip super-resolution optical microscopy is an emerging field relying on waveguide excitation with visible light. Here, we investigate two commonly used high-refractive index waveguide platforms, tantalum pentoxide (Ta2O5) and silicon nitride (Si3N4), with respect to their background with excitation in the range 488-640 nm. The background strength from these waveguides were estimated by imaging fluorescent beads. The spectral dependence of the background from these waveguide platforms was also measured. For 640 nm wavelength excitation both the materials had a weak background, but the background increases progressively for shorter wavelengths for Si3N4. We further explored the effect of the waveguide background on localization precision of single molecule localization for direct stochastic optical reconstruction microscopy (dSTORM). An increase in background for Si3N4 at 488 nm is shown to reduce the localization precision and thus the resolution of the reconstructed images. The localization precision at 640nm was very similar for both the materials. Thus, for shorter wavelength applications Ta2O5 is preferable. Reducing the background from Si3N4 at shorter wavelengths via improved fabrication will be worth pursuing.

Two-dimensional TIRF-SIM-traction force microscopy (2D TIRF-SIM-TFM).

Quantifying small, rapidly evolving forces generated by cells is a major challenge for the understanding of biomechanics and mechanobiology in health and disease. Traction force microscopy remains one of the most broadly applied force probing technologies but typically restricts itself to slow events over seconds and micron-scale displacements. Here, we improve >2-fold spatially and >10-fold temporally the resolution of planar cellular force probing compared to its related conventional modalities by combining fast two-dimensional total internal reflection fluorescence super-resolution structured illumination microscopy and traction force microscopy. This live-cell 2D TIRF-SIM-TFM methodology offers a combination of spatio-temporal resolution enhancement relevant to forces on the nano- and sub-second scales, opening up new aspects of mechanobiology to analysis.

Object detection neural network improves Fourier ptychography reconstruction.

High resolution microscopy is heavily dependent on superb optical elements and superresolution microscopy even more so. Correcting unavoidable optical aberrations during post-processing is an elegant method to reduce the optical system's complexity. A prime method that promises superresolution, aberration correction, and quantitative phase imaging is Fourier ptychography. This microscopy technique combines many images of the sample, recorded at differing illumination angles akin to computed tomography and uses error minimisation between the recorded images with those generated by a forward model. The more precise knowledge of those illumination angles is available for the image formation forward model, the better the result. Therefore, illumination estimation from the raw data is an important step and supports correct phase recovery and aberration correction. Here, we derive how illumination estimation can be cast as an object detection problem that permits the use of a fast convolutional neural network (CNN) for this task. We find that faster-RCNN delivers highly robust results and outperforms classical approaches by far with an up to 3-fold reduction in estimation errors. Intriguingly, we find that conventionally beneficial smoothing and filtering of raw data is counterproductive in this type of application. We present a detailed analysis of the network's performance and provide all our developed software openly.

Visualizing ultrastructural details of placental tissue with super-resolution structured illumination microscopy.

Super-resolution fluorescence microscopy is a widely employed technique in cell biology research, yet remains relatively unexplored in the field of histopathology. Here, we describe the sample preparation steps and acquisition parameters necessary to obtain fluorescent multicolor super-resolution structured illumination microscopy (SIM) images of both formalin-fixed paraffin-embedded and cryo-preserved placental tissue sections. We compare super-resolved images of chorionic villi against diffraction-limited deconvolution microscopy and show the significant contrast and resolution enhancement attainable with SIM, demonstrating the applicability of this imaging technique for both clinical diagnosis and biological research.

MusiJ: an ImageJ plugin for video nanoscopy.

We present an open-source implementation of the fluctuation-based nanoscopy method MUSICAL for ImageJ. This implementation improves the algorithm's computational efficiency and takes advantage of multi-threading to provide orders of magnitude faster reconstructions than the original MATLAB implementation. In addition, the plugin is capable of generating super-resolution videos from large stacks of time-lapse images via an interleaved reconstruction, thus enabling easy-to-use multi-color super-resolution imaging of dynamic systems.

Automatic fringe pattern enhancement using truly adaptive period-guided bidimensional empirical mode decomposition.

Fringe patterns encode the information about the result of a measurement performed via widely used optical full-field testing methods, e.g., interferometry, digital holographic microscopy, moiré techniques, structured illumination etc. Affected by the optical setup, changing environment and the sample itself fringe patterns are often corrupted with substantial noise, strong and uneven background illumination and exhibit low contrast. Fringe pattern enhancement, i.e., noise minimization and background term removal, at the pre-processing stage prior to the phase map calculation (for the measurement result decoding) is therefore essential to minimize the jeopardizing effect the mentioned error sources have on the optical measurement outcome. In this contribution we propose an automatic, robust and highly effective fringe pattern enhancement method based on the novel period-guided bidimensional empirical mode decomposition algorithm (PG-BEMD). The spatial distribution of the fringe period is estimated using the novel windowed approach and then serves as an indicator for the truly adaptive decomposition with the filter size locally adjusted to the fringe pattern density. In this way the fringe term is successfully extracted in a single (first) decomposition component alleviating the cumbersome mode mixing phenomenon and greatly simplifying the automatic signal reconstruction. Hence, the fringe term is dissected without the need for modes selection nor summation. The noise removal robustness is ensured employing the block matching 3D filtering of the fringe pattern prior to its decomposition. Performance validation against previously reported modified empirical mode decomposition techniques is provided using numerical simulations and experimental data verifying the versatility and effectiveness of the proposed approach.

A waveguide imaging platform for live-cell TIRF imaging of neurons over large fields of view.

Large fields of view (FOVs) in total internal reflection fluorescence microscopy (TIRFM) via waveguides have been shown to be highly beneficial for single molecule localisation microscopy on fixed cells [1,2] and have also been demonstrated for short-term live-imaging of robust cell types [3-5], but not yet for delicate primary neurons nor over extended periods of time. Here, we present a waveguide-based TIRFM set-up for live-cell imaging of demanding samples. Using the developed microscope, referred to as the ChipScope, we demonstrate successful culturing and imaging of fibroblasts, primary rat hippocampal neurons and axons of Xenopus retinal ganglion cells (RGCs). The high contrast and gentle illumination mode provided by TIRFM coupled with the exceptionally large excitation areas and superior illumination homogeneity offered by photonic waveguides have potential for a wide application span in neuroscience applications.

Liver sinusoidal endothelial cells contribute to the uptake and degradation of entero bacterial viruses.

The liver is constantly exposed to dietary antigens, viruses, and bacterial products with inflammatory potential. For decades cellular uptake of virus has been studied in connection with infection, while the few studies designed to look into clearance mechanisms focused mainly on the role of macrophages. In recent years, attention has been directed towards the liver sinusoidal endothelial cells (LSECs), which play a central role in liver innate immunity by their ability to scavenge pathogen- and damage-associated molecular patterns. Every day our bodies are exposed to billions of gut-derived pathogens which must be efficiently removed from the circulation to prevent inflammatory and/or immune reactions in other vascular beds. Here, we have used GFP-labelled Enterobacteria phage T4 (GFP-T4-phage) as a model virus to study the viral scavenging function and metabolism in LSECs. The uptake of GFP-T4-phages was followed in real-time using deconvolution microscopy, and LSEC identity confirmed by visualization of fenestrae using structured illumination microscopy. By combining these imaging modalities with quantitative uptake and inhibition studies of radiolabelled GFP-T4-phages, we demonstrate that the bacteriophages are effectively degraded in the lysosomal compartment. Due to their high ability to take up and degrade circulating bacteriophages the LSECs may act as a primary anti-viral defence mechanism.

Super-condenser enables labelfree nanoscopy.

Labelfree nanoscopy encompasses optical imaging with resolution in the 100 nm range using visible wavelengths. Here, we present a labelfree nanoscopy method that combines coherent imaging techniques with waveguide microscopy to realize a super-condenser featuring maximally inclined coherent darkfield illumination with artificially stretched wave vectors due to large refractive indices of the employed Si3N4 waveguide material. We produce the required coherent plane wave illumination for Fourier ptychography over imaging areas 400 µm2 in size via adiabatically tapered single-mode waveguides and tackle the overlap constraints of the Fourier ptychography phase retrieval algorithm two-fold: firstly, the directionality of the illumination wave vector is changed sequentially via a multiplexed input structure of the waveguide chip layout and secondly, the wave vector modulus is shortend via step-wise increases of the illumination light wavelength over the visible spectrum. We test the method in simulations and in experiments and provide details on the underlying image formation theory as well as the reconstruction algorithm. While the generated Fourier ptychography reconstructions are found to be prone to image artefacts, an alternative coherent imaging method, rotating coherent scattering microscopy (ROCS), is found to be more robust against artefacts but with less achievable resolution.

Nanoscopy on-a-chip: super-resolution imaging on the millimeter scale.

Optical nanoscopy techniques can image intracellular structures with high specificity at sub-diffraction limited resolution, bridging the resolution gap between optical microscopy and electron microscopy. So far conventional nanoscopy lacks the ability to generate high throughput data, as the imaged region is small. Photonic chip-based nanoscopy has demonstrated the potential for imaging large areas, but at a lateral resolution of 130 nm. However, all the existing super-resolution methods provide a resolution of 100 nm or better. In this work, chip-based nanoscopy is demonstrated with a resolution of 75 nm over an extraordinarily large area of 0.5 mm × 0.5 mm, using a low magnification and high N.A. objective lens. Furthermore, the performance of chip-based nanoscopy is benchmarked by studying the localization precision and illumination homogeneity for different waveguide widths. The advent of large field-of-view chip-based nanoscopy opens up new routes in diagnostics where high throughput is needed for the detection of non-diffuse disease, or rare events such as the early detection of cancer.

Live-cell imaging of human spermatozoa using structured illumination microscopy.

Structural details of spermatozoa are interesting from the perspectives of fundamental biology and growing reproductive health problems. Studies of nanostructural details of these extremely motile cells have been limited to fixed cells, largely using electron microscopy. Here we provide the protocols for and demonstrate live-cell multi-color super-resolution imaging of human spermatozoa using structured illumination microscopy (SIM). By using patches of agarose for immobilization, we achieved four-channel 3D SIM imaging of the plasma membrane, nucleus, mitochondria and microtubulin in the same living sperm cells. We expect that high-resolution imaging of living spermatozoa will be implemented for research on fundamental cellular mechanisms together with morphological aberrations involved in male infertility for a future improved cell selection process in in vitro fertilization treatments.