Title: Full-field spectral-domain optical interferometry for snapshot three-dimensional microscopy
Prevalent techniques in label-free linear optical microscopy are either confined to imaging in two dimensions or rely on scanning, both of which restrict their applications in imaging subtle biological dynamics. In this paper, we present the theoretical basis along with demonstrations supporting that full-field spectral-domain interferometry can be used for imaging samples in 3D with no moving parts in a single shot. Consequently, we propose a novel optical imaging modality that combines low-coherence interferometry with hyperspectral imaging using a light-emitting diode and an image mapping spectrometer, called Snapshot optical coherence microscopy (OCM). Having first proved the feasibility of Snapshot OCM through theoretical modeling and a comprehensive simulation, we demonstrate an implementation of the technique using off-the-shelf components capable of capturing an entire volume in 5 ms. The performance of Snapshot OCM, when imaging optical targets, shows its capability to axially localize and section images over an axial range of ±10 µm, while maintaining a transverse resolution of 0.8 µm, an axial resolution of 1.4 µm, and a sensitivity of up to 80 dB. Additionally, its performance in imaging weakly scattering live cells shows its capability to not only localize the cells in a densely populated culture but also to generate detailed phase profiles of the structures at each depth for long durations. Consolidating the advantages of several widespread optical microscopy modalities, Snapshot OCM has the potential to be a versatile imaging technique for a broad range of applications.
Non-destructive measurements of internal morphological structures in plant materials such as seeds are of high interest in agricultural research. The estimation of pericarp thickness is important to understand the grain quality and storage stability of seeds and can play a crucial role in improving crop yield. In this study, we demonstrate the applicability of fiber-based Bessel beam Fourier domain (FD) optical coherence microscopy (OCM) with a nearly constant high lateral resolution maintained at over ~400 µm for direct non-invasive measurement of the pericarp thickness of two different sorghum genotypes. Whereas measurements based on axial profiles need additional knowledge of the pericarp refractive index, en-face views allow for direct distance measurements. We directly determine pericarp thickness from lateral sections with a 3 µm resolution by taking the width of the signal corresponding to the pericarp at the 1/e threshold. These measurements enable differentiation of the two genotypes with 100% accuracy. We find that trading image resolution for acquisition speed and view size reduces the classification accuracy. Average pericarp thicknesses of 74 µm (thick phenotype) and 43 µm (thin phenotype) are obtained from high-resolution lateral sections, and are in good agreement with previously reported measurements of the same genotypes. Extracting the morphological features of plant seeds using Bessel beam FD-OCM is expected to provide valuable information to the food processing industry and plant breeding programs.
Light field microscopy (LFM) is an emerging technology for high-speed wide-field 3D imaging by capturing 4D light field of 3D volumes. However, its 3D imaging capability comes at a cost of lateral resolution. In addition, the lateral resolution is not uniform across depth in the light field dconvolution reconstructions. To address these problems, here, we propose a snapshot multifocal light field microscopy (MFLFM) imaging method. The underlying concept of the MFLFM is to collect multiple focal shifted light fields simultaneously. We show that by focal stacking those focal shifted light fields, the depth-of-field (DOF) of the LFM can be further improved but without sacrificing the lateral resolution. Also, if all differently focused light fields are utilized together in the deconvolution, the MFLFM could achieve a high and uniform lateral resolution within a larger DOF. We present a house-built MFLFM system by placing a diffractive optical element at the Fourier plane of a conventional LFM. The optical performance of the MFLFM are analyzed and given. Both simulations and proof-of-principle experimental results are provided to demonstrate the effectiveness and benefits of the MFLFM. We believe that the proposed snapshot MFLFM has potential to enable high-speed and high resolution 3D imaging applications.
We present a robust fiber-based setup for Bessel-like beam extended depth-of-focus Fourier-domain optical coherence microscopy, where the Bessel-like beam is generated in a higher order mode fiber module. In this module a stable guided LP02core mode is selectively excited by a long period grating written in the higher order mode fiber. Imaging performance of this system in terms of lateral resolution and depth of focus was analyzed using samples of suspended microbeads and compared to the case where illumination is provided by the fundamental LP01mode of a single mode fiber. Illumination with the LP02mode allowed for a lateral resolution down to 2.5 µm as compared to 4.5 µm achieved with the LP01mode of the single mode fiber. A three-fold enhancement of the depth of focus compared to a Gaussian beam with equally tight focus is achieved with the LP02mode. Analysis of the theoretical lateral point spread functions for the case of LP01and LP02illumination agrees well with the experimental data. As the design space of waveguides and long-period gratings allows for further optimization of the beam parameters of the generated Bessel-like beams in an all-fiber module, this approach offers a robust and yet flexible alternative to free-space optics approaches or the use of conical fiber tips.
Mapping 3D plasma membrane topology in live cells can bring unprecedented insights into cell biology. Widefield-based super-resolution methods such as 3D-structured illumination microscopy (3D-SIM) can achieve twice the axial ( ~ 300 nm) and lateral ( ~ 100 nm) resolution of widefield microscopy in real time in live cells. However, twice-resolution enhancement cannot sufficiently visualize nanoscale fine structures of the plasma membrane. Axial interferometry methods including fluorescence light interference contrast microscopy and its derivatives (e.g., scanning angle interference microscopy) can determine nanoscale axial locations of proteins on and near the plasma membrane. Thus, by combining super-resolution lateral imaging of 2D-SIM with axial interferometry, we developed multi-angle-crossing structured illumination microscopy (MAxSIM) to generate multiple incident angles by fast, optoelectronic creation of diffraction patterns. Axial localization accuracy can be enhanced by placing cells on a bottom glass substrate, locating a custom height-controlled mirror (HCM) at a fixed axial position above the glass substrate, and optimizing the height reconstruction algorithm for noisy experimental data. The HCM also enables imaging of both the apical and basal surfaces of a cell. MAxSIM with HCM offers high-fidelity nanoscale 3D topological mapping of cell plasma membranes with near-real-time ( ~ 0.5 Hz) imaging of live cells and 3D single-molecule tracking.
Cannon, Taylor M.; Bouma, Brett E.; Uribe-Patarroyo, Néstor(
, Biomedical Optics Express)
Structural optical coherence tomography (OCT) images of tissue stand to benefit from greater functionalization and quantitative interpretation. The OCT attenuation coefficientµ, an analogue of the imaged sample’s scattering coefficient, offers potential functional contrast based on the relationship ofµto sub-resolution physical properties of the sample. Attenuation coefficients are computed either by fitting a representativeµover several depth-wise pixels of a sample’s intensity decay, or by using previously-developed depth-resolved attenuation algorithms by Girardet al.[Invest. Ophthalmol. Vis. Sci.52,7738(2011).10.1167/iovs.10-6925] and Vermeeret al.[Biomed. Opt. Express5,322(2014).10.1364/BOE.5.000322]. However, the former method sacrifices axial information in the tomogram, while the latter relies on the stringent assumption that the sample’s backscattering fraction, another optical property, does not vary along depth. This assumption may be violated by layered tissues commonly observed in clinical imaging applications. Our approach preserves the full depth resolution of the attenuation map but removes its dependence on backscattering fraction by performing signal analysis inside individual discrete layers over which the scattering properties (e.g., attenuation and backscattering fraction) vary minimally. Although this approach necessitates the detection of these layers, it removes the constant-backscattering-fraction assumption that has constrained quantitative attenuation coefficient analysis in the past, and additionally yields a layer-resolved backscattering fraction, providing complementary scattering information to the attenuation coefficient. We validate our approach using automated layer detection in layered phantoms, for which the measured optical properties were in good agreement with theoretical values calculated with Mie theory, and show preliminary results in tissue alongside corresponding histological analysis. Together, accurate backscattering fraction and attenuation coefficient measurements enable the estimation of both particle density and size, which is not possible from attenuation measurements alone. We hope that this improvement to depth-resolved attenuation coefficient measurement, augmented by a layer-resolved backscattering fraction, will increase the diagnostic power of quantitative OCT imaging.
Iyer, Rishyashring R., Žurauskas, Mantas, Cui, Qi, Gao, Liang, Theodore Smith, R., and Boppart, Stephen A. Full-field spectral-domain optical interferometry for snapshot three-dimensional microscopy. Biomedical Optics Express 11.10 Web. doi:10.1364/BOE.402796.
Iyer, Rishyashring R., Žurauskas, Mantas, Cui, Qi, Gao, Liang, Theodore Smith, R., and Boppart, Stephen A.
"Full-field spectral-domain optical interferometry for snapshot three-dimensional microscopy". Biomedical Optics Express 11 (10). Country unknown/Code not available: Optical Society of America. https://doi.org/10.1364/BOE.402796.https://par.nsf.gov/biblio/10194952.
@article{osti_10194952,
place = {Country unknown/Code not available},
title = {Full-field spectral-domain optical interferometry for snapshot three-dimensional microscopy},
url = {https://par.nsf.gov/biblio/10194952},
DOI = {10.1364/BOE.402796},
abstractNote = {Prevalent techniques in label-free linear optical microscopy are either confined to imaging in two dimensions or rely on scanning, both of which restrict their applications in imaging subtle biological dynamics. In this paper, we present the theoretical basis along with demonstrations supporting that full-field spectral-domain interferometry can be used for imaging samples in 3D with no moving parts in a single shot. Consequently, we propose a novel optical imaging modality that combines low-coherence interferometry with hyperspectral imaging using a light-emitting diode and an image mapping spectrometer, called Snapshot optical coherence microscopy (OCM). Having first proved the feasibility of Snapshot OCM through theoretical modeling and a comprehensive simulation, we demonstrate an implementation of the technique using off-the-shelf components capable of capturing an entire volume in 5 ms. The performance of Snapshot OCM, when imaging optical targets, shows its capability to axially localize and section images over an axial range of ±10 µm, while maintaining a transverse resolution of 0.8 µm, an axial resolution of 1.4 µm, and a sensitivity of up to 80 dB. Additionally, its performance in imaging weakly scattering live cells shows its capability to not only localize the cells in a densely populated culture but also to generate detailed phase profiles of the structures at each depth for long durations. Consolidating the advantages of several widespread optical microscopy modalities, Snapshot OCM has the potential to be a versatile imaging technique for a broad range of applications.},
journal = {Biomedical Optics Express},
volume = {11},
number = {10},
publisher = {Optical Society of America},
author = {Iyer, Rishyashring R. and Žurauskas, Mantas and Cui, Qi and Gao, Liang and Theodore Smith, R. and Boppart, Stephen A.},
}
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