Structured illumination microscopy (SIM) is a most popular super-resolution technique used in cell biology and bio-imaging. Here, we present a novel approach to realize multiscale super-resolution SIM by swapping the non-linearity between instrumentation and reconstruction algorithm to achieve super-resolution. Our goal is to overcome two conventional limitations of SIM i.e., fixed resolution and the need of precise knowledge of illumination pattern. The optical system encodes higher order frequencies of the sample by projecting PSF-modulated binary patterns for illuminating the sample plane, which do not have clean Fourier peaks conventionally used in SIM. These patterns fold high frequency content of sample into the measurements in an obfuscated manner, which are de-obfuscated using multiple signal classification algorithm. Our approach eliminates the need of clean peaks in the illumination pattern, which have multiple advantages i.e., simple instrumentation and the flexibility of using different collection lenses. The reconstruction algorithm used in the proposed work does not require known illumination. Finally, we reduce the sensitivity of reconstruction algorithm to the signal to background ratio. Here, we acquired patterned illumination images of the same sample using different collection objective lenses, and obtained diffraction limited as well as super-resolved images, supporting 4 different resolution in the same system through SIM. Our experimental results with multiple collection objective lens show wider applicability of the proposed system at signal to background ration as small as <3.
White light phase shifting is an important technique in interferometry to extract the high-resolution quantitative phase images with high spatial phase sensitivity i.e., of the order of sub nanometer. The dynamical information about a biological sample is limited in white light phase-shifting interferometry (WL-PSI) due to multiple frame requirement. The multiple frame requirement with controlled phase-shift is the key limitation for the high-resolution phase information extraction about the sample. A high-cost piezoelectric transducer (PZT) is required to introduce equal phase-shifts between the frames in WL-PSI. Here, we introduce a deep learning (DL)-based phase-shifter instead of PZT to introduce equal phase-shift in WL-PSI. We use deep neural network to introduce the equal phase-shift between the data frames. The idea is to train the network with multiple equal phase-shifted frames for training and learn the basic application of a PZT. After sufficient training of the network, it will generate multiple phase-shifted frames. Our study is validated by simulating step-like object with equal phase-shifts for training and testing of the network. The network is trained for a total 4 phase-shifted frame generation from a single interferogram. Further, the line profile of DL-based phase-shifter generated data frames are compared with the line profile of simulated data frames. Finally, generated phase-shifted data frames are used for the final phase reconstruction of the sample and compared with the phase reconstruction from simulated data frames.
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.
Optical coherence tomography (OCT) is low temporal coherence gated cross-sectional imaging modality which is noncontact, non-invasive and in vivo technology. In conventional OCT mainly a broad-band light source is used to achieve high axial resolution which is inversely proportional to spectral bandwidth of the light source. The most preferred light source in OCT is super-luminescent diode (SLD) or a supercontinuum light source, but they are bulky and costly. In addition, use of broad band light in OCT systems require dispersion compensation system because most of biological tissues are birefringence, therefore, it further adds complexity to the system. Further, use of highly spatially coherent light leads to speckle noise which degrades the image quality.
We report longitudinal spatial coherence (LSC) gated line-field optical coherence tomography (LF-OCT) of multilayer structures using partially spatially coherent monochromatic light with speckle-free and reduced crosstalk. The LSC properties of low-cost diode laser source was engineered to significantly reduce LSC length to achieve high axial-resolution. The OCT system was operated both in full-field and line-field mode and it was found that line-field version has better contrast and less crosstalk. Experimental results of LSC gated OCT of multi-layer samples both biological and industrial objects along with speckle contrast, interferometric fringe contrast and LSC length measurement will be presented. The use of a low-cost diode laser in the wavelength range 800 nm for high resolution OCT is important for many biological applications. In addition, the system does not require a dispersion compensation mechanism.
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.
Motility of cells plays an important role to determine the cell growth, health and to monitor the gene transformation. The aim of the current study is to propose a combination of a high-contrast label-free imaging method and a computational approach (conventionally used for super-resolution) which can be used as a tool in tracing the motion of the cells and organelles. Here, we integrate quantitative phase microscopy (QPM) with waveguide-based trapping (WT) and applied multiple signal classification algorithm (MUSICAL) to analyze the motion of the trapped particle. We successfully trapped and displaced a 1 µm polystyrene bead particle on a strip waveguide using a 1064 nm laser beam. While propelling the polystyrene bead particle, we recorded time-lapsed interferometric images using a partially spatially coherent (PSC) light-based off-axis QPM system. The reconstruction of time-lapsed phase images of the trapped particle is accomplished using the Fourier transform and transport of intensity algorithm, which further used in MUSICAL for the motion trace analysis. Here, we traced the motion of a trapped bead particle with scale finer than the size of the object i.e., diffraction limit of the system. We show super-resolved motion trace even though the particle’s image is itself diffraction limited in each frame. The proposed study could be useful in different biological applications such as cell monitoring, cell tracking, manipulation, and classification between healthy and unhealthy cells.
Multiple equal phase-shifted interferograms are the key for accurate phase measurement in phase-shifting interferometry (PSI). Here, we present single-shot PSI technique assisted with deep learning to extract the phase map of biological specimens.
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 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.
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.
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.
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.
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