Advanced Interferometry with 3-D Structured Illumination Reveals the Surface Fine Structure of Complex Biospecimens.

Interference reflection microscopy (IRM) is a powerful, label-free technique to visualize the surface structure of biospecimens. However, stray light outside a focal plane obscures the surface fine structures beyond the diffraction limit (dxy ≈ 200 nm). Here, we developed an advanced interferometry approach to visualize the surface fine structure of complex biospecimens, ranging from protein assemblies to single cells. Compared to 2-D, our unique 3-D structure illumination introduced to IRM enabled successful visualization of fine structures and the dynamics of protein crystal growth under lateral (dx-y ≈ 110 nm) and axial (dx-z ≤ 5 nm) resolutions and dynamical adhesion of microtubule fiber networks with lateral resolution (dx-y ≈ 120 nm), 10 times greater than unstructured IRM (dx-y ≈ 1000 nm). Simultaneous reflection/fluorescence imaging provides new physical fingerprints for studying complex biospecimens and biological processes such as myogenic differentiation and highlights the potential use of advanced interferometry to study key nanostructures of complex biospecimens.

Observing Membrane and Cell Adhesion via Reflection Interference Contrast Microscopy.

Reflection interference contrast microscopy (RICM) is an optical microscopy technique ideally suited for imaging adhesion. While RICM (and the closely related interference reflection microscopy (IRM)) has been extensively used qualitatively or semiquantitatively to image cells, including immune cells, it can also be used quantitatively to measure membrane to surface distance, especially for model membranes. Here, we present a protocol for RICM and IRM imaging and the details of semiquantitative and quantitative analysis.

Reflectional quantitative differential phase microscopy using polarized wavefront phase modulation.

Quantitative phase microscopy (QPM), as a label-free and nondestructive technique, has been playing an indispensable tool in biomedical imaging and industrial inspection. Herein, we introduce a reflectional quantitative differential phase microscopy (termed RQDPM) based on polarized wavefront phase modulation and partially coherent full-aperture illumination, which has high spatial resolution and spatio-temporal phase sensitivity and is applicable to opaque surfaces and turbid biological specimens. RQDPM does not require additional polarized devices and can be easily switched from reflectional mode to transmission mode. In addition, RQDPM inherits the characteristic of high axial resolution of differential interference contrast microscope, thereby providing topography for opaque surfaces. We experimentally demonstrate the reflectional phase imaging ability of RQDPM with several samples: semiconductor wafer, thick biological tissues, red blood cells, and Hela cells. Furthermore, we dynamically monitor the flow state of microspheres in a self-built microfluidic channel by using RQDPM converted into the transmission mode.

3D differential interference contrast microscopy using polarisation-sensitive tomographic diffraction microscopy.

Tomographic diffraction microscopy (TDM) is a generalisation of digital holographic microscopy (DHM), for which the illumination angle onto the sample is fully controlled, which has become a tool of choice for 3D, high-resolution imaging of unlabelled samples. TDM makes it possible to obtain the optical field in both amplitude and phase for each illumination angle. Proper information reallocation eventually allows for 3D reconstruction of the complex refractive index map. On the other hand, polarisation array sensors (PAS) paves new way for TDM, as vectorial information assessment about the investigated sample. In this contribution, we show an alternative use of this polarisation information based on the phase sensitive nature of TDM. Here, we demonstrated that TDM coupled with PAS can lead to a 3D differential interference contrast (DIC) microscope with almost no experimental configuration modification.

Demystifying speckle field interference microscopy.

Dynamic speckle illumination (DSI) has recently attracted strong attention in the field of biomedical imaging as it pushes the limits of interference microscopy (IM) in terms of phase sensitivity, and spatial and temporal resolution compared to conventional light source illumination. To date, despite conspicuous advantages, it has not been extensively implemented in the field of phase imaging due to inadequate understanding of interference fringe formation, which is challenging to obtain in dynamic speckle illumination interference microscopy (DSI-IM). The present article provides the basic understanding of DSI through both simulation and experiments that is essential to build interference microscopy systems such as quantitative phase microscopy, digital holographic microscopy and optical coherence tomography. Using the developed understanding of DSI, we demonstrated its capabilities which enables the use of non-identical objective lenses in both arms of the interferometer and opens the flexibility to use user-defined microscope objective lens for scalable field of view and resolution phase imaging. It is contrary to the present understanding which forces us to use identical objective lenses in conventional IM system and limits the applicability of the system for fixed objective lens. In addition, it is also demonstrated that the interference fringes are not washed out over a large range of optical path difference (OPD) between the object and the reference arm providing competitive edge over low temporal coherence light source based IM system. The theory and explanation developed here would enable wider penetration of DSI-IM for applications in biology and material sciences.

Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins.

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. Total-internal-reflection-fluorescence (TIRF) microscopy has a high signal-to-background ratio, but it suffers from photobleaching and photodamage of the fluorescent proteins. Label-free techniques such as interference reflection microscopy (IRM) and interferometric scattering microscopy (iSCAT) circumvent the problem of photobleaching but cannot readily visualize single molecules. This paper presents a protocol for combining IRM with a commercial TIRF microscope for the simultaneous imaging of microtubule-associated proteins (MAPs) and dynamic microtubules in vitro. This protocol allows for high-speed observation of MAPs interacting with dynamic microtubules. This improves on existing two-color TIRF setups by eliminating both the need for microtubule labeling and the need for several additional optical components, such as a second excitation laser. Both channels are imaged on the same camera chip to avoid image registration and frame synchronization problems. This setup is demonstrated by visualizing single kinesin molecules walking on dynamic microtubules.

In Vitro Reconstitution of Microtubule Dynamics and Severing Imaged by Label-Free Interference-Reflection Microscopy.

The dynamic architecture of the microtubule cytoskeleton is crucial for cell division, motility and morphogenesis. The dynamic properties of microtubules-growth, shrinkage, nucleation, and severing-are regulated by an arsenal of microtubule-associated proteins (MAPs). The activities of many of these MAPs have been reconstituted in vitro using microscope assays. As an alternative to fluorescence microscopy, interference-reflection microscopy (IRM) has been introduced as an easy-to-use, wide-field imaging technique that allows label-free visualization of microtubules with high contrast and speed. IRM circumvents several problems associated with fluorescence microscopy including the high concentrations of tubulin required for fluorescent labeling, the potential perturbation of function caused by the fluorophores, and the risks of photodamage. IRM can be implemented on a standard epifluorescence microscope at low cost and can be combined with fluorescence techniques like total-internal-reflection-fluorescence (TIRF) microscopy. Here we describe the experimental procedure to image microtubule dynamics and severing using IRM , providing practical tips and guidelines to resolve possible experimental hurdles.

Sizing individual dielectric nanoparticles with quantitative differential interference contrast microscopy.

We report a method to measure the size of single dielectric nanoparticles with high accuracy and precision using quantitative differential interference contrast (DIC) microscopy. Dielectric nanoparticles are detected optically by the conversion of the optical phase change into an intensity change using DIC. Phase images of individual nanoparticles were retrieved from DIC by Wiener filtering, and a quantitative methodology to extract nanoparticle sizes was developed. Using polystyrene beads of 100 nm radius as size standard, we show that the method determines this radius within a few nm accuracy. The smallest detectable polystyrene bead is limited by background and shot-noise, which depend on acquisition and analysis parameters, including the objective numerical aperture, the DIC phase offset, and the refractive index contrast between particles and their surrounding. Measurements on small beads of 15 nm nominal radius are shown, and a sensitivity limit potentially reaching down to 1.8 nm radius was inferred. As application example, individual nanodiamonds with nominal sizes below 50 nm were measured, and were found to have a nearly exponential size distribution with 28 nm mean value. Considering the importance of dielectric nanoparticles in many fields, from naturally occurring virions to polluting nanoplastics, the proposed method could offer a powerful quantitative tool for nanoparticle analysis, combining accuracy, sensitivity and high-throughput with widely available and easy-to-use DIC microscopy.

Automatic Colorectal Cancer Screening Using Deep Learning in Spatial Light Interference Microscopy Data.

The surgical pathology workflow currently adopted by clinics uses staining to reveal tissue architecture within thin sections. A trained pathologist then conducts a visual examination of these slices and, since the investigation is based on an empirical assessment, a certain amount of subjectivity is unavoidable. Furthermore, the reliance on external contrast agents such as hematoxylin and eosin (H&E), albeit being well-established methods, makes it difficult to standardize color balance, staining strength, and imaging conditions, hindering automated computational analysis. In response to these challenges, we applied spatial light interference microscopy (SLIM), a label-free method that generates contrast based on intrinsic tissue refractive index signatures. Thus, we reduce human bias and make imaging data comparable across instruments and clinics. We applied a mask R-CNN deep learning algorithm to the SLIM data to achieve an automated colorectal cancer screening procedure, i.e., classifying normal vs. cancerous specimens. Our results, obtained on a tissue microarray consisting of specimens from 132 patients, resulted in 91% accuracy for gland detection, 99.71% accuracy in gland-level classification, and 97% accuracy in core-level classification. A SLIM tissue scanner accompanied by an application-specific deep learning algorithm may become a valuable clinical tool, enabling faster and more accurate assessments by pathologists.

Visualizing bleb mass dynamics in single cells using quantitative phase microscopy.

Understanding biological responses to directed energy (DE) is critical to ensure the safety of personnel within the Department of Defense. At the Air Force Research Laboratory, we have developed or adapted advanced optical imaging systems that quantify biophysical responses to DE. One notable cellular response to DE exposure is the formation of blebs, or semi-spherical protrusions of the plasma membrane in living cells. In this work, we demonstrate the capacity of quantitative phase imaging (QPI) to both visualize and quantify the formation of membrane blebs following DE exposure. QPI is an interferometric imaging tool that uses optical path length as a label-free contrast mechanism and is sensitive to the non-aqueous mass density, or dry mass, of living cells. Blebs from both CHO-K1 and U937 cells were generated after exposure to a series of 600 ns, 21.2 kV/cm electric pulses. These blebs were visualized in real time, and their dry mass relative to the rest of the cell body was quantified as a function of time. It is our hope that this system will lead to an improved understanding of both DE-induced and apoptotic blebbing.

Single-shot quantitative phase imaging as an extension of differential interference contrast microscopy.

Various studies have been conducted to obtain quantitative phase information based on differential interference contrast (DIC) microscopy. As one such attempt, we propose in this study a single-shot quantitative phase imaging (QPI) method by combining two developments. First, an add-on optical system to a commercialized DIC microscope was developed to perform quantitative phase gradient imaging (QPGI) with single image acquisition using a polarization camera. Second, an algorithm was formulated to reconstitute QPI from the obtained QPGI by reducing linear artifacts, which arise in simply integrated QPGI images. To demonstrate the applicability of the developed system in cell biology, the system was used to measure various cell lines and compared with fluorescence microscopy images of the same field of view. Consistent with previous studies, nucleoli and lipid droplets can be imaged by the system with greater optical path lengths (OPL). The results also implied that combining fluorescence microscopy and the developed system might be more informative for cell biology research than using these methods individually. Exploiting the single-shot performance of the developed system, time-lapse imaging was also conducted to visualize the dynamics of intracellular granules in monocyte-/macrophage-like cells. Our proposed approach may accelerate the implementation of QPI in standard biomedical laboratories.

Real-time imaging of cellular forces using optical interference.

Important dynamic processes in mechanobiology remain elusive due to a lack of tools to image the small cellular forces at play with sufficient speed and throughput. Here, we introduce a fast, interference-based force imaging method that uses the illumination of an elastic deformable microcavity with two rapidly alternating wavelengths to map forces. We show real-time acquisition and processing of data, obtain images of mechanical activity while scanning across a cell culture, and investigate sub-second fluctuations of the piconewton forces exerted by macrophage podosomes. We also demonstrate force imaging of beating neonatal cardiomyocytes at 100 fps which reveals mechanical aspects of spontaneous oscillatory contraction waves in between the main contraction cycles. These examples illustrate the wider potential of our technique for monitoring cellular forces with high throughput and excellent temporal resolution.

Experimental Evaluation of an Interferometric Light Microscopy Particle Counter for Titering and Characterization of Virus Preparations.

Virus particle concentration is a critical piece of information for virology, viral vaccines and gene therapy research. We tested a novel nanoparticle counting device, "Videodrop", for its efficacy in titering and characterization of virus particles. The Videodrop nanoparticle counter is based on interferometric light microscopy (ILM). The method allows the detection of particles under the diffraction limit capabilities of conventional light microscopy. We analyzed lenti-, adeno-, and baculovirus samples in different concentrations and compared the readings against traditional titering and characterization methods. The tested Videodrop particle counter is especially useful when measuring high-concentration purified virus preparations. Certain non-purified sample types or small viruses may be impossible to characterize or may require the use of standard curve or background subtraction methods, which increases the duration of the analysis. Together, our testing shows that Videodrop is a reasonable option for virus particle counting in situations where a moderate number of samples need to be analyzed quickly.

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.

Photonic resonator interferometric scattering microscopy.

Interferometric scattering microscopy is increasingly employed in biomedical research owing to its extraordinary capability of detecting nano-objects individually through their intrinsic elastic scattering. To significantly improve the signal-to-noise ratio without increasing illumination intensity, we developed photonic resonator interferometric scattering microscopy (PRISM) in which a dielectric photonic crystal (PC) resonator is utilized as the sample substrate. The scattered light is amplified by the PC through resonant near-field enhancement, which then interferes with the <1% transmitted light to create a large intensity contrast. Importantly, the scattered photons assume the wavevectors delineated by PC's photonic band structure, resulting in the ability to utilize a non-immersion objective without significant loss at illumination density as low as 25 W cm-2. An analytical model of the scattering process is discussed, followed by demonstration of virus and protein detection. The results showcase the promise of nanophotonic surfaces in the development of resonance-enhanced interferometric microscopies.

Interference Resolution in Nonfluent Variant Primary Progressive Aphasia: Evidence From a Picture-Word Interference Task.

BACKGROUND: Picture-word interference tasks have been used to investigate (a) the time course of lexical access in individuals with primary progressive aphasia (PPA) and (b) how these individuals resolve competition during lexical selection. OBJECTIVE: To investigate the time course of Greek-speaking individuals with PPA to produce grammatical gender-marked determiner phrases by examining their picture-naming latencies in the context of distractor words. METHOD: Eight individuals with nonfluent variant PPA (nfv-PPA; M age = 62.8 years) and eight cognitively intact controls (M age = 61.1 years) participated in our study. In a picture-word interference task, the study participants named depicted objects by producing determiner + noun sequences. Interference was generated by manipulating the grammatical gender of the depicted objects and distractor words. Two stimulus onset asynchronies were used: +200 ms and +400 ms. RESULTS: The individuals with nfv-PPA exhibited longer picture-naming latencies than the controls (P = 0.003). The controls exhibited interference from incongruent distractors at both asynchronies (P < 0.001); the individuals with PPA exhibited interference from incongruent distractors only at the +400-ms interval (P = 0.002). The gender-congruency effect was stronger for the individuals with PPA than for the controls at the +400-ms interval (P = 0.05); the opposite pattern was observed at the +200-ms interval (P = 0.024). CONCLUSION: Gender interference resolution was abnormal in the individuals with nfv-PPA. The results point to deficits in lexicosyntactic networks that compromised the time course of picture-naming production.

CD8 Co-Receptor Enhances T-Cell Activation without Any Effect on Initial Attachment.

The scanning of surrounding tissues by T lymphocytes to detect cognate antigens requires high speed, sensitivity and specificity. T-cell receptor (TCR) co-receptors such as CD8 increase detection performance, but the exact mechanism remains incompletely understood. Here, we used a laminar flow chamber to measure at the single molecule level the kinetics of bond formation and rupture between TCR- transfected CD8+ and CD8- Jurkat cells and surfaces coated with five peptide-exposing major histocompatibility antigens (pMHCs) of varying activating power. We also used interference reflection microscopy to image the spreading of these cells dropped on pMHC-exposing surfaces. CD8 did not influence the TCR-pMHC interaction during the first few seconds following cell surface encounter, but it promoted the subsequent spreading responses, suggesting that CD8 was involved in early activation rather than binding. Further, the rate and extent of spreading, but not the lag between contact and spreading initiation, depended on the pMHC. Elucidating T-lymphocyte detection strategy may help unravel underlying signaling networks.

Direct evaluation of self-quenching behavior of fluorophores at high concentrations using an evanescent field.

The quantum yield of a fluorophore is reduced when two or more identical fluorophores are in close proximity to each other. The study of protein folding or particle aggregation is can be done based on this above-mentioned phenomenon-called self-quenching. However, it is challenging to characterize the self-quenching of a fluorophore at high concentrations because of the inner filter effect, which involves depletion of excitation light and re-absorption of emission light. Herein, a novel method to directly evaluate the self-quenching behavior of fluorophores was developed. The evanescent field from an objective-type total internal reflection fluorescence (TIRF) microscope was used to reduce the path length of the excitation and emission light to ~100 nm, thereby supressing the inner filter effect. Fluorescence intensities of sulforhodamine B, fluorescein isothiocyanate (FITC), and calcein solutions with concentrations ranging from 1 µM to 50 mM were directly measured to evaluate the concentration required for 1000-fold degree of self-quenching and to examine the different mechanisms through which the fluorophores undergo self-quenching.

Large field-of-view nanometer-sectioning microscopy by using metal-induced energy transfer and biexponential lifetime analysis.

Total internal reflection fluorescence (TIRF) microscopy, which has about 100-nm axial excitation depth, is the method of choice for nanometer-sectioning imaging for decades. Lately, several new imaging techniques, such as variable angle TIRF microscopy, supercritical-angle fluorescence microscopy, and metal-induced energy transfer imaging, have been proposed to enhance the axial resolution of TIRF. However, all of these methods use high numerical aperture (NA) objectives, and measured images inevitably have small field-of-views (FOVs). Small-FOV can be a serious limitation when multiple cells need to be observed. We propose large-FOV nanometer-sectioning microscopy, which breaks the complementary relations between the depth of focus and axial sectioning by using MIET. Large-FOV imaging is achieved with a low-magnification objective, while nanometer-sectioning is realized utilizing metal-induced energy transfer and biexponential fluorescence lifetime analysis. The feasibility of our proposed method was demonstrated by imaging nanometer-scale distances between the basal membrane of human aortic endothelial cells and a substrate.