Shwetadwip Chowdhury

Structured oblique illumination microscopy for enhanced resolution imaging of non-fluorescent, coherently scattering samples.

Many biological structures of interest are beyond the diffraction limit of conventional microscopes and their visualization requires application of super-resolution techniques. Such techniques have found remarkable success in surpassing the diffraction limit to achieve sub-diffraction limited resolution; however, they are predominantly limited to fluorescent samples. Here, we introduce a non-fluorescent analogue to structured illumination microscopy, termed structured oblique illumination microscopy (SOIM), where we use simultaneous oblique illuminations of the sample to multiplex high spatial-frequency content into the frequency support of the system. We introduce a theoretical framework describing how to demodulate this multiplexed information to reconstruct an image with a spatial-frequency support exceeding that of the system’s classical diffraction limit. This approach allows enhanced-resolution imaging of non-fluorescent samples. Experimental confirmation of the approach is obtained in a reflection test target with moderate numerical aperture.

Structured illumination quantitative phase microscopy for enhanced resolution amplitude and phase imaging

Structured illumination microscopy (SIM) is an established microscopy technique typically used to image samples at resolutions beyond the diffraction limit. Until now, however, achieving sub-diffraction resolution has predominantly been limited to intensity-based imaging modalities. Here, we introduce an analogue to conventional SIM that allows sub-diffraction resolution, quantitative phase-contrast imaging of optically transparent objects. We demonstrate sub-diffraction resolution amplitude and quantitative-phase imaging of phantom targets and enhanced resolution quantitative-phase imaging of cells. We report a phase accuracy to within 5% and phase noise of 0.06 rad.

Refractive index tomography with structured illumination

To probe biological questions with significant biophysical, biochemical, and molecular components, an imaging solution compatible with both endogenous and molecular 3D imaging may be necessary. In this work, we show that structured illumination (SI) microscopy, popularly associated with 3D fluorescent super-resolution, can allow 3D refractive index (RI) reconstructions when operated in the coherent realm. We introduce a novel reinterpretation of coherent SI, which mathematically equates it to a superposition of angled illuminations. Raw acquisitions for standard SI-enhanced quantitative-phase images can be processed into electric field maps of the sample under angled illuminations. Standard diffraction tomography (DT) computation can then be used to reconstruct the sample’s 3D RI distribution at sub-diffraction resolutions. We demonstrate this concept by using SI to computationally reconstruct 3D RI distributions of human breast (MCF-7) and colorectal (HT-29) adenocarcinoma cells. Our experimental setup generates SI patterns using broadband illumination with a spatial light modulator and detects angle-dependent sample diffraction through a common-path, off-axis interferometer with no moving components. This technique may easily pair with SI fluorescence microscopy and important future extensions may include multimodal, sub-diffraction resolution, 3D RI, and fluorescent visualizations.

Structured illumination diffraction phase microscopy for broadband, subdiffraction resolution, quantitative phase imaging

Structured illumination microscopy (SIM) is an established technique that allows subdiffraction resolution imaging by heterodyning high sample frequencies into the systems passband via structured illumination. However, until now, SIM has been typically used to achieve subdiffraction resolution for intensity-based imaging. Here, we present a novel optical setup that uses structured illumination with a broadband light source to obtain noise-reduced, subdiffraction resolution, quantitative phase imaging (QPM) of cells. We compare this with a previous work for subdiffraction QPM imaging via SIM that used a laser source, and was thus still corrupted by coherent noise.

Structured illumination multimodal 3D-resolved quantitative phase and fluorescence sub-diffraction microscopy

Sub-diffraction resolution imaging has played a pivotal role in biological research by visualizing key, but previously unresolvable, sub-cellular structures. Unfortunately, applications of far-field sub-diffraction resolution are currently divided between fluorescent and coherent-diffraction regimes, and a multimodal sub-diffraction technique that bridges this gap has not yet been demonstrated. Here we report that structured illumination (SI) allows multimodal sub-diffraction imaging of both coherent quantitative-phase (QP) and fluorescence. Due to SIs conventionally fluorescent applications, we first demonstrate the principle of SI-enabled three-dimensional (3D) QP sub-diffraction imaging with calibration microspheres. Image analysis confirmed enhanced lateral and axial resolutions over diffraction-limited QP imaging, and established striking parallels between coherent SI and conventional optical diffraction tomography. We next introduce an optical system utilizing SI to achieve 3D sub-diffraction, multimodal QP/fluorescent visualization of A549 biological cells fluorescently tagged for F-actin. Our results suggest that SI has a unique utility in studying biological phenomena with significant molecular, biophysical, and biochemical components.

Spatial frequency-domain multiplexed microscopy for simultaneous, single-camera, one-shot, fluorescent, and quantitative-phase imaging

Multimodal imaging is a crucial tool when imaging biological phenomena that cannot be comprehensively captured by a single modality. Here, we introduce a theoretical framework for spatial-frequency-multiplexed microscopy via off-axis interference as a novel wide-field imaging technique that enables true simultaneous multimodal and multichannel wide-field imaging. We experimentally demonstrate this technique for single-camera, simultaneous two-channel fluorescence and one-channel quantitative-phase imaging for fluorescent microspheres and fixed cells stained for F-actin and nuclear fluorescence.

Structured illumination microscopy for dual-modality 3D sub-diffraction resolution fluorescence and refractive-index reconstruction

Though structured illumination (SI) microscopy is a popular imaging technique conventionally associated with fluorescent super-resolution, recent works have suggested its applicability towards sub-diffraction resolution coherent imaging with quantitative endogenous biological contrast. Here, we demonstrate that SI can efficiently integrate together the principles of fluorescent super-resolution and coherent synthetic aperture to achieve 3D dual-modality sub-diffraction resolution, fluorescence and refractive-index (RI) visualizations of biological samples. We experimentally demonstrate this framework by introducing a SI microscope capable of 3D sub-diffraction resolution fluorescence and RI imaging, and verify its biological visualization capabilities by experimentally reconstructing 3D RI/fluorescence visualizations of fluorescent calibration microspheres as well as alveolar basal epithelial adenocarcinoma (A549) and human colorectal adenocarcinmoa (HT-29) cells, fluorescently stained for F-actin. This demonstration may suggest SI as an especially promising imaging technique to enable future biological studies that explore synergistically operating biophysical/biochemical and molecular mechanisms at sub-diffraction resolutions.

Structured illumination for 3D subdiffraction reconstruction of refractive-index and fluorescence (Conference Presentation)

Refractive-index (RI) is an inherent optical property of materials that can provide important biochemical and biophysical information about a biological sample. Optical-diffraction-tomography (ODT) is a current standard to obtain quantitative three-dimensional RI distributions, by measuring optical fields diffracted from the sample by rotated illumination beams. This method for ODT also synthetically enlarges the microscope’s lateral spatial-frequency support, and thus images the RI distribution with lateral resolution beyond the microscopes coherent diffraction limit. Fluorescence microscopy offers a complementary set of biological insights by offering imaging capabilities with molecular specificity. Analogous to ODT, super-resolution fluorescence techniques can offer these insights at spatial resolutions beyond the microscopes incoherent diffraction limit. Unfortunately, such super-resolution techniques are generally incompatible with ODT and a generalized sub-diffraction technique has been difficult to find, which hinders a cohesive, high-resolution, multimodal analysis of biological samples. We experimentally introduce, for the first time to our knowledge, a novel, high resolution, optical system that uses structured illumination (SI) to enable 3D sub-diffraction resolution imaging for both fluorescence and RI. We demonstrate sub-diffraction resolution, multimodal SI imaging of HT29 and MCF7 cells fluorescently stained for F-actin, such that the 3D RI and fluorescent distributions may offer unique, but complementary, insights into the biological samples.

Simultaneous fluorescence and phase imaging with extensions toward sub-diffraction resolution via structured-illumination(Conference Presentation)

In the biological sciences, there is much emphasis on elucidating the functions of various biological components and processes. To do so, advances in general microscopy have yielded various imaging modalities to probe such processes under specific visualization and contrast requirements. Examples of modalities that are popularly integrated into conventional biological studies include fluorescent, dark-field, phase-contrast, and polarization-sensitive microscopies, with each modality offering unique insights into the biological function of the sample. Often times, however, a comprehensive understanding of biological phenomena requires the integration of the unique and separate visualizations of various modalities. Unfortunately, conventional microscopes typically support only one modality and rarely allow multiple modalities to be used in conjunction. Though high-end microscopes may support multimodal visualization, they often require either mechanical (and often manual) toggling, which obstruct real-time multimodal imaging, or simultaneous detection via multiple cameras, which dramatically increases the microscope’s cost. Here, we present a one-shot technique that allows multiple imaging channels, of potentially different modalities, to be simultaneously detected by a single camera. We experimentally demonstrate this method on transparent cells that have been tagged for F-actin and nuclear fluorescence. Our multimodal system consists of 2-channel fluorescence and 1-channel quantitative-phase (QP) imaging, and clearly demonstrates ability for simultaneous fluorescent and QP visualization. Though we experimentally verify our framework using dual fluorescent/QP imaging, we emphasize that our framework for single-shot, simultaneous single-camera detection is applicable to an arbitrary number of widefield imaging modalities so long as they fulfill criteria for Fourier spectra separation, SNR, and detector dynamic range

Multiplexed structured illumination microscopy for simultaneous, sub-diffraction resolution fluorescent and quantitative-phase imaging

We introduce a variant technique to structured illumination microscopy (SIM) that multiplexes fluorescent and quantitative-phase imaging for simultaneous, sub-diffraction resolution, multimodal imaging.