High-resolution cell imaging using white light phase shifting interferometry and iterative phase deconvolution.

An optimization algorithm is presented for the deconvolution of a complex field to improve the resolution and accuracy of quantitative phase imaging (QPI). A high-resolution phase map can be recovered by solving a constrained optimization problem of deconvolution using a complex gradient operator. The method is demonstrated on phase measurements of samples using a white light based phase shifting interferometry (WLPSI) method. The application of the algorithm on real and simulated objects shows a significant resolution and contrast improvement. Experiments performed on Escherichia coli bacterium have revealed its sub-cellular structures that were not visible in the raw WLPSI images obtained using a five phase shifting method. These features can give valuable insights into the structures and functioning of biological cells. The algorithm is simple in implementation and can be incorporated into other QPI modalities .

Label-free high-resolution white light quantitative phase nanoscopy system.

We present a high-resolution white light quantitative phase nanoscopy (WLQPN) system that can be utilized to visualize nanoparticles and subcellular features of the biological specimens. The five-phase shifting technique, along with deconvolution, is adopted to obtain super-resolution in phase imaging. The phase shifting technique can provide full detector resolution, making it beneficial as compared to the well-known Fourier analysis method. The Fourier transform method requires minimum angle of sin - 1 3 f x λ , where f x is maximum achievable spatial frequency. It limits the highest achievable resolution to much below the actual diffraction limit of the system. Thus, to obtain a high-resolution phase map of the biological specimen, a two-step process is adopted. First, the phase map is recovered using the five-phase shifting algorithm, with full detector spatial resolution. Second, the complex field is obtained from the recovered phase map and further processed using the Richardson Lucy total variation deconvolution algorithm to obtain super-resolution phase images. The present technique was tested on 1951 USAF resolution chart, 200 nm polystyrene beads and Escherichia coli bacteria using a 50×, 0.55NA objective lens. The 200 nm polystyrene beads are visually resolvable and subcellular features of the E. coli bacteria are also observed, suggesting a significant improvement in the resolution.

White light phase shifting interferometric microscopy with whole slide imaging for quantitative analysis of biological samples.

In this paper, we demonstrate the white light phase shifting interferometer employed as whole slide scanner and phase profiler for determining qualitative and quantitative information over large field-of-view (FOV). Experiments were performed on human erythrocytes and MG63 Osteosarcoma cells. Here, we have recorded microscopic images and phase shifted white light interferograms simultaneously in a stepped manner. Sample slide is translated in transverse direction such that there exists a correlation between the adjacent frames, and they were stitched together using correlation functions. Final stitched image has a FOV of 0.24 × 1.14 mm with high resolution ~0.8 µm. Circular Hough transform algorithm is implemented to the resulting image for cell counting and five-step phase shifting algorithm is utilised to retrieve the phase profiles over a large FOV. Further, this technique is utilised to study the difference between normal and anaemic erythrocytes. Significant changes are observed in anaemic cells as compared to normal cells.

Multimodal biomicroscopic system for the characterization of cells with high spatial phase sensitivity and sub-pixel accuracy.

Multimodal analysis is highly advantageous for various biomedical applications including cancer and brain studies. Simultaneous measurement of quantitative phase with sub-pixel accuracy and fluorescence image is difficult to achieve in single measurement. Conventionally, off-axis interferograms are analyzed using the Fourier-transform method which limits the accuracy of the phase maps by pixel size, and usually the location of the carrier peak is in sub-pixel. We report a multimodal microscopic system consisting of high-resolution (HR) quantitative phase interferometer to retrieve sub-pixel accuracy in phase imaging and an oblique-illumination-based fluorescence imaging system which decouples the excited light from emitted signal light to avoid saturation of the camera, both integrated into a single unit. Here, highly resolved phase maps are obtained using a two-step process. First, using a speckle-free illumination which offers high spatial phase sensitivity. Second, using a hamming window for accurate estimation of original signal frequency information and HR discrete Fourier transform (DFT) which offers sub-pixel accuracy in phase measurements. HR-DFT has computational load of OABß , where A×B is the size of the interferogram and ß is the upsampling factor, making system computationally more robust and efficient compared to zero-padded FFT. The experiment is conducted on MG63 osteosarcoma and human mesenchymal stem cells (hMSCs) and their quantitative parameters are extracted with significantly improved accuracy. The average phase for MG63 cells and hMSCs, for nucleus is obtained to be 8.02 rad ± 0.80 rad and 4.29 rad ± 0.43 rad, respectively, and for cytoplasm is obtained to be 2.63 rad ± 0.96 rad and 1.73 rad ± 0.57 rad, respectively.

Development of multimodal micro-endoscopic system with oblique illumination for simultaneous fluorescence imaging and spectroscopy of oral cancer.

Multimodality of an optical system implies the use of one or more optical techniques to improve the system's overall performance and maximum utility. In this article, we demonstrate a multimodal system with oblique illumination that combines two different techniques; fluorescence micro-endoscopy and spectroscopy simultaneously and can be utilized to obtain diverse information from the same location of biological sample. In present system, use of graded index (GRIN) rod-lens makes it highly compact and oblique incidence decouples illumination geometry with collection geometry, preventing CCD cameras from saturation and reduces number of optical elements, thereby making system further miniaturized and field-portable. It also overcomes the disadvantages of undesired reflections from different optical elements. The experimental results of simultaneous imaging and spectroscopy of the biological samples are presented along with quantitative spectroscopic parameters; peak wavelength shift, area under the curve and full width half maximum (FWHM). The spatial resolution, spectral resolution and field of view of the system are found to be 4.38 µm, 0.5 nm and 2.071×1.548mm2 , respectively. Furthermore, we have obtained the red shift for cancerous oral tissue with respect to normal oral tissue 5.79 ± 1.071 nm. This could be important indicator for oral cancer screening.

Simultaneous fluorescence and quantitative phase imaging of MG63 osteosarcoma cells to monitor morphological changes with time using partially spatially coherent light source.

Quantitative phase imaging (QPI) technique is used to determine various biophysical parameters, such as refractive index, cell thickness, morphology, etc. On the other hand, fluorescence microscopy is used to acquire information regarding molecular specificity of the biological cells and tissues. Conventionally, a fully coherent light source such as laser is used in QPI technique to obtain the interference fringes with ease; however, its high coherence is also responsible for the generation of speckle and spurious fringes, which results in degraded image quality and affects the phase measurement results too. In this paper, we report a multi-modal system that can be effectively utilized to acquire time varied diverse information about the biological specimen with high spatial phase sensitivity. Herein, a single unit comprising of a fluorescence microscope and the Linnik based interferometer specially equipped with a partially spatially coherent light source illumination was developed. The integrated system is capable to procure molecular specificity and phase information of biological specimen, in a single shot, utilizing a single-chip color CCD camera. Here, we performed experiments on MG63 osteosarcoma cells, and the composite interferometric-fluorescence images were obtained and then digitally decomposed into red and green colors; and, the phase maps were reconstructed using the Fourier fringe analysis method. Furthermore, the cultured cells were monitored over a time-span to observe and investigate the time dependent morphological changes along with the quantification of cellular adhesion and spreading. Hence, the proposed system can be utilized to quantify time dependent changes in the cell's morphology and in cell adhesion which can be an indicator for the detection of various range of diseases such as arthritis, cancer, osteoporosis and atherosclerosis.