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Controlled illumination at targetted locations (targeted illumination) and imaging at high switching speed have found significant applications in different frontiers of science and technology such as optogenetics, photodynamic therapy (PDT), neurophotonics, and medical imaging. Although there are numerous optical systems reported with targeted illumination ability, most of these are modified standard (bench top) microscopes, hence not suitable for in vivo applications. Therefore, there is a critical need to develop optical imaging systems with targeted illumination suitable for in vivo studies. The small diameter, user-defined length and high mechanical flexibility of fiber optic imaging bundle (FOIB) facilitates its positioning at remote and difficult-toaccess in vivo sites. Recently, few FOIB based studies are reported, which have shown adaptation of targeted illumination. However, image resolution and switching speed achieved with these methods are found to be limited to 10 11m and 20 Hz, respectively. In order to investigate biological or chemical processes, the methodology adopted or equipment used should be able to provide higher resolution, faster-switching speed and an option for targeted illumination and imaging. Moreover, images obtained with FOIB probes are always affected by pixelation noise which deteriorates the image resolution and contrast. Though there are many algorithms to remove pixelation noise, objective comparison of these algorithms are not possible due to the non-availability of a common test image database. Also, there are no theoretical models available currently to simulate the image guidance through FOm. From this perspective, one of the objectives of this thesis is aimed at developing a high-speed imaging probe using a combination of FOffi and Digital Micromirror Device (DMD) with targeted illumination and imaging feature for potential in vivo applications. Compared to the earlier FOffi based probes, the newly developed probe has shown improved lateral and axial image resolution of2.7 Jlm and 5.5 J.Lm, respectively. This imaging system also provides a larger field of view (200 J.Lm X 200 J.Lm) at high resolution compared to earlier reported targeted FOffi probes. The developed FOffi probe's illumination switching speed is defined by the DMD, which is 10,000 Hz. The multiline digitally controlled laser source allows the FOffi probe to illuminate the targeted regions with different wavelengths. Further, an objective comparison of different depixelation methods for FOIB imaging is also performed as part of the investigation. A theoretical model for FOffi based imaging is developed and used to generate images of simulated fiber pixelated 1mages. The parameters such as packing fraction, fiberlet to fiberlet distance, fiberlet core diameter, core-cladding properties and light guiding properties of fiberlet are considered in the theoretical formulations. These studies have led to the development of a Fiber Pixelated Image Database (FPID), which now serves as a free open source common test image sample database for researchers working on the development of novel depixelation methods. The effect of variable pinhole size on the imaging properties is also studied using the proposed probe. An illustrative demonstration is carried out for four different imaging approaches using this targeted FOffi probe. These approaches are 'targeted confocal imaging', 'multi-directional scanning in targeted sample regions', 'targeted time averaged imaging for contrast enhancement', and 'single shot multitarget multispectral imaging', which were found to improve resolution, contrast, and imaging speed. The potential of the FOffi probe is demonstrated by synchronous multispectral spatiotemporal illumination of targeted mouse kidney cells. Additionally, the efficiency of this FOffi probe for tracking and targeted illumination of dynamic (moving) particles is demonstrated as a proof of concept. This developed FOIB probe ·has also been demonstrated as a portable targeted illumination source for the standard bench-top microscope. The second major objective of this thesis is aimed at imaging around opaque obstacles. Since there are numerous injuries (cuts) reported due to blind injection or improper position of the surgical tool, caused due to the blocking of the field of view of the sample by opaque surgical tools during surgery, an imaging system capable of imaging around obstacle would be helpful in avoiding such injuries. Hence in this thesis, a detailed research is carried out to come up with an optical system to image around obstacles. In this thesis, use of an axicon lens is explored for imaging around opaque obstacles. The simulation of axicon lens to perform imaging around an opaque obstacle is demonstrated using Zemax software. This is further validated experimentally by imaging around different thick opaque obstacles of different shape and thickness, such as Allen key, syringe needle, metallic pin, hair, and thread. Finally, the proof of concept of this method is demonstrated by imaging around a surgical needle during needle injection procedure. It is envisaged that the invaluable advantages provided by the targeted probe along with its specialty features can make a great impact in the research and developmental arena such as optogenetics to selectively activate neuronal cells or cell organelles. Further the work done on the imaging round opaque obstacles are expected to contribute significantly during surgical procedures in the near future. Doctor of Philosophy (MAE)

Breaking the diffraction limit in imaging microscopes with far-field imaging options has always been the thrust challenge for optical engineers and biologists over the years. Although structured illumination microscopy and Bessel beam assisted imaging has shown the capability of imaging with sub-diffraction resolutions, they rely on the use of objective lenses with large numerical apertures (NA). Hence, they fail to sustain resolutions at larger working distances. In this context, we demonstrate a method for nanoscale resolution imaging at longer working distances, named as Structured Illumination Bessel Microscopy (SIBM). The proposed method is envisaged for both biological and engineering applications that necessitate high imaging resolutions at large working distances. EDB (Economic Devt. Board, S’pore) MOE (Min. of Education, S’pore) Published version

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. MOE (Min. of Education, S’pore) Published version

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.

An optical imaging probe with targeted multispectral and spatiotemporal illumination features has applications in many diagnostic biomedical studies. However, these systems are mostly adapted in conventional microscopes, limiting their use for in vitro applications. We present a variable resolution imaging probe using a digital micromirror device (DMD) with an achievable maximum lateral resolution of 2.7 μm and an axial resolution of 5.5 μm, along with precise shape selective targeted illumination ability. We have demonstrated switching of different wavelengths to image multiple regions in the field of view. Moreover, the targeted illumination feature allows enhanced image contrast by time averaged imaging of selected regions with different optical exposure. The region specific multidirectional scanning feature of this probe has facilitated high speed targeted confocal imaging.

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. MOE (Min. of Education, S’pore) Published version

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. MOE (Min. of Education, S’pore) Published version

Direct visualization and assessment of the iridocorneal angle (ICA) region with high resolution is important for the clinical evaluation of glaucoma. However, the current clinical imaging systems for ICA do not provide sufficient structural details due to their poor resolution. The key challenges in achieving high quality ICA imaging are its location in the anterior region of the eye and the occurrence of total internal reflection due to refractive index difference between cornea and air. Here, we report an indirect axicon assisted gonioscopy imaging probe with white light illumination. The illustrated results with this probe shows significantly improved visualization of structures in the ICA including TM region, compared to the current available tools. It could reveal critical details of ICA and expected to aid management by providing information that is complementary to angle photography and gonioscopy.

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. Published version

We report a simple real time optical imaging concept using an axicon lens to image the object kept behind opaque obstacles in free space. The proposed concept underlines the importance and advantages of using an axicon lens compared to a conventional lens to image behind the obstacle. The potential of this imaging concept is demonstrated by imaging the insertion of surgical needle in biological specimen in real time, without blocking the field of view. It is envisaged that this proposed concepts and methodology can make a telling impact in a wide variety of areas especially for diagnostics, therapeutics and microscopy applications.