Quantitative phase microscopy (QPM) has recently become indispensable technology for label-free quantitative analysis of various biological cells and tissues, such as, sperm cells, liver sinusoidal cells, cancerous cells, red blood cells etc. The key parameters controlling measurement accuracy and capability of QPM system depends on its spatial and temporal phase sensitivity. The spatial phase sensitivity of QPM is governed by coherence properties of light source and temporal stability depends on optical interferometric configuration. Most of the QPM techniques utilize highly coherent light sources like lasers benefited by their high spatial and temporal coherence, and brightness. But high spatio-temporal coherence leads to occurrence of speckle noise and spurious fringes leading to inhomogeneous illumination and poor spatial phase sensitivity. We have developed QPM systems using partially spatially coherent monochromatic (PSCM) light sources which guarantees high contrast interferograms over large field-of-view to increase space-bandwidth product of QPM system by ten-times and demonstrated ten-fold improvement in spatial-phase sensitivity and phase measurement accuracy compared to coherent laser light. By means of using PSCM with common path configuration we could also achieve ten-fold temporal phase stability. We have demonstrated advantages of PSCM based QPM in various industrial and bio-imaging applications. Experimental results of reduced speckle noise, free-from spurious fringes, spatial phase sensitivity using industrial objects are demonstrated and compared with highly coherent light using single mode fiber. Finally, phase map of biological samples is also presented with high accuracy in phase measurement. Thus, the use of PSCM light in phase microscopy, holography of realistic objects, i.e., industrial and biological samples leads to high accuracy in the measurement of quantitative information.
We describe field-portable GRIN lens based micro-endoscope with oblique-illumination for cancer screening. Fluorescence microscopic images of different samples were recorded with micro-endoscope which provides molecular information about the sample.
We demonstrate a multi-modal system to obtain diverse information about biological specimen in single-shot. The quantitative analysis of MG63 osteosarcoma cells is presented which are cultured on Si substrate and stained using sodium fluorescein dye.
We report classification between normal and anemic erythrocytes by determining cell counts computationally using Circular Hough transform algorithm in matlab and quantifying phase map which are important for early diagnosis of diseases.
We report the development of field-portable multi-modal chip-based fluorescence, bright field and quantitative phase microscopy using smartphone detecting system. Fluorescence microscopy provide molecular information of the specimen with excellent specificity, while phase microscopy provides quantitative information of the specimen. Quantifying the optical phase shifts associated with biological structures gives access to information about morphology and dynamics at the nanometer scale. Here, we propose an integrated waveguide chip-based total internal reflection fluorescence (TIRF) microscopy and quantitative phase microscopy (QPM). We have developed microLED with cylindrical beam profile to couple excitation light into the edges of glass slide easily and efficiently. The evanescent field present on top of a waveguide surface is used to excite the fluorescence and a mobile phone microscope is used to collect the signal. 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. Light for bright field imaging and QPM integrated in the transmission mode. A microscope objective is used for collecting the fluorescence excited by evanescent field and transmitted light for bright field and quantitative phase microscopy (QPM). A compact and common path interferometer is used for QPM. The entire device is fabricated using three-D printer and integrated into one, which is compact and field portable. Images are recorded using a smart phone. Experimental results of onion epithelial cells, polystyrene microspheres and normal breast tissue are presented. The cost of entire system is very less.
Quantitative phase microscopy (QPM) is a label-free imaging technique to quantify various biophysical parameters, such as refractive index, optical thickness, cell dry mass, and dynamic membrane fluctuations. Accurate determination of these parameters requires the use of a QPM system with high temporal phase stability and high spatial phase sensitivity. We report a QPM system based on a common-path interferometer with high temporal phase stability and high spatial phase sensitivity. The proposed QPM system is highly temporally stable, compact and easy to align and implement. The interference pattern can be obtained quickly even with a low coherent light source. In order to realize high spatial phase sensitivity, we used partially spatially coherent (pseudo-thermal) light source for illumination. Due to the partial spatial coherent nature of the light source, a speckle-free interferogram/hologram is recorded over the entire field-of-view. Two types of speckle free QPM systems are implemented using common path Fresnel biprism as well as lateral shearing interferometers. A Fresnel biprism is used in the self-referencing mode, thus offering the advantage of no optical power loss in addition to high temporal stability and the least speckle artifacts. Furthermore, it is very easy to implement, as the system completely replaces the need for spatial filtering at the source end as well as for the reference beam generation. In another configuration, we used a lateral shearing interferometer. The scattered light from the object is collected by the microscope objective lens and passes through a 4mm thick optically flat parallel plate to generate the interference pattern. Phase maps of human RBCs are reconstructed and the results are compared for fully and partially coherent light illumination.
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