Spectral Domain Phase Microscopy Research Articles

Spectral-Domain Phase Microscopy for Thickness Encoded Suspension Array

A system containing spectral-domain phase microscopy and fluorescence microscopy has been developed to decode and detect thickness-encoded suspension arrays. The spectral-domain phase imaging path is used for decoding the thickness of the micro glass pieces while the fluorescence imaging path is used for quantifying the targeted analytes. The spectral-domain phase microscopy uses a spectral domain phase retrieval technique so that it has a high-dynamic thickness imaging range and submicron-scale thickness resolution, increasing the encoding efficiency and decoding precision. The thickness decoding capability of the system has been demonstrated by multiplexed immunoassay experiments. The quantitative analysis capability has been verified by concentration gradient experiments.

Scanning optical coherence tomography probe for in vivo imaging and displacement measurements in the cochlea.

We developed a spectral domain optical coherence tomography (SDOCT) fiber optic probe for imaging and sub-nanometer displacement measurements inside the mammalian cochlea. The probe, 140 μm in diameter, can scan laterally up to 400 μm by means of a piezoelectric bender. Two different sampling rates are used, 10 kHz for high-resolution B-scan imaging, and 100 kHz for displacement measurements in order to span the auditory frequency range of gerbil (~50 kHz). Once the cochlear structures are recognized, the scanning range is gradually decreased and ultimately stopped with the probe pointing at the selected angle to measure the simultaneous displacements of multiple structures inside the organ of Corti (OC). The displacement measurement is based on spectral domain phase microscopy. The displacement noise level depends on the A-scan signal of the structure within the OC and we have attained levels as low as ~0.02 nm in in vivo measurements. The system's broadband infrared light source allows for an imaging depth of ~2.7 mm, and axial resolution of ~3 μm. In future development, the probe can be coupled with an electrode for time-locked voltage and displacement measurements in order to explore the electro-mechanical feedback loop that is key to cochlear processing. Here, we describe the fabrication of the laterally-scanning optical probe, and demonstrate its functionality with in vivo experiments.

Quantitative phase imaging using spectral domain phase microscopy without phase wrapping ambiguity.

We demonstrate a method for quantitative phase (QP) imaging without 2π ambiguity using a spectral domain phase microscopy system. The method is capable of QP measurement of a large dynamic range with a high sensitivity. We determine an integer multiple of 2π to correct wrapped phases by calculating the phase shift difference between the detected interference fringe and simulated fringes. The presented method is quantitatively verified by measuring the vibration generated by a piezo linear stage and mapping the surface topography of a slanted mirror. QP imaging of red blood cells is also performed to demonstrate the method's capacity and application in biological imaging.

Optical coherence tomography reveals complex motion between the basilar membrane and organ of corti in the gerbil cochlea

The mechanical basis of auditory transduction within the mammalian cochlea is not fully understood but relies on complex mechanical interactions between different structures within the Organ of Corti (OoC). We used spectral domain phase microscopy, a functional extension of optical coherence tomography (OCT), to measure the sound-evoked motion in response to “Zwuis” frequency complexes, in vivo, in the basal region of the gerbil cochlea. Because OCT allows for measurements from multiple axial positions along the optical path, we are able to simultaneously record vibration at different points within the OoC between the basilar membrane (BM) and tectorial membrane. Imaging through the round window membrane, we could record from several longitudinal locations along the cochlea and at each location, we measured from several radial positions. Both the BM and intra-OoC vibrations are tuned and show nonlinear amplification, or compressive growth, with the applied sound pressure level. Similar to recent reports (Ren et al, PNAS 2016; Ren and He, MoH 2017), we find that the motions of surfaces within the OoC can exhibit a greater degree of compressive nonlinearity (amplification) and physiological vulnerability than the motion of the BM. Additionally, some responses show sharp notches near the onset of the nonlinear regime.

Fourier phase in Fourier-domain optical coherence tomography

Phase of an electromagnetic wave propagating through a sample-of-interest is well understood in the context of quantitative phase imaging in transmission-mode microscopy. In the past decade, Fourier-domain optical coherence tomography has been used to extend quantitative phase imaging to the reflection-mode. Unlike transmission-mode electromagnetic phase, however, the origin and characteristics of reflection-mode Fourier phase are poorly understood, especially in samples with a slowly varying refractive index. In this paper, the general theory of Fourier phase from first principles is presented, and it is shown that Fourier phase is a joint estimate of subresolution offset and mean spatial frequency of the coherence-gated sample refractive index. It is also shown that both spectral-domain phase microscopy and depth-resolved spatial-domain low-coherence quantitative phase microscopy are special cases of this general theory. Analytical expressions are provided for both, and simulations are presented to explain and support the theoretical results. These results are further used to show how Fourier phase allows the estimation of an axial mean spatial frequency profile of the sample, along with depth-resolved characterization of localized optical density change and sample heterogeneity. Finally, a Fourier phase-based explanation of Doppler optical coherence tomography is also provided.

High-sensitive and broad-dynamic-range quantitative phase imaging with spectral domain phase microscopy

Spectral domain phase microscopy for high-sensitive and broad-dynamic-range quantitative phase imaging is presented. The phase retrieval is realized in the depth domain to maintain a high sensitivity, while the phase information obtained in the spectral domain is exploited to extend the dynamic range of optical path difference. Sensitivity advantage of phase retrieved in the depth domain over that in the spectral domain is thoroughly investigated. The performance of the proposed depth domain phase based approach is illustrated by phase imaging of a resolution target and an onion skin.

High-sensitive quantitative phase imaging with averaged spectral domain phase microscopy

We present an averaged spectral domain phase microscopy (A-SDPM) for high-sensitive quantitative phase imaging, where multiple spectral phases from different wavenumbers are fully exploited to determine the final optical path difference (OPD). Uncertainties of recovered spectral phases and OPDs at all wavenumbers are investigated, and a threshold value for the uncertainty of OPD is set to obtain the optimized averaging. A sensitivity of 17.84pm is achieved under signal to noise ratio (SNR) of 61dB, resulting in a 4.11 fold reduction in noise compared with the single spectral phase approach. The performance of the proposed A-SDPM is further illustrated by phase imaging of a coverslip with continuous changes in OPD.

Joint spectral and depth domain spectral domain phase microscopy

We present a joint spectral and depth domain spectral domain phase microscopy for high-sensitive and high-dynamic-range quantitative phase imaging, where phase information retrieved in spectral domain is used to overcome the limitation of 2π ambiguity and phase information in depth domain is used to achieve a high phase sensitivity. By theoretical derivation and simulation, the sensitivity advantage of phase information in depth domain over phase information in spectral domain is investigated. The theoretical derivation of joint spectral and depth domain spectral domain phase microscopy is presented in detail. The performance of the proposed joint spectral and depth domain spectral domain phase microscopy is illustrated by phase imaging of a coverslip and a resolution target.

Optical phase nanoscopy in red blood cells using low-coherence spectroscopy

We propose a low-coherence spectral-domain phase microscopy (SDPM) system for accurate quantitative phase measurements in red blood cells (RBCs) for the prognosis and monitoring of disease conditions that affect the visco-elastic properties of RBCs. Using the system, we performed time-recordings of cell membrane fluctuations, and compared the nano-scale fluctuation dynamics of healthy and glutaraldehyde-treated RBCs. Glutaraldehyde-treated RBCs possess lower amplitudes of fluctuations, reflecting an increased membrane stiffness. To demonstrate the ability of our system to measure fluctuations of lower amplitudes than those measured by the commonly used holographic phase microscopy techniques, we also constructed wide-field digital interferometry (WFDI) system and compared the performances of both systems. Due to its common-path geometry, the optical-path-delay stability of SDPM was found to be less than 0.3 nm in liquid environment, at least three times better than WFDI under the same conditions. In addition, due to the compactness of SDPM and its inexpensive and robust design, the system possesses a high potential for clinical applications.

Depth-encoded spectral domain phase microscopy for simultaneous multi-site nanoscale optical measurements

Spectral domain phase microscopy (SDPM) is an extension of spectral domain optical coherence tomography (SDOCT) that exploits the extraordinary phase stability of spectrometer-based systems with common-path geometry to resolve sub-wavelength displacements within a sample volume. This technique has been implemented for high resolution axial displacement and velocity measurements in biological samples, but since axial displacement information is acquired serially along the lateral dimension, it has been unable to measure fast temporal dynamics in extended samples. Depth-encoded SDPM (DESDPM) uses multiple sample arms with unevenly spaced common path reference reflectors to multiplex independent SDPM signals from separate lateral positions on a sample simultaneously using a single interferometer, thereby reducing the time required to detect unique optical events to the integration period of the detector. Here, we introduce DESDPM and demonstrate the ability to acquire useful phase data concurrently at two laterally separated locations in a phantom sample as well as a biological preparation of spontaneously beating chick cardiomyocytes. DESDPM may be a useful tool for imaging fast cellular phenomena such as nervous conduction velocity or contractile motion.

Spectral domain fluorescence coherence phase microscopy

Spectral domain phase microscopy (SDPM) has been reported in the literature as a functional extension to low-coherence interferometry, which enables nanoscale measurement of a scatter's displacement. The signal in SDPM is generated from structural images that lack molecular specificity. This paper investigates the expansion of phase analysis to fluorescence self-interference signals to provide functional information about a sample. Spectral domain fluorescence coherence phase microscopy is demonstrated for nanoscale resolution motion detection of fluorescent particles with a signal-to-noise ratio limited resolution of ~10 nm. This paper demonstrates the feasibility of combining phase processing with fluorescence self-interference, which may be useful for future applications such as cell rheology.

Spectral-domain phase microscopy with improved sensitivity using two-dimensional detector arrays

In this work we demonstrate the use of two-dimensional detectors to improve the signal-to-noise ratio (SNR) and sensitivity in spectral-domain phase microscopy for subnanometer accuracy measurements. We show that an increase in SNR can be obtained, from 82 dB to 105 dB, using 150 pixel lines of a low-cost CCD camera as compared to a single line, to compute an averaged axial scan. In optimal mechanical conditions, phase stability as small as 92 μrad, corresponding to 6 pm displacement accuracy, could be obtained. We also experimentally demonstrate the benefit of spatial-averaging in terms of the reduction of signal fading due to an axially moving sample. The applications of the improved system are illustrated by imaging live cells in culture.

High-dynamic-range quantitative phase imaging with spectral domain phase microscopy

Phase microscopy for high-dynamic-range quantitative phase-contrast imaging of a transparent phase object was demonstrated. Using a common path Fourier domain optical coherence tomography system, this technique is capable of displacement measurement with a sensitivity of 34 pm. The limitation of 2pi ambiguity restriction was overcome by the use of a phase retrieval approach performed in spectral domain. Two-dimensional quantitative phase imaging of human neonatal dermal keratinocyte cells was demonstrated to evaluate the performance of the system for cell imaging.

Improved phase sensitivity in spectral domain phase microscopy using line-field illumination and self phase-referencing

We report a quantitative phase microscope based on spectral domain optical coherence tomography and line-field illumination. The line illumination allows self phase-referencing method to reject common-mode phase noise. The quantitative phase microscope also features a separate reference arm, permitting the use of high numerical aperture (NA > 1) microscope objectives for high resolution phase measurement at multiple points along the line of illumination. We demonstrate that the path-length sensitivity of the instrument can be as good as 41 pm/square root of Hz, which makes it suitable for nanometer scale study of cell motility. We present the detection of natural motions of cell surface and two-dimensional surface profiling of a HeLa cell.

Investigating nanoscale cellular dynamics with cross-sectional spectral domain phase microscopy

We report on cross-sectional imaging of dynamic biological specimens using a spectral domain phase microscopy (SDPM) system capable of operating at a line rate of 19 kHz. This system combines the time-sensitive capabilities of SDPM with the multi-point acquisition features of related phase-sensitive techniques. The presented phase portraits and B-scan phase images of spontaneously beating embryonic cardiomyocytes and cytoplasmic flow in A. proteus offer insight into the nature and timing of the observed cellular phenomena, demonstrating the utility of this technique for dynamic cell studies.

Phase retrieval in low-coherence interferometric microscopy

We present compensating methods that address inherent errors in quantitative phase reporting for low-coherence interferometric techniques. A brief theoretical treatment of the problem and experimental validation using spectral domain phase microscopy demonstrate mitigation of the degrading effects of phase leakage on accurate measurement of optical path length in the vicinity of closely spaced reflectors. This result has direct implications for phase-sensitive interferometry techniques, such as Doppler imaging, as well as amplitude-based quantitative reporting. Corrected phase retrieval is demonstrated for conversion of interferometric phase to optical path length in cell surface deflections of beating cardiomyocytes.

Spectral domain phase microscopy for local measurements of cytoskeletal rheology in single cells

We present spectral domain phase microscopy (SDPM) as a new tool for measurements at the cellular scale. SDPM is a functional extension of spectral domain optical coherence tomography that allows for the detection of cellular motions and dynamics with nanometer-scale sensitivity in real time. Our goal was to use SDPM to investigate the mechanical properties of the cytoskeleton of MCF-7 cells. Magnetic tweezers were designed to apply a vertical force to ligand-coated magnetic beads attached to integrin receptors on the cell surfaces. SDPM was used to resolve cell surface motions induced by the applied stresses. The cytoskeletal response to an applied force is shown for both normal cells and those with compromised actin networks due to treatment with Cytochalasin D. The cell response data were fit to several models for cytoskeletal rheology, including one- and two-exponential mechanical models, as well as a power law. Finally, we correlated displacement measurements to physical characteristics of individual cells to better compare properties across many cells, reducing the coefficient of variation of extracted model parameters by up to 50%.

Optical coherence‐based imaging and sensing in biomedicine and biotechnology

Optical coherence‐based imaging techniques including optical coherence tomography (OCT), optical coherence microscopy (OCM), and spectral domain phase microscopy (SDPM) use low‐coherence spectral interferometry to obtain nanometer to micron‐scale measurements of structure, motion, and molecular composition in living cells, tissues, and organisms. OCT has become a standard diagnostic tool in clinical ophthalmology and is under investigation for other human diagnostic applications including cancer detection and evaluation of cardiovascular disease. Within the past few years, dramatic technology advances have increased the performance of OCT and OCM systems manyfold, and they are now capable of micron‐scale two‐ and three‐dimensional functional and molecular imaging noninvasively in living systems. Applications of these new technologies for high‐throughput noninvasive phenotyping and rapid 3D imaging in small animals and developmental biology models is particularly compelling. Related technology advances hav...

Doppler flow imaging of cytoplasmic streaming using spectral domain phase microscopy

Spectral domain phase microscopy (SDPM) is a function extension of spectral domain optical coherence tomography. SDPM achieves exquisite levels of phase stability by employing common-path interferometry. We discuss the theory and limitations of Doppler flow imaging using SDPM, demonstrate monitoring the thermal contraction of a glass sample with nanometer per second velocity sensitivity, and apply this technique to measurement of cytoplasmic streaming in an Amoeba proteus pseudopod. We observe reversal of cytoplasmic flow induced by extracellular CaCl2, and report results that suggest parabolic flow of cytoplasm in the A. proteus pseudopod.

Spectral-domain phase microscopy

Broadband interferometry is an attractive technique for the detection of cellular motions because it provides depth-resolved phase information via coherence gating. We present a phase-sensitive technique called spectral-domain phase microscopy (SDPM). SDPM is a functional extension of spectral-domain optical coherence tomography that allows for the detection of nanometer-scale motions in living cells. The sensitivity of the technique is demonstrated, and its calibration is verified. A shot-noise limit to the displacement sensitivity of this technique is derived. Measurement of cellular dynamics was performed on spontaneously beating cardiomyocytes isolated from chick embryos.