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.

MAxSIM: multi-angle-crossing structured illumination microscopy with height-controlled mirror for 3D topological mapping of live cells.

Mapping 3D plasma membrane topology in live cells can bring unprecedented insights into cell biology. Widefield-based super-resolution methods such as 3D-structured illumination microscopy (3D-SIM) can achieve twice the axial ( ~ 300 nm) and lateral ( ~ 100 nm) resolution of widefield microscopy in real time in live cells. However, twice-resolution enhancement cannot sufficiently visualize nanoscale fine structures of the plasma membrane. Axial interferometry methods including fluorescence light interference contrast microscopy and its derivatives (e.g., scanning angle interference microscopy) can determine nanoscale axial locations of proteins on and near the plasma membrane. Thus, by combining super-resolution lateral imaging of 2D-SIM with axial interferometry, we developed multi-angle-crossing structured illumination microscopy (MAxSIM) to generate multiple incident angles by fast, optoelectronic creation of diffraction patterns. Axial localization accuracy can be enhanced by placing cells on a bottom glass substrate, locating a custom height-controlled mirror (HCM) at a fixed axial position above the glass substrate, and optimizing the height reconstruction algorithm for noisy experimental data. The HCM also enables imaging of both the apical and basal surfaces of a cell. MAxSIM with HCM offers high-fidelity nanoscale 3D topological mapping of cell plasma membranes with near-real-time ( ~ 0.5 Hz) imaging of live cells and 3D single-molecule tracking.

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.

Computational Portable Microscopes for Point-of-Care-Test and Tele-Diagnosis.

In bio-medical mobile workstations, e.g., the prevention of epidemic viruses/bacteria, outdoor field medical treatment and bio-chemical pollution monitoring, the conventional bench-top microscopic imaging equipment is limited. The comprehensive multi-mode (bright/dark field imaging, fluorescence excitation imaging, polarized light imaging, and differential interference microscopy imaging, etc.) biomedical microscopy imaging systems are generally large in size and expensive. They also require professional operation, which means high labor-cost, money-cost and time-cost. These characteristics prevent them from being applied in bio-medical mobile workstations. The bio-medical mobile workstations need microscopy systems which are inexpensive and able to handle fast, timely and large-scale deployment. The development of lightweight, low-cost and portable microscopic imaging devices can meet these demands. Presently, for the increasing needs of point-of-care-test and tele-diagnosis, high-performance computational portable microscopes are widely developed. Bluetooth modules, WLAN modules and 3G/4G/5G modules generally feature very small sizes and low prices. And industrial imaging lens, microscopy objective lens, and CMOS/CCD photoelectric image sensors are also available in small sizes and at low prices. Here we review and discuss these typical computational, portable and low-cost microscopes by refined specifications and schematics, from the aspect of optics, electronic, algorithms principle and typical bio-medical applications.

Analysis of micro-relief of biofilm of yeast Candida albicans of basic plastics by the method of laser modulation interference microscopy.

The development of mycotic colonization of the base surface with further biodegradation of acrylic plastics is currently of undoubted interest. The oral cavity is a favorable ecological niche for colonization by fungi and their subsequent possible invasion into the epithelium of the oral mucosa. The method of modulation interference laser microscopy is of considerable interest to researchers in medicine in the context of obtaining the necessary information about the morphological characteristics of microbial cells and the microbiome community as a whole during the colonization of a certain ecological niche in the human body. Purpose of the study: to analyze the microrelief of the biofilm of yeast-like fungi of the species Candida albicans of base plastics of the hot type of polymerization using the method of laser modulation interference microscopy. An experimental study was carried out in order to study biofilms of yeast-like fungi of the genus Candida on samples of basic plastics, an image of a biofilm of yeast-like fungi of the species Candida albicans was obtained on the surface of a plastic of a hot type of polymerization (polymethyl methacrylate) in the visualization of the phase portrait, a description of its horizontal and vertical bioprofile. As a result of the research, the heterogeneous structure of the biofilm was determined, due to the different density and accumulation of cells along the surface, the characteristics of the surface were established in accordance with the roughness criteria. The microrelief parameters on a separately arbitrarily selected section line allow one to determine the characteristics of the biofilm in the required area and make it possible to judge the nature of its formation in a certain biological niche.

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.

Characterizing the Number of Kinesin Motors Bound to Microtubules in the Gliding Motility Assay Using FLIC Microscopy.

Intracellular transport by kinesin motors moving along their associated cytoskeletal filaments, microtubules, is essential to many biological processes. This active transport system can be reconstituted in vitro with the surface-adhered motors transporting the microtubules across a planar surface. In this geometry, the kinesin-microtubule system has been used to study active self-assembly, to power microdevices, and to perform analyte detection. Fundamental to these applications is the ability to characterize the interactions between the surface tethered motors and microtubules. Fluorescence Interference Contrast (FLIC) microscopy can illuminate the height of the microtubule above a surface, which, at sufficiently low surface densities of kinesin, also reveals the number, locations, and dynamics of the bound motors.

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.

KIAA0319 influences cilia length, cell migration and mechanical cell-substrate interaction.

Following its association with dyslexia in multiple genetic studies, the KIAA0319 gene has been extensively investigated in different animal models but its function in neurodevelopment remains poorly understood. We developed the first human cellular knockout model for KIAA0319 in RPE1 retinal pigment epithelia cells via CRISPR-Cas9n to investigate its role in processes suggested but not confirmed in previous studies, including cilia formation and cell migration. We observed in the KIAA0319 knockout increased cilia length and accelerated cell migration. Using Elastic Resonator Interference Stress Microscopy (ERISM), we detected an increase in cellular force for the knockout cells that was restored by a rescue experiment. Combining ERISM and immunostaining we show that RPE1 cells exert highly dynamic, piconewton vertical pushing forces through actin-rich protrusions that are surrounded by vinculin-rich pulling sites. This protein arrangement and force pattern has previously been associated to podosomes in other cells. KIAA0319 depletion reduces the fraction of cells forming these actin-rich protrusions. Our results suggest an involvement of KIAA0319 in cilia biology and cell-substrate force regulation.

Label-Free Dynamic Imaging of Chromatin in Live Cell Nuclei by High-Speed Scattering-Based Interference Microscopy.

Chromatin is a DNA-protein complex that is densely packed in the cell nucleus. The nanoscale chromatin compaction plays critical roles in the modulation of cell nuclear processes. However, little is known about the spatiotemporal dynamics of chromatin compaction states because it remains difficult to quantitatively measure the chromatin compaction level in live cells. Here, we demonstrate a strategy, referenced as DYNAMICS imaging, for mapping chromatin organization in live cell nuclei by analyzing the dynamic scattering signal of molecular fluctuations. Highly sensitive optical interference microscopy, coherent brightfield (COBRI) microscopy, is implemented to detect the linear scattering of unlabeled chromatin at a high speed. A theoretical model is established to determine the local chromatin density from the statistical fluctuation of the measured scattering signal. DYNAMICS imaging allows us to reconstruct a speckle-free nucleus map that is highly correlated to the fluorescence chromatin image. Moreover, together with calibration based on nanoparticle colloids, we show that the DYNAMICS signal is sensitive to the chromatin compaction level at the nanoscale. We confirm the effectiveness of DYNAMICS imaging in detecting the condensation and decondensation of chromatin induced by chemical drug treatments. Importantly, the stable scattering signal supports a continuous observation of the chromatin condensation and decondensation processes for more than 1 h. Using this technique, we detect transient and nanoscopic chromatin condensation events occurring on a time scale of a few seconds. Label-free DYNAMICS imaging offers the opportunity to investigate chromatin conformational dynamics and to explore their significance in various gene activities.

Azimuthal Beam Scanning Microscope Design and Implementation for Axial Localization with Scanning Angle Interference Microscopy.

Azimuthal beam scanning, also referred to as circle scanning, is an effective way of eliminating coherence artifacts with laser illumination in widefield microscopy. With a static excitation spot, dirt on the optics and internal reflections can produce an uneven excitation field due to interference fringes. These artifacts become more pronounced in TIRF microscopy, where the excitation is confined to an evanescent field that extends a few hundred nanometers above the coverslip. Unwanted intensity patterns that arise from these imperfections vary with path of the excitation beam through the microscope optical train, so by rapidly rotating the beam through its azimuth the uneven illumination is eliminated by averaging over the camera exposure time. In addition to being useful from TIRF microscopy, it is also critical for scanning angle interference microscopy (SAIM), an axial localization technique with nanometer-scale precision that requires similar instrumentation to TIRF microscopy. For robust SAIM localization, laser excitation with a homogeneous profile over a range of polar angles is required. We have applied the circle scanning principle to SAIM, constructing an optimized instrument configuration and open-source hardware, enabling high-precision localization and significantly higher temporal resolution than previous implementations. In this chapter, we detail the design and construction of the SAIM instrument, including the optical configuration, required peripheral devices, and system calibration.

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.

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.

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.