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Photonic-chip based TIRF illumination has been used to demonstrate several on-chip optical nanoscopy methods. The sample is illuminated by the evanescent field generated by the electromagnetic wave modes guided inside the optical waveguide. In addition to the photokinetics of the fluorophores, the waveguide modes can be further exploited for introducing controlled intensity fluctuations for exploitation by techniques such as super-resolution optical fluctuation imaging (SOFI). However, the problem of non-uniform illumination pattern generated by the modes contribute to artifacts in the reconstructed image. To alleviate this problem, we propose to perform Haar wavelet kernel (HAWK) analysis on the original image stack prior to the application of (SOFI). HAWK produces a computational image stack with higher spatio-temporal sparsity than the original stack. In the case of multimoded non-uniform illumination patterns, HAWK processing breaks the mode pattern while introducing spatio-temporal sparsity, thereby differentially affecting the non-uniformity of the illumination. Consequently, this assists nanoscopy methods such as SOFI to better support super-resolution, which is otherwise compromised due to spatial correlation of the mode patterns in the raw image. Furthermore, applying HAWK prior to SOFI alleviates the problem of artifacts due to non-uniform illumination without degrading temporal resolution. Our experimental results demonstrate resolution enhancement as well as reduction in artifacts through the combination of HAWK and SOFI.

Quantitative phase microscopy (QPM) is a label-free technique that enables monitoring of morphological changes at the subcellular level. The performance of the QPM system in terms of spatial sensitivity and resolution depends on the coherence properties of the light source and the numerical aperture (NA) of objective lenses. Here, we propose high space-bandwidth quantitative phase imaging using partially spatially coherent digital holographic microscopy (PSC-DHM) assisted with a deep neural network. The PSC source synthesized to improve the spatial sensitivity of the reconstructed phase map from the interferometric images. Further, compatible generative adversarial network (GAN) is used and trained with paired low-resolution (LR) and high-resolution (HR) datasets acquired from the PSC-DHM system. The training of the network is performed on two different types of samples, i.e. mostly homogenous human red blood cells (RBC), and on highly heterogeneous macrophages. The performance is evaluated by predicting the HR images from the datasets captured with a low NA lens and compared with the actual HR phase images. An improvement of 9× in the space-bandwidth product is demonstrated for both RBC and macrophages datasets. We believe that the PSC-DHM + GAN approach would be applicable in single-shot label free tissue imaging, disease classification and other high-resolution tomography applications by utilizing the longitudinal spatial coherence properties of the light source.

Multiple signal classification algorithm (MUSICAL) exploits temporal fluctuations in fluorescence intensity to perform super-resolution microscopy by computing the value of a super-resolving indicator function across a fine sample grid. A key step in the algorithm is the separation of the measurements into signal and noise subspaces, based on a single user-specified parameter called the threshold. The resulting image is strongly sensitive to this parameter and the subjectivity arising from multiple practical factors makes it difficult to determine the right rule of selection. We address this issue by proposing soft thresholding schemes derived from a new generalized framework for indicator function design. We show that the new schemes significantly alleviate the subjectivity and sensitivity of hard thresholding while retaining the super-resolution ability. We also evaluate the trade-off between resolution and contrast and the out-of-focus light rejection using the various indicator functions. Through this, we create significant new insights into the use and further optimization of MUSICAL for a wide range of practical scenarios.

Phase shifting interferometric (PSI) techniques are among the most sensitive phase measurement methods. Owing to its high sensitivity, any minute phase change caused due to environmental instability results into, inaccurate phase measurement. Consequently, a well calibrated piezo electric transducer (PZT) and highly-stable environment is mandatory for measuring accurate phase map using PSI implementation. Here, we present an inverse approach, which can retrieve phase maps of the samples with negligible errors under environmental fluctuations. The method is implemented by recording a video of continuous temporally phase shifted interferograms and phase shifts were calculated between all the data frames using Fourier transform algorithm with a high accuracy ≤ 5.5 × 10 -4 π rad. To demonstrate the robustness of the proposed method, a manual translation of the stage was employed to introduce continuous temporal phase shift between data frames. The developed algorithm is first verified by performing quantitative phase imaging of optical waveguide and red blood cells using uncalibrated PZT under the influence of vibrations/air turbulence and compared with the well calibrated PZT results. Furthermore, we demonstrated the potential of the proposed approach by acquiring the quantitative phase imaging of an optical waveguide with a rib height of only 2 nm and liver sinusoidal endothelial cells (LSECs). By using 12-bit CMOS camera the height of shallow rib waveguide is measured with a height sensitivity of 4 Å without using PZT and in presence of environmental fluctuations.vn.

The advantages of quantitative phase microscopy (QPM) such as label-free imaging with high spatial sensitivity, live cell compatibility and high-speed imaging makes it viable for various biological applications. The measurement accuracy of QPM strongly relies on the shape of the recorded interferograms, whether straight or curved fringes are recorded during the data acquisition. Moreover, for a single shot phase recovery high fringe density is required. The wavefront curvature for the high-density fringes over the entire field of view is difficult to be discerned with the naked eye. As a consequence, there is a quadratic phase aberration in the recovered phase images due to curvature mismatch. In the present work, we have implemented sampling moiré method for real-time sensing of the wavefront curvature mismatch between the object and the reference wavefronts and further for its correction. By zooming out the interferogram, moiré fringes are generated which helps to easily identify the curvature of the fringes. The wavefront curvature mismatch correction accuracy of the method is tested with the help of low temporal coherent light source such as a white light (temporal coherence ∼ 1.6 µm). The proposed scheme is successfully demonstrated to remove the quadratic phase aberration caused due to wavefront mismatch from an USAF resolution target and the biological tissue samples. The phase recovery accuracy of the current scheme is further compared with and found to better than the standard method called principle component analysis. The proposed method enables recording of the corrected wavefront interferogram without needing any additional optical components or modification and also does not need any post-processing correction algorithms. The proposed method of curvature compensation paves the path for a high-throughput and accurate quantitative phase imaging.

Multi-spectral quantitative phase imaging (QPI) is an emerging imaging modality for wavelength dependent studies of several biological and industrial specimens. Simultaneous multi-spectral QPI is generally performed with color CCD cameras. Here, we present a new approach for accurately measuring the color crosstalk of 2D area detectors, without needing prior information about camera specifications. Color crosstalk is systematically studied and compared using compact interference microscopy on two different cameras commonly used in QPI, single chip CCD (1-CCD) and three chip CCD (3-CCD). The influence of color crosstalk on the fringe width and the visibility of the monochromatic constituents corresponding to three color channels of white light interferogram are studied both through simulations and experiments. It is observed that presence of color crosstalk changes the fringe width and visibility over the imaging field of view. This leads to an unwanted non-uniform background error in the multi-spectral phase imaging of the specimens. The color crosstalk of the detector is observed to be the limiting factor for phase measurement accuracy of simultaneous multi-spectral QPI systems.

Total internal reflection fluorescence (TIRF) microscopy benefits from high-sensitivity, low background noise, low photo-toxicity and high-contrast imaging of sub-cellular structures close to the membrane surface. Although, TIRF microscopy provides high-contrast imaging it does not provide quantitative information about morphological features of the biological cells. Here, we propose an integrated waveguide chip-based TIRF microscopy and label-free quantitative phase imaging (QPI). The evanescent field present on top of a waveguide surface is used to excite the fluorescence and an upright microscope is used to collect the signal. The upright microscope is converted into a Linnik-type interferometer to sequentially extract both the quantitative phase information and TIRF images of the cells. Waveguide chip-based TIRF microscopy benefits from decoupling of illumination and collection light path, large field of view imaging and pre-aligned configuration for multi-color TIRF imaging. The proposed multi-modal microscopy is used to study inflammation caused by lipopolysaccharide (LPS) on rat macrophages. The TIRF microscopy showed that LPS inflammatory molecule disrupts the cell membrane and causes cells to significantly expand across a substrate. While, QPI module quantified changes in the sub-cellular content of the LPS challenged macrophages, showing a net decrease in its maximum phase values.

Waveguide chip-based microscopy reduces the complexity of total internal reflection fluorescence (TIRF) microscopy, and adds features like large field of view illumination, decoupling of illumination and collection path and easy multimodal imaging. However, for the technique to become widespread there is a need of low-loss and affordable waveguides made of high-refractive index material. Here, we develop and report a low-loss silicon nitride (Si 3 N 4 ) waveguide platform for multi-color TIRF microscopy. Single mode conditions at visible wavelengths (488-660 nm) were achieved using shallow rib geometry. To generate uniform excitation over appropriate dimensions waveguide bends were used to filter-out higher modes followed by adiabatic tapering. Si 3 N 4 material is finally shown to be biocompatible for growing and imaging living cells.

Rib waveguides are investigated as an alternative to strip waveguides for planar trapping and transport of microparticles. Microparticles are successfully propelled along the surface of rib waveguides and trapped in the gap between opposing rib waveguides. The trapping capabilities of waveguide end facets formed by a single and opposing waveguide geometries are investigated. The slab beneath a rib waveguide continues to guide light after the end facet of a rib waveguide. Thus particles can be trapped in wider gaps formed by opposing rib waveguides than with strip waveguides. Rib waveguides were found more efficient in trapping a collection of particles in the gap and particles could be moved to different locations in the gap by changing the relative power in the two opposing rib waveguides. Numerical simulations are used to show that the trapping efficiency on the surface of rib and strip waveguides is comparable. The simulations also confirm the advantage of opposing rib waveguides for trapping particles in wide gaps. The low sidewalls of rib waveguides give low propagation losses and make it easy to integrate rib waveguides with other functions in a lab-on-a-chip where particle trapping and transport is required.

Optical waveguides can be used to trap and transport micro-particles. The particles are held close to the waveguide surface by the evanescent field and propelled forward. We propose a new technique to lift and trap particles above the surface of the waveguides. This is made possible by a gap between two opposing, planar waveguides. The field emitted from each of the waveguide ends diverge fast, away from the substrate and into the cover-medium. By combining two fields propagating at an angle upwards and coming from opposite sides of a gap, particles can be stably lifted and trapped at the crossing of the two fields. Thus, particles are transported by waveguides leading to a gap, where they are lifted away from the substrate and trapped. The experiments are supported by numerical simulations of the forces on the micro-particles. Fluorescence imaging is used to track the particles in 3D with a precision of 50 nm.

We demonstrate that Raman spectroscopy can be used to characterize and identify particles that are trapped and propelled along optical waveguides. To accomplish this, microscopic particles on a waveguide are moved along the waveguide and then individually addressed by a focused laser beam to obtain their characteristic Raman signature within 1 second acquisition time. The spectrum is used to distinguish between glass and polystyrene particles. After the characterization, the particles continue to be propelled along the straight waveguide. Alternatively, a waveguide loop with a gap is also investigated, and in this case particles are held in the gap for characterization before they are released.

We have studied optical trapping and propulsion of red blood cells in the evanescent field of optical waveguides. Cell propulsion is found to be highly dependent on the biological medium and serum proteins the cells are submerged in. Waveguides made of tantalum pentoxide are shown to be efficient for cell propulsion. An optical propulsion velocity of up to 
6 µm/s on a waveguide with a width of ~6 µm is reported. Stable optical trapping and propulsion of cells during transverse flow is also reported.