Differential Interference Contrast (DIC) microscopy is a label-free technique crucial for visualizing transparent biological specimens. In this study, we introduce a novel DIC interferometry imaging technique based on calcite beam displacer (CBD), significantly enhancing resolution and contrast. Our system, requires only two polarizers and a calcite beam displacer, offers a practical and straightforward implementation on standard microscopes. We demonstrate its efficacy by imaging human red blood cells (RBCs) and bacteria, retrieving differential phase information, and recording the dynamics of U2OS cells over an hour at 15 frames per second (fps). This advancement in DIC microscopy holds promise for broader applications in high-resolution imaging of transparent specimens in cell biology research.
Quantitative phase microscope (QPM) is used for the quantitative information and dynamic phase imaging of biological specimen, which provides wide application in biomedical sciences. High temporal phase stability of the QPM system is the primary requirement for accurate phase measurement. We have developed a common-path QPM geometry based on beam displacer and pinhole unit to achieve high temporal stability. The convenient adjustment of reference and object beams makes optical system compact and low-cost. The membrane fluctuations and qualitative phase are measured to demonstrate the capability and applicability of the system.
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 have investigated the enhanced Raman spectra of AMR bacteria strains of E. coli using silver coated silicon nanowires SERS assay. Three different E. coli strains, E. coli CCUG17620, NCTC 13441, and A239, were detected using two different excitation laser wavelengths. We found stable and enhanced SERS spectrum using 785 nm laser as opposed to 532 nm. Future development of SERS-chip could offer a reliable platform for direct identification of the pathogen in bio-fluid samples at strains level.
We report high-speed and highly sensitive quantitative phase microscopy (QPM) using dynamic speckle illumination (DSI). The DSI-QPM is used for real-time analyses of highly motile human spermatozoa. The DSI-QPM supports high-speed and high spatial phase sensitivity, that are crucial for imaging tail (nanoscale) of living spermatozoa during motion. The scalable FoV and high temporal coherence offered by DSI-QPM is harnessed for histopathology and marine biology. Further, by integrating the single molecule localization microscopy (SMLM) with QPM, nanoscale imaging and quantification in lateral (via SMLM) and axial (via QPM) directions was achieved on liver cells.
Histology is a well-known examination technique to study the biological cell and tissue structures. For histological assessment, imaging throughput, contrast, resolution, and quantification of morphology are crucial parameters. Although, there are techniques available which can scan the whole slide, but they lack specificity and quantification. In present study, we introduce a photonic chip based platform for multimodal imaging of FFPE tissue sections. Here, the photonic chip platform was integrated with Linnik type QPM module, which enables high contrast TIRF imaging and optical thickness of the specimen over scalable FOV. The proposed system has been used as a high throughput microscopy platform to study the functional and morphological features of FFPE human placenta tissue sections. The investigation of the tissue sections facilitates the identification and diagnosis of the various diseases, which can provide direction for treatment and can assist the prognosis of clinical outcome.
Significance: High temporal stability, wavelength independency, and scalable field of view (FOV) are the primary requirements of a quantitative phase microscopy (QPM) system. The high temporal stability of the system provides accurate measurement of minute membrane fluctuations of the biological cells that can be an indicator of disease diagnosis.
Aim: The main aim of this work is to develop a high temporal stable technique that can accurately quantify the cell’s dynamics such as membrane fluctuations of human erythrocytes. Further, the technique should be capable of acquiring scalable FOV and resolution at multiple wavelengths to make it viable for various biological applications.
Approach: We developed a single-element nearly common path, wavelength-independent, and scalable resolution/FOV QPM system to obtain temporally stable holograms/interferograms of the biological specimens.
Results: With the proposed system, the temporal stability is obtained ∼15 mrad without using any vibration isolation table. The capability of the proposed system is first demonstrated on USAF resolution chart and polystyrene spheres (4.5-μm diameter). Further, the system is implemented for single shot, wavelength-independent quantitative phase imaging of human red blood cells (RBCs) with scalable resolution using color CCD camera. The membrane fluctuation of healthy human RBCs is also measured and was found to be around 47 nm.
Conclusions: Contrary to its optical counterparts, the present system offers an energy efficient, cost effective, and simple way of generating object and reference beam for the development of common-path QPM. The present system provides the flexibility to the user to acquire multi-wavelength quantitative phase images at scalable FOV and resolution.
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.
We report longitudinal spatial coherence (LSC) gated high-resolution tomography and quantitative phase microscopy of biological cells and tissues with uniform illumination using laser as a light source. To accomplish this a pseudo thermal light source was synthesized by passing laser beams through an optical system, which is basically a speckle reduction system with combined effect of spatial, temporal, angular and polarisation diversity. The longitudinal spatial coherence length of such light was significantly reduced by synthesizing a pseudo thermal source with the combined effect of spatial, angular and temporal diversity. This results in a low spatially coherent (i.e., broad angular frequency spectrum) light source with narrow temporal frequency spectrum. Light from such a pseudo thermal light source was passed through an interference microscope with varying magnification, such as, 10X and 50X. The interference microscope was used for full-field OCT imaging of multilayer objects and topography of industrial objects. Experimental results of optical sectioning of multilayer biological objects with high axial-resolution less than 10μm was achieved which is comparable to broadband white light source. The synthesized light source with reduced speckles having uniform illumination on the sample, which can be very useful for fluorescence microscopy as well as quantitative phase microscopy with less phase noise. The present system does not require any dispersion compensation optical system for biological samples as a highly monochromatic light source is used.
In digital holographic interferometry (DHI), coherent noise degrades accuracy of phase
information. We present multi-beam polarization DHI in which two cross polarized interferograms are
recorded. Fourier analysis of interferograms reduces coherent noise and increases accuracy.
A spectrally resolved white light interferometry is demonstrated using a discrete
spectrum light sources (i.e., RGB LEDs) and monochrome CCD camera for the multi-color
quantitative phase imaging of biological cells without color cross talk.
We demonstrate results for phase maps of biological cells using white-light and multi-spectral
interference microscopy. Study on comparison of phase maps reconstructed using 1-CCD and 3-
CCD is presented to reduce color cross-talk and improved resolution.
A spatially low coherent light source is synthesized to reduce the spatial phase noise in
the laser based digital holographic microscope, which otherwise introduces unwanted spatial
phase, subsequently, the height measurement error of the biological objects.
We present a single-shot white light interference microscopy for the quantitative phase imaging (QPI) of biological cells and tissues. A common path white light interference microscope is developed and colorful white light interferogram is recorded by three-chip color CCD camera. The recorded white light interferogram is decomposed into the red, green and blue color wavelength component interferograms and processed it to find out the RI for different color wavelengths. The decomposed interferograms are analyzed using local model fitting (LMF)" algorithm developed for reconstructing the phase map from single interferogram. LMF is slightly off-axis interferometric QPI method which is a single-shot method that employs only a single image, so it is fast and accurate. The present method is very useful for dynamic process where path-length changes at millisecond level. From the single interferogram a wavelength-dependent quantitative phase imaging of human red blood cells (RBCs) are reconstructed and refractive index is determined. The LMF algorithm is simple to implement and is efficient in computation. The results are compared with the conventional phase shifting interferometry and Hilbert transform techniques.
This paper describes a new and novel phase shifting technique for qualitative as well as quantitative measurement in microscopy. We have developed a phase shifting device which is robust, inexpensive and involves no mechanical movement. In this method, phase shifting is implemented using LED array, beam splitters and defocused projection of Ronchi grating. The light from the LEDs are made incident on the beam splitters at spatially different locations. Due to variation in the geometrical distances of LEDs from the Ronchi grating and by sequentially illuminating the grating by switching on one LED at a time the phase shifted grating patterns are generated. The phase shifted structured patterns are projected onto the sample using microscopic objective lens. The phase shifted deformed patterns are recorded by a CCD camera. The initial alignment of the setup involves a simple procedure for the calibration for equal fringe width and intensity such that the phase shifted fringes are at equal phase difference. Three frame phase shifting algorithm is employed for the reconstruction of the phase map. The method described here is fully automated so that the phase shifted images are recorded just by switching of LEDs and has been used for the shape measurement of microscopic industrial objects. The analysis of the phase shifted images provides qualitative as well as quantitative information about the sample. Thus, the method is simple, robust and low cost compared to PZT devices commonly employed for phase shifting.
We report white light phase shifting interferometry in conjunction with color fringe analysis for the detection of contaminants in water such as Escherichia coli (E.coli), Campylobacter coli and Bacillus cereus. The experimental setup is based on a common path interferometer using Mirau interferometric objective lens. White light interferograms are recorded using a 3-chip color CCD camera based on prism technology. The 3-chip color camera have lesser color cross talk and better spatial resolution in comparison to single chip CCD camera. A piezo-electric transducer (PZT) phase shifter is fixed with the Mirau objective and they are attached with a conventional microscope. Five phase shifted white light interferograms are recorded by the 3-chip color CCD camera and each phase shifted interferogram is decomposed into the red, green and blue constituent colors, thus making three sets of five phase shifted intererograms for three different colors from a single set of white light interferogram. This makes the system less time consuming and have lesser effect due to surrounding environment. Initially 3D phase maps of the bacteria are reconstructed for red, green and blue wavelengths from these interferograms using MATLAB, from these phase maps we determines the refractive index (RI) of the bacteria. Experimental results of 3D shape measurement and RI at multiple wavelengths will be presented. These results might find applications for detection of contaminants in water without using any chemical processing and fluorescent dyes.
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