Current clinical optical imaging systems do not provide sufficient structural information of trabecular meshwork (TM) in the iridocorneal angle (ICA) of the eye due to their low resolution. Increase in the intraocular pressure (IOP) can occur due to the abnormalities in TM, which could subsequently lead to glaucoma. Here, we present an indirect gonioscopy based imaging probe with significantly improved visualization of structures in the ICA including TM region, compared to the currently available tools. Imaging quality of the developed system was tested in porcine samples. Improved direct high quality visualization of the TM region through this system can be used for Laser trabeculoplasty, which is a primary treatment of glaucoma. This system is expected to be used complementary to angle photography and gonioscopy.
Time averaged imaging is one of the widely used methods to achieve improved image quality, used in different types of microscopic methods. Time averaged imaging refers to adjusting the exposure time of the imaging system to obtain optimal images. In state of the art microscopes, the region of interest (ROI) of illumination beam for time averaged imaging can be selected to be of regular shapes such as circle or rectangle. This forces smallest possible ROI to be larger than the actual sample’s ROI which can be of a specific shape with complex contours. In this context, we present a flexible fiber bundle based illumination probe capable of illuminating samples of irregular shapes for time averaged imaging. Further, this probe is capable of multi-wavelength illumination, hence can be used for multi-fluorescence imaging. The fiber probe with features such as region selective and multi- wavelength illumination allows it to be used for optimal imaging of multi-fluorescence sample.
Axicon lenses are conical prisms, which are known to focus a light source to a line comprising of multiple points along the optical axis. In this study, we analyze the potential of axicon lenses to view, image and record the object behind opaque obstacles in free space. The advantage of an axicon lens over a regular lens is demonstrated experimentally. Parameters such as obstacle size, object and the obstacle position in the context of imaging behind obstacles are tested using Zemax optical simulation. This proposed concept can be easily adapted to most of the optical imaging methods and microscopy modalities.
Spatially non-uniform illumination patterns have shown significant potential to improve the imaging. Recent developments in the patterned illumination microscopy have demonstrated that the use of an optical speckle as an illumination pattern significantly improves the imaging resolution at the same time reducing the computational overheads. We present a DMD based method for generation of digital speckle pattern. The generated digital speckle and uniform white light illumination are used as two illuminations to acquire images. The image reconstruction algorithm for blind structured illumination microscopy is used to get the high resolution image. Our approach does not require any calibration step or stringent control of the illumination, and dramatically simplifies the experimental set-up.
It is well known for structured illumination microscopy (SIM) that the lateral resolution by a factor of two beyond the classical diffraction limit is achieved using spatially structured illumination in wide-field fluorescence microscope. In the state of art SIM systems, grating patterns are generally generated by physical gratings or by spatial light modulators such as digital micro mirrors (DMD), liquid crystal displays (LCD). In this study, using a combination of LCD and ground glasses, size controlled randomized speckle patterns are generated as an illumination source for the microscope. Proof of concept of using speckle illumination in SIM configuration is tested by imaging fixed BPAE cells.
Imaging of physically inaccessible parts of the body such as the colon at micron-level resolution is highly important in diagnostic medical imaging. Though flexible endoscopes based on the imaging fiber bundle are used for such diagnostic procedures, their inherent honeycomb-like structure creates fiber pixelation effects. This impedes the observer from perceiving the information from an image captured and hinders the direct use of image processing and machine intelligence techniques on the recorded signal. Significant efforts have been made by researchers in the recent past in the development and implementation of pixelation removal techniques. However, researchers have often used their own set of images without making source data available which subdued their usage and adaptability universally. A database of pixelated images is the current requirement to meet the growing diagnostic needs in the healthcare arena. An innovative fiber pixelated image database is presented, which consists of pixelated images that are synthetically generated and experimentally acquired. Sample space encompasses test patterns of different scales, sizes, and shapes. It is envisaged that this proposed database will alleviate the current limitations associated with relevant research and development and would be of great help for researchers working on comb structure removal algorithms.
Automated and unbiased methods of non-invasive cell monitoring able to deal with complex biological heterogeneity are fundamentally important for biology and medicine. Label-free cell imaging provides information about endogenous fluorescent metabolites, enzymes and cofactors in cells. However extracting high content information from imaging of native fluorescence has been hitherto impossible. Here, we quantitatively characterise cell populations in different tissue types, live or fixed, by using novel image processing and a simple multispectral upgrade of a wide-field fluorescence microscope. Multispectral intrinsic fluorescence imaging was applied to patient olfactory neurosphere-derived cells, cell model of a human metabolic disease MELAS (mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like syndrome). By using an endogenous source of contrast, subtle metabolic variations have been detected between living cells in their full morphological context which made it possible to distinguish healthy from diseased cells before and after therapy. Cellular maps of native fluorophores, flavins, bound and free NADH and retinoids unveiled subtle metabolic signatures and helped uncover significant cell subpopulations, in particular a subpopulation with compromised mitochondrial function. The versatility of our method is further illustrated by detecting genetic mutations in cancer, non-invasive monitoring of CD90 expression, label-free tracking of stem cell differentiation, identifying stem cell subpopulations with varying functional characteristics, tissue diagnostics in diabetes, and assessing the condition of preimplantation embryos. Our optimal discrimination approach enables statistical hypothesis testing and intuitive visualisations where previously undetectable differences become clearly apparent.
There was a renewed interest, during the recent years, in the imaging and tracking of targeted cells or organelles for
a variety of biomedical and lab-on a chip applications that include particles movement. However, nonspecific
illumination during tracking can have adverse effects such as heating, reduced image contrast and photo bleaching.
In fact, current available tracking and imaging systems are unable to selectively illuminate the particle being
tracked. To fill this void, we have developed a fiber optics based probe system incorporating a spatial light
modulator (SLM) and an imaging fiber bundle for selective illumination on the targeted particle. A GRIN lens is
attached at the distal endface of the image fiber bundle for optimised illumination and collection. A tracking
algorithm is developed in order to enable controlled illumination through SLM to target the illumination point or
location in accordance with the particle movement and size variation. Further with this probe, particles can be
illuminated with light pulses of controllable duty cycle and frequency. The proposed methodology and developed
probe have good significance and expected to find potential applications areas such as optogenetics, cell signalling
studies, and lab-on a chip systems.
Flexible fiber optic imaging systems including fiber optic confocal probes have found tremendous significance in the recent past for its applications in high resolution imaging. However, motorized stage is required for scanning the sample or tip of the fiber in fiber based confocal probes. In this context, we propose a fiber probe confocal system using digital spatial light modulator devoid of using a mechanical scanning stage. Each fiberlet in the image fiber acts not only as a light conduit but also as a confocal pinhole. The paper also introduces the variation in the contrast by varying the number of illuminated fiberlets which effectively implies variation in the effective pinhole size. This approach has enabled the probe to act as an imaging unit with resolution that can be controlled and varied from a wide-field to a confocal.