Kavya Mohan

Three Dimensional Fluorescence Imaging Using Multiple Light-Sheet Microscopy

We developed a multiple light-sheet microscopy (MLSM) system capable of 3D fluorescence imaging. Employing spatial filter in the excitation arm of a SPIM system, we successfully generated multiple light-sheets. This improves upon the existing SPIM system and is capable of 3D volume imaging by simultaneously illuminating multiple planes in the sample. Theta detection geometry is employed for data acquisition from multiple specimen layers. This detection scheme inherits many advantages including, background reduction, cross-talk free fluorescence detection and high-resolution at long working distance. Using this technique, we generated equi-intense light-sheets of thickness approximately with an inter-sheet separation of . Moreover, the light-sheets generated by MLSM is found to be 2 times thinner than the state-of-art SPIM system. Imaging of fluorescently coated yeast cells of size (encaged in Agarose gel-matrix) is achieved. Proposed imaging technique may accelerate the field of fluorescence microscopy, cell biology and biophotonics.

High resolution light-sheet based high-throughput imaging cytometry system enables visualization of intra-cellular organelles

Visualization of intracellular organelles is achieved using a newly developed high throughput imaging cytometry system. This system interrogates the microfluidic channel using a sheet of light rather than the existing point-based scanning techniques. The advantages of the developed system are many, including, single-shot scanning of specimens flowing through the microfluidic channel at flow rate ranging from micro- to nano- lit./min. Moreover, this opens-up in-vivo imaging of sub-cellular structures and simultaneous cell counting in an imaging cytometry system. We recorded a maximum count of 2400 cells/min at a flow-rate of 700 nl/min, and simultaneous visualization of fluorescently-labeled mitochondrial network in HeLa cells during flow. The developed imaging cytometry system may find immediate application in biotechnology, fluorescence microscopy and nano-medicine.

Light sheet based imaging flow cytometry on a microfluidic platform

We propose a light sheet based imaging flow cytometry technique for simultaneous counting and imaging of cells on a microfluidic platform. Light sheet covers the entire microfluidic channel and thus omits the necessity of flow focusing and point scanning based technology. Another advantage lies in the orthogonal detection geometry that totally cuts‐off the incident light, thereby substantially reducing the background in the detection. Compared to the existing state‐of‐art techniques the proposed technique shows marked improvement. Using fluorescently‐coated Saccharomyces cerevisiae cells we have recorded cell counting with throughput as high as 2,090 cells/min in the low flow rate regime and were able to image the individual cells on‐the‐go. Overall, the proposed system is cost‐effective and simple in channel geometry with the advantage of efficient counting in operational regime of low laminar flow. This technique may advance the emerging field of microfluidic based cytometry for applications in nanomedicine and point of care diagnostics. Microsc. Res. Tech. 76:1101–1107, 2013.

Generation of multiple sheets of light using spatial-filtering technique.

We develop an optical system for generating multiple light sheets. This is enabled by employing a special class of spatial filters in a cylindrical lens geometry. The proposed binary filter placed at the back aperture of the cylindrical lens results in the generation of a periodic transverse pattern extending along the z axis (i.e., multiple light sheets). Experimental results confirm the generation of multiple light sheets of thickness 6.6 μm with an intersheet spacing of 13.4 μm. The proposed imaging technique may facilitate three-dimensional imaging in nano-optics, fluorescence microscopy, and nanobiology.

Spatial filter based light-sheet laser interference technique for three-dimensional nanolithography

We propose a laser interference technique for the fabrication of 3D nano-structures. This is possible with the introduction of specialized spatial filter in a 2 pi cylindrical lens system (consists of two opposing cylindrical lens sharing a common geometrical focus). The spatial filter at the back-aperture of a cylindrical lens gives rise to multiple light-sheet patterns. Two such interfering counter-propagating light-sheet pattern result in periodic 3D nano-pillar structure. This technique overcomes the existing slow point-by-point scanning, and has the ability to pattern selectively over a large volume. The proposed technique allows large-scale fabrication of periodic structures. Computational study shows a field-of-view (patterning volume) of approximately 12: 2mm(3) with the pillar-size of 80 nm and inter-pillar separation of 180 nm. Applications are in nano-waveguides, 3D nano-electronics, photonic crystals, and optical microscopy

Light‐sheet based lithography technique for patterning an array of microfluidic channels

We propose a Light‐sheet laser interference lithography technique for fabricating periodic microfluidic channels. This technique uses multiple light‐sheet illumination pattern that is generated using a spatial filter at the back‐aperture of the cylindrical lens. Specially designed spatial filter is used that give rise to a periodic pattern at the focal plane which is essentially a 1D Fourier transform of the spatial filter transfer function. One‐dimensional focusing property of the cylindrical lens result in the generation of line shaped channel geometry. To design microfluidic channels, the illumination pattern is exposed to the glass substrate coated with a photopolymer sensitized to 532 nm and subsequently developed using standard chemical protocols. Experimentally, the 1D periodic channel structure has an approximate width and periodicity of approximately 11.25 microns. Light‐sheets based lithography technique offer a fast and single‐shot process to generate microfluidic channels.

Note: Experimental observation of nano-channel pattern in light sheet laser interference nanolithography system

We experimentally observed nano-channel-like pattern in a light-sheet based interference nanolithography system. The optical system created nano-channel-like patterned illumination. Coherent counter-propagating light sheets are made to interfere at and near geometrical focus along the propagation z-axis. This results in the formation of nano-channel-like pattern (of size ≈ 300 nm and inter-channel periodicity of ≈337.5 nm) inside the sample due to constructive and destructive interference. In addition, the technique has the ability to generate large area patterning using larger light-sheets. Exciting applications are in the broad field of nanotechnology (nano-electronics and nano-fluidics).

Efficient generation of diffraction-limited multi-sheet pattern for biological imaging.

We demonstrate a new technique to generate multiple light-sheets for fluorescence microscopy. This is possible by illuminating the cylindrical lens using multiple copies of Gaussian beams. A diffraction grating placed just before the cylindrical lens splits the incident Gaussian beam into multiple beams traveling at different angles. Subsequently, this gives rise to diffraction-limited light-sheets after the Gaussian beams pass through the combined cylindrical lens-objective sub-system. Direct measurement of field at and around the focus of objective lens shows multi-sheet pattern with an average thickness of 7.5 μm and inter-sheet separation of 380 μm. Employing an independent orthogonal detection sub-system, we successfully imaged fluorescently-coated yeast cells (≈4  μm) encaged in agarose gel-matrix. Such a diffraction-limited sheet-pattern equipped with dedicated detection system may find immediate applications in the field of optical microscopy and fluorescence imaging.

Limited-view light sheet fluorescence microscopy for three dimensional volume imaging

We propose and demonstrate a limited-view light sheet microscopy (LV-LSM) for three dimensional (3D) volume imaging. Realizing that longer and frequent image acquisition results in significant photobleaching, we have taken limited angular views (18 views) of the macroscopic specimen and integrated with maximum likelihood (ML) technique for reconstructing high quality 3D volume images. Existing variants of light-sheet microscopy require both rotation and translation with a total of approximately 10-fold more views to render a 3D volume image. Comparatively, LV-LSM technique reduces data acquisition time and consequently minimizes light-exposure by many-folds. Since ML is a post-processing technique and highly parallelizable, this does not cost precious imaging time. Results show noise-free and high contrast volume images when compared to the state-of-the-art selective plane illumination microscopy.

Directional bilateral filters for smoothing fluorescence microscopy images

Images obtained through fluorescence microscopy at low numerical aperture (NA) are noisy and have poor resolution. Images of specimens such as F-actin filaments obtained using confocal or widefield fluorescence microscopes contain directional information and it is important that an image smoothing or filtering technique preserve the directionality. F-actin filaments are widely studied in pathology because the abnormalities in actin dynamics play a key role in diagnosis of cancer, cardiac diseases, vascular diseases, myofibrillar myopathies, neurological disorders, etc. We develop the directional bilateral filter as a means of filtering out the noise in the image without significantly altering the directionality of the F-actin filaments. The bilateral filter is anisotropic to start with, but we add an additional degree of anisotropy by employing an oriented domain kernel for smoothing. The orientation is locally adapted using a structure tensor and the parameters of the bilateral filter are optimized for within the framework of statistical risk minimization. We show that the directional bilateral filter has better denoising performance than the traditional Gaussian bilateral filter and other denoising techniques such as SURE-LET, non-local means, and guided image filtering at various noise levels in terms of peak signal-to-noise ratio (PSNR). We also show quantitative improvements in low NA images of F-actin filaments