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.
With applications in quantitative metabolomics and label-free digital pathology, Quantitative Phase Microscopy (QPM) measures refractive index maps of thin transparent specimens like live cells or tissue sections. In QPM, refractive index maps are usually reconstructed from interference measurements of the object’s light field with respect to a reference field. To this end, many previous works focused on designing stable full-field interferometers from the bottom up. In this work, we present an alternative strategy to design a QPM system top-down, starting from the desired measurement outcomes, with no explicit knowledge about interferometry. We call our inverse design strategy Differentiable Microcopy. To this end, our Differentiable Microcopy approach designed a range of Fourier-filter-based QPM systems that do not require computational post-reconstruction. Our designs are superior to existing similar designs in numerical benchmarks. We also experimentally validated one design using a spatial light modulator. Finally, to fabricate these custom designs in the future, we also propose a new fabrication-aware Differentiable Microcopy pipeline.
The ubiquitous nature of microplastics in marine environments poses a significant threat to Atlantic salmon aquaculture, and fish in general. Mucosal surfaces are continuously exposed to microparticles – such as the skin epithelial layer with highly migrating keratocyte cells. This study applied the capabilities of Linnik-based Quantitative Phase Microscopy (QPM) equipped with a pseudo-thermal light source (PTLS) to examine the interactions between polystyrene microplastics (MPs) and Atlantic salmon keratocytes. The PTLS enables the QPM system to achieve high spatial phase sensitivity, allowing for the observation of precise morphological changes within the cell and migration in real time. Our study shows that cells that were exposed to polystyrene MPs exhibited dynamic fluctuations of the lamellipodia and provide evidence for early-stage phase membrane invaginations around the beads indicative of phagocytic activity. Implementation of QPM based on PTLS is a high-speed, non-invasive, label-free approach that provides vital insights into the morphological dynamics of MP exposure. This research not only contributes to the understanding of cellular morphological responses to microplastics in Atlantic salmon but also highlights the utility of advanced QPM imaging techniques from an environmental toxicology perspective.
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.
With applications ranging from metabolomics to histopathology, quantitative phase microscopy (QPM) is a powerful label-free imaging modality. However, the speed of current QPM systems is limited by electronic hardware. To improve throughput further, here we propose differentiable optical-electronic quantitative phase microscopy (∂μ) that acquires images in a compressed form such that more information can be transferred beyond the electronic hardware bottleneck. The proposed microscopy uses optical feature extractors as image compressors. The resultant intensity distribution is then decompressed into QPM images by a reconstruction network. By optimizing optical-electronic parameters in an end-to-end manner, our method can improve the QPI throughput from Hours to Seconds (more than an order of magnitude).
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.
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.
Chip-based optical nanoscopy, relying on single molecule localization microscopy has recently been demonstrated to reach 70 nm lateral resolution over wide fields of view (500 µm x 500 µm). To make this technique more sustainable for live-cell imaging we embedded a photonic chip into a microfluidic support that is able to perfuse and thermalize the samples. In this way specimens are maintained under physiological conditions during the imaging which can be a timeconsuming process. The system consists of a multilayer chip with the size of a glass coverslip (60 mm × 24 mm). The sample is illuminated using waveguides that are fabricated from high refractive index material. The waveguide hosts a chamber (17.3 μl) where cells are seeded and perfused with medium. A thin layer (188 µm) of cyclic olefin polymer (COP) seals the chamber and allows optical image acquisition. A thermalizing solution is perfused from the bottom to accurately warm up/cool down the waveguide in a range of 5°C - 45°C. Thus, samples are kept at the proper temperature. As proof of concept and verification of super-resolution imaging, we imaged fluorescent beads perfused across the coated surface (fibronectin 0.2 mg/ml) of the chip, which is needed to guarantee proper cell-to-substrate adhesion.
In this work we have explored the live-cell friendly nanoscopy method Multiple Signal Classification Algorithm (MUSICAL) for multi-colour imaging of various organelles and sub-cellular structures in the cardiomyoblast cell line H2c9. We have tested MUSICAL for fast (up to 230Hz), multi-colour time-lapse sequences of various sub-cellular structures (mitochondria, endoplasmic reticulum, microtubules, endosomes and nuclei) in living cells using low excitation-light dose. Challenges and opportunities with applying MUSICAL for studying rapid sub-cellular dynamics are discussed.
In this project it was found that Fourier ptychographic microscopy can be improved far beyond its conventional limits via waveguide-based optical systems. Extensive in silico studies showed that images obtained on high-refractive index material waveguide chips in conjunction with hyperspectral illumination light and finely designed waveguide geometries can be combined via a modified phase-retrieval algorithm to yield a resolution below 150 nm.
Super-resolution optical microscopy, commonly referred to as optical nanoscopy, has enabled imaging of biological
samples with a resolution that was only achievable previously using electron microscopy. Optical nanoscopy is a rapidly
growing field, with several different techniques and implementations that overcome the diffraction limit of light.
However, the common nanoscope continues to be a rather complex, expensive and bulky instrument. Direct stochastic
optical reconstruction microscopy (dSTORM) imaging was recently demonstrated using a waveguide platform for
excitation in combination with a simple microscope for imaging. High refractive index waveguide materials have a high
intensity evanescent field stretching around 100-200 nm outside the guiding material, which is ideally suited for total
internal reflection fluorescence (TIRF) excitation over large areas. We demonstrate dSTORM imaging of the plasma
membrane of liver sinusoidal endothelial cells (LSECs) and trophoblasts (HTR-8 cells) using waveguide excitation, with
resolution down to around 70 nm. Additionally, we present TIRF imaging of LSEC micro-tubules over a 500 μm x 500
μm area, laying the foundation for large field of view (f-o-v) nanoscopy.
The evanescent field on top of optical waveguides is used to image membrane network and sieve-plates of liver
endothelial cells. In waveguide excitation, the evanescent field is dominant only near the surface (~100-150 nm)
providing a default optical sectioning by illuminating fluorophores in close proximity to the surface and thus benefiting
higher signal-to-noise ratio. The sieve plates of liver sinusoidal endothelial cells are present on the cell membrane, thus
near-field waveguide chip-based microscopy configuration is preferred over epi-fluorescence. The waveguide chip is
compatible with optical fiber components allowing easy multiplexing to different wavelengths. In this paper, we will
discuss the challenges and opportunities provided by integrated optical microscopy for imaging cell membranes.
Optical nanoscopy allows to study biological and functional processes of sub-cellular organelles. In structured
illumination microscopy (SIM) the sample is illuminated with a grid-like interference pattern to encode higher spatial
frequency information into observable Moiré patterns. By acquiring multiple images and a computation trick a superresolved
image is obtained. SIM provides resolution enhancement of 2X in each axis as compared to conventional
microscopes. For a visible light, SIM provides an optical resolution of 100 nm. The challenges associated with optical
nanoscopy of a living cell are photo-toxicity, special dye requirements and artifacts due to cell movement. SIM works
with conventional dyes and is a wide-field technique making it suitable for imaging living cells. In this work, we will
discuss the opportunities and challenges of imaging living cells using SIM. Two applications of optical nanoscopy of
living cells will be discussed; a) imaging of mitochondria in a keratinocyte cell and
Optical microscopy based on fluorescence has emerged as a vital tool in modern bio-medical imaging and diagnosis.
Super-resolution bio-imaging allows gathering information from sub-cellular organelles. In structured illumination
microscopy (SIM) the sample is illuminated with a grid-like interference patterns to encode higher spatial frequencies
information into observable images (Moiré fringes). A super-resolved image is then decoded using computational trick.
In this work, we used SIM to acquired super-resolved optical images of mitochondria from a live keratinocyte cell (see
Fig 1). SIM provides resolution enhancement of 2X in each axis and contrast enhancement of 8X on a projected image.
Time-lapsed imaging was used to study the dynamics of mitochondria in a live cell.
Reduced blood deformability is clinically linked to several diseases. It is important to develop sensitive tools to measure the loss of blood deformability. The evanescent field of an optical waveguide can trap and propel red blood cells along the waveguide. Here we propose to use the evanescent field from a narrow optical waveguide to trap and deform red blood cells. We demonstrate that the intensity gradient of the evanescent field at the edge of narrow waveguides (1-3 μm) can be used to squeeze a blood cell. The RBCs are squeezed to a size comparable to the waveguide width. When the laser is switched on the cell is attracted towards the waveguide and is held in place. Subsequently, the part of the cell not on the waveguide is pulled in across the waveguide. The result is a cell (7-8 μm in diameter) squeezed down to a significantly smaller width (typically 3 μm). The cell regains its original shape when laser is switched-off.
An integrated optical sensor is developed for measuring gas concentration for subsea and atmospheric applications. The optical sensor is based on a waveguide Mach-Zehnder interferometer (MZI). In MZI, the light is spilt into a sensing and a reference arm and after a certain distance the branches are recombined. The sensing branch is covered with a sensitive layer that has high affinity towards a specified gas. The presence of the gas gives a change in the refractive index of the sensing arm, which is translated into a change in the output signal. With a prior calibration, the change in the output signal is correlated to the gas concentration. The waveguide should be single-mode and it is desirable to have high intensity in
the evanescent field. By using a high refractive index material and a thin waveguide core, the intensity of the evanescent field can be enhanced. Simulations are performed to obtain waveguide parameters with low losses and high sensitivity. The maximum sensitivity at wavelength 785 nm was obtained for a waveguide of core thickness 150 nm, rib height 5 nm and width 1 m for TM polarization. The first measurements of phase sensitivity of 12456π rad/RIU was obtain by the Hydrogen Chlorine (HCl) measurement. This is comparable to the phase sensitivity of 14268π rad/RIU obtained by the simulation.μ
The evanescent field from an optical waveguide is used for near-field trapping and transporting of fluorescent microspheres. Out-of-focus fluorescence imaging is used to track the trapped particle in 3-D with nanometer precision (<100 nm). A prior calibration is done to determine the relationship between the z-coordinate and the radius of the outermost diffraction ring in the image of the sphere. This gives precise information about how much the particle moves up and down during propulsion along the waveguide. Results are presented for trapping and tracking a 1 μm fluorescent particle on a strip waveguide.
The evanescent field from a waveguide can be used to trap and propel a particle. An optical waveguide loop with an
intentional gap at the center is used for planar transport and stable trapping of particles. The waveguide acts as a
conveyor belt to trap and deliver spheres towards the gap. At the gap, the counter-diverging light fields hold the sphere
at a fixed position. Numerical simulation based on the finite element method was performed in three dimensions using a
computer cluster. The field distribution and optical forces for rib and strip waveguide designs are compared and
discussed. The optical force on a single particle was computed for various positions of the particle in the gap. Simulation
predicted stable trapping of particles in the gap. Depending on the gap separation (2-50 μm) a single or multiple spheres
and red blood cells were trapped at the gap. Waveguides were made of tantalum pentaoxide material. The waveguides
are only 180 nm thick and thus could be integrated with other functions on the chip.
A three dimensional finite element method is used to model the forces acting on red blood cells trapped on
an optical waveguide surface. The parameters are chosen to correspond to strip waveguides made of tantalum
pentoxide (Ta2O5). A wavelength of 1070 nm is used and the cells are taken to be spherical. Gradient and
scattering forces experienced by the cells are studied and found to be highly dependent on the refractive index
of the cells. Gradient forces are found to be one order of magnitude larger than scattering forces. Only the lower
part of the cells is in contact with the evanescent field of the waveguide. For low refractive indices, we find that
the lower 0.5-1 μm of the cells is sufficient to determine the optical forces. For the cell sizes considered, all forces
increase with the size.
Three dimensional finite element method is employed to determine optical trapping forces on hollow glass spheres on
an optical waveguide. The evanescent field from the waveguide interacts mostly with the shell of the hollow glass
sphere. We describe how the optical forces vary with shell thickness and particle size, and find the minimum shell
thickness allowing trapping and propulsion of hollow glass spheres. Hollow glass spheres with shell thickness less than
the minimum are repelled away from the optical waveguide. The simulation results are compared with analytical Mie
calculations and experimental data.
Design, fabrication and optimization of high refractive index (2.1 @ 1070 nm), sub-micron thickness (200 nm) Tantalum
Pentoxide waveguides is reported. Optimization of fabrication parameters reduces the propagation loss to ~ 1 dB/cm @
1070 nm for Ta2O5 waveguides. Ta2O5 waveguides were found to be stable for high power application with no significant
absorption peaks over a large range of wavelengths (600-1700 nm). Ta2O5 waveguides provide high intensity in the
evanescent field, which is useful for efficient optical propelling of micro-particles. We have employed Ta2O5 waveguide
to propel polystyrene micro-particles with 50 μm/s velocity.
The high-refractive index contrast (▵n ~0.65 as compared to silicon oxide) of Tantalum pentoxide (Ta2O5) waveguide
allows strong confinement of light in waveguides of sub-micron thickness (200 nm). This enhances the intensity in the
evanescent field, which we have employed for efficient propelling of micro-particles. The feasibility of opto-fluidics
sorting of different sized micro-particles based on their varying optical propulsion velocity is suggested. Optical
propulsion of fixed red blood cells (RBC) with velocity higher than previously obtained is also reported. The optical
propulsion velocities of RBC in isotonic solution (0.25 M sucrose) and water have been compared.
Non-diffracting laser modes and interfering non-diffracting beams have been extensively studied. Interfering nondiffracting
beams generate novel laser modes. In this paper we accumulate various interfering conditions for nondiffracting
beams and discuss the properties of the resultant beam. Contrasting intensity profiles and topological charge
distribution are obtained on varying interfering conditions. Collinear propagation of non-diffracting laser beams is also
reported.
Light assisted micro-manipulation techniques have provided a non-invasive technique to investigate microscopic world. Employing micro-optical elements for laser beam shaping can aggrandize the trapping capabilities of conventional optical tweezers system. In the paper we report the design parameters and fabrication techniques of diversified micro-optical elements. High quality Laguerre-Gaussian beams, Bessel beams, self-imaged bottle beams and fractional Bessel beams were achieved using such micro-optical elements. We further integrated these micro-elements in conventional optical tweezers systems for various trapping applications. Such micro-elements were high efficient (in terms of power conversion) which is an important criteria to be incorporated with the optical trapping systems. Due to its micro-size such micro-elements are potential candidates to be integrated with lab-on-a-chip techniques to realize next generation "miniaturized optical trap".
Optical tweezers technique has aggrandized the light-matter interaction, whereby researchers have employed light forces from colloidal sciences to biomedical applications. There have been constant quests to integrate novel beams in tweezers system for myriads of novel applications. In this paper, we report optical tweezers system based on self-imaged optical bottle beams, which has some distinct advantages over other conventional beams like Gaussian, Laguerre-Gaussian and Bessel beams in few applications. A self-imaged bottle beam is obtained by superimposing two Bessel beams with varying radial wavevectors. A self-imaged bottle beam possesses three-dimensional intensity-null points along the propagation axis and by varying the radial wavevectors of the Bessel beams number of bottles; location of bottles and the dimension of the bottles can be pre-determined. Moreover, by dynamically varying the parameters of radial wavevector of interfering Bessel beams, the dynamic potential-well obtained are capable of transporting both high and low indices microparticle in conveyor-belt arrangement along the longitudinal axis. Bottle beams belongs to the family of propagation-invariant beams and hence inherent the propagation-invariance and self-reconstruction properties of Bessel beam together with three-dimensional trapping potential contrary to Bessel beam whereby the particles trapped in the central core are loosing defined in the longitudinal axis. Such bottle beams also holds promise applications in blue-detuned optical taps for the optical confinement of laser-cooled atoms in three-dimensional dark region.
The quest for applying optical tweezers system for novel applications has aggrandized its trapping capabilities since its inception. Researchers have proposed and applied light based micro-manipulation technique in the field of colloidal sciences, bioscience, MEMS and the count is limitless. In this paper we report the self-imaged optical bottle beam based optical tweezers system. A self-imaged bottle beam possesses three-dimensional intensity-null points along the propagation axis. The transverse intensity profile of the self-imaged bottle beam oscillates along the propagation axis, hence providing three-dimensional trapping potential for high and low indices microparticles at constructive and destructive interference points, respectively. Bottle beam based optical tweezer system adds the beneficial property of Gaussian and Bessel beam based trapping systems by providing three-dimensional trapping potential and self-reconstruction ability, respectively. As self-imaged bottle beam belong to the family of propagation-invariant beams, it can be used to trap chain of high and low indices microparticles three-dimensionally along the propagation directions, which can be used to periodically stack microparticles (of different refractive index) longitudinally.
Optical trapping and manipulation are based on three types of the force and momentum, in the range of pN, to grab, lift and rotate microparticles due to the light intensity or phase distribution. In the first type, particles are trapped in the highest intensity region of the beam due to optical gradient force for high refractive index particles with respective to its surrounding medium. Secondly, orbital angular momentum (OAM) of a beam can be transferred to a particle due to phase singularity within a beam. Thirdly, spin angular momentum (SAM) can be transferred to the particles due to the circular polarization of the beam. In recent years, microfabricated optical elements have been used to modulate the amplitude and phase of the optical beam to create new generation of optical tweezers with additional manipulation dimensions. In this paper, a review of our recent discoveries of optical trapping and manipulation using micro-beam shaping and micro-optical elements will be presented.
The Laguerre-Gaussian beams or higher-order Bessel beams, which possess a full-doughnut shaped intensity pattern at the center, repel the low index particles away from the dark central spot due to radiation pressure. Optical beams with fractional helical phase are possible solutions to this problem, as they possess a clear opening slit. By using a spatial light modulator, we demonstrate the control of an opening slit by embedding different fractional helical phase into Bessel beams and Gaussian beams. This dynamic opening and closing of the slit will allow a possible selective process of the micro-particles. This is also a new way to control the guiding of particles in the transverse plane instead of the propagation direction.
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