KEYWORDS: 3D image processing, Biological imaging, Tissues, Stereoscopy, Real time imaging, Light sources and illumination, Biomedical applications, Phase contrast, In vivo imaging, 3D applications
Quantitative oblique-back-illumination microscopy (qOBM) enables quantitative phase imaging (QPI) with epi-illumination, and thus permits the use of phase contrast in applications that were previously out-of-reach for QPI, including clinical medicine. Here, I will discuss our latest efforts to apply qOBM for clinical applications, specifically tissue imaging for non-invasive diagnostics and image guided therapy. Our approach uses an unsupervised cycle generative adversarial networks to translate 3D phase images of thick fresh tissues to appear like H&E-stained tissue sections. This work paves the way for non-invasive, label-free, real-time 3D H&E imaging which can be transformative for disease detection and guided therapy.
Quantitative phase imaging (QPI) has emerged as a valuable method in biomedical research by providing label-free, high-resolution phase distribution of transparent cells and tissues. While QPI is limited to transparent samples, quantitative oblique back-illumination microscopy (qOBM) is a novel imaging technology that enables epi-mode 3D quantitative phase imaging and refractive index (RI) tomography of thick scattering samples. This technology employs four oblique back illumination images taken at the same focal planes, along with a rapid 2D deconvolution reconstruction algorithm, to generate 2D phase cross-sections of thick samples. Alternatively, a through-focus z-stack of oblique back illumination images can be utilized to produce 3D RI tomograms, offering enhanced RI quantitative accuracy. However, 3D RI generation requires a more computationally intensive reconstruction process, preventing its potential of a real-time 3D RI tomography. In this paper, we propose a neural network-involved reconstruction technique that significantly reduces the processing time to a third while maintaining high fidelity compared to the deconvolution-based results.
Quantitative oblique back illumination microscopy (qOBM) is a recently developed phase imaging modality that enables 3D quantitative phase imaging and refractive index (RI) tomography of thick scattering samples. The approach uses four oblique illumination images (acquired in epi-mode) at a given focal plane to obtain cross sectional quantitative information. In order to quantify the information, qOBM uses a deconvolution algorithm which requires an estimate of the angular distribution of light at the focal plane to obtain the system’s optical transfer function (OTF). This information is obtained using Monte Carlo numerical simulations which uses published scattering parameters of tissues. While this approach has shown robust results with high quantitative fidelity, the reliance on available published scattering parameters is not optimal. Here we present an experimental approach to measure the angular distribution of the back-scattered light at the focal plane. The approach simultaneously obtains information from the imaging plane and the Fourier plane to provide insight into the overall angular distribution of light at the focal plane. Together with the pupil function, given by the known numerical aperture of the system, this approach directly yields the OTF. A theoretical analysis and experimental results will be presented. This approach has the potential to widen the utility of qOBM to also include tissues and samples whose scattering properties are not well documented in the literature.
Phase imaging and fluorescence microscopy provide valuable complementary information, and individually form the basis for a significant portion of the routing biological and biomedical optical imaging performed today. While multimodal phase and fluorescence microscopy has been explored for thin transparent samples to obtain structural information based on the refractive index distribution (with phase contrast) and molecular content (with fluorescence), combining these complementary technologies to study thick samples has been challenging and remains largely unexplored. This work presents the results of a study that combines quantitative phase imaging (QPI) and refractive index (RI) tomography in thick samples—using quantitative oblique back illumination—and bright field fluorescence deconvolution microscopy. The two technologies use a simple bright field microscope configuration with epi-illumination and through-focus z-stack acquisition, along with a deconvolution algorithm, to achieve 3D imaging. Phase and RI information is acquired nearly simultaneously with the fluorescence information with inherent co-registration of the two modalities. In this work, we will present the theoretical underpinning of this multimodal approach, describe the simple multimodal system, and show imaging results of thick tissues, such as labeled mice brains. This multimodal imaging approach could help biologists and clinicians gain a more comprehensive understanding of the tissue’s morphology and molecular composition, and can be widely applied across a number of biological and biomedical disciplines, including neuroscience, pathology, and oncology.
KEYWORDS: Biological samples, Phase imaging, Light sources and illumination, In vivo imaging, Imaging systems, Biological imaging, Brain, Tumors, Real time imaging, Design
Quantitative phase imaging (QPI) offers label-free access to refractive index information of biological samples, which can achieve nanometer-level optical-path-length sensitivity with cellular/sub-cellular biophysical and histological details. Recently we introduced quantitative oblique back-illumination microscopy (qOBM) which works in epi-mode and uses multiply scattered photons within thick samples to yield quantitative phase in thick scattering tissues, thus overcoming QPI’s long-standing limitation to thin transparent samples. qOBM provides real-time quantitative phase in 3D, and can be configured in a compact form factor. Here we describe a handheld qOBM probe, suitable for in-vivo diagnostic applications such as brain tumor assessment, dermatology, and more.
Quantitative oblique back-illumination microscopy (qOBM) enables quantitative phase imaging (QPI) in thick samples using epi-illumination. While qOBM offers unprecedented access to refractive index (RI) information in arbitrarily thick scattering samples, QPI-based (or RI index based) imaging still suffers from low cell nuclear contrast, which important for disease detection, including cancer. In this work, we use the acetowhitening effect of acetic acid to enhance the nuclear phase contrast of thick fresh tissue samples. Imaging results from brain samples are presented. Acetic acid phase staining may have important implications for in-vivo QPI-based disease detection
Quantitative Phase Imaging (QPI) has become a mainstay imaging technique in the biomedical sciences to study cells and other biological processes. Traditional QPI techniques are transmission-based and, thus, limited to thin samples. Over the past few years, multiple 3D QPI tools have emerged attempting to overcome this limitation and provide cross-sectional phase information of thicker samples. However, most of these techniques remain transmission-based, which constrains their ability to image samples thicker than a few mean free scattering lengths. Recently, we have developed quantitative oblique back-illumination microscopy (qOBM) as an epimode technique that enables label-free quantitative phase imaging of thick samples with tomographic crosssectioning. Like in most 3D QPI instances, qOBM requires multiple captures to render a quantitative phase image. Specifically, qOBM requires four raw captures, obtained by illuminating the sample obliquely from four different directions, to reconstruct the quantitative phase. This muti-capture scheme hinders qOBM’s ability to investigate valuable fast dynamic processes, such as blood flow, as well as its usability in some in-vivo applications. Here, we present a deep-learning enabled single-capture version of qOBM that quadruples the system’s imaging speed and prevents motion artifacts. To this end, we have trained a U-Net GAN to learn the qOBM reconstruction from a single capture obtained with oblique illumination. We show the capabilities and limitations of this approach, as well as some of the novel applications that this system enables, such as in-vivo high-resolution non-invasive blood flow quantitative phase imaging.
Quantitative phase imaging (QPI) enables label-free optical-path-length measurement of biological samples with nanometer-scale sensitivity, which offers unparalleled access to important histological and biophysical properties of cells and tissues. However, traditional QPI methods require a transmission-based optical geometry and are thus restricted to thin samples, which prevents the use of QPI for in-vivo applications. In this work, we present the design, characterization, and experimental validation of a handheld rigid probe for QPI with epi-illumination, using an optimized lighting configuration to achieve high phase-contrast sensitivity. The approach is based on a recently developed technology called quantitative oblique back illumination microscopy (qOBM). We demonstrate the real-time operation of our system with the future goal of applying it to help guide human brain tumor margin assessment intraoperatively in vivo, among many other potential applications.
As the repetition rates of high-intensity, ultrashort laser systems increase, diagnostics with matching data collection speeds must be developed. We have previously developed a spatiotemporal laser diagnostic, STRIPED FISH, which measures the complete laser electric field on a single shot. To provide rapid feedback, we now introduce a high-repetition-rate compatible adaptation of the STRIPED FISH retrieval algorithm which condenses the key electric field quantities into a handful of scalars for rapid assessment of the pulse’s first-order spatiotemporal distortions, and we validate this novel retrieval method with an experimental data trace.
Significance: In neurosurgery, it is essential to differentiate between tumor and healthy brain regions to maximize tumor resection while minimizing damage to vital healthy brain tissue. However, conventional intraoperative imaging tools used to guide neurosurgery are often unable to distinguish tumor margins, particularly in infiltrative tumor regions and low-grade gliomas.
Aim: The aim of this work is to assess the feasibility of a label-free molecular imaging tool called stimulated Raman scattering-spectroscopic optical coherence tomography (SRS-SOCT) to differentiate between healthy brain tissue and tumor based on (1) structural biomarkers derived from the decay rate of signals as a function of depth and (2) molecular biomarkers based on relative differences in lipid and protein composition extracted from the SRS signals.
Approach: SRS-SOCT combines the molecular sensitivity of SRS (based on vibrational spectroscopy) with the spatial and spectral multiplexing capabilities of SOCT to enable fast, spatially and spectrally resolved molecular imaging. SRS-SOCT is applied to image a 9L gliosarcoma rat tumor model, a well-characterized model that recapitulates human high-grade gliomas, including high proliferative capability, high vascularization, and infiltration at the margin. Structural and biochemical signatures acquired from SRS-SOCT are extracted to identify healthy and tumor tissues.
Results: Data show that SRS-SOCT provides light-scattering-based signatures that correlate with the presence of tumors, similar to conventional OCT. Further, nonlinear phase changes from the SRS interaction, as measured with SRS-SOCT, provide an additional measure to clearly separate tumor tissue from healthy brain regions. We also show that the nonlinear phase signals in SRS-SOCT provide a signal-to-noise advantage over the nonlinear amplitude signals for identifying tumors.
Conclusions: SRS-SOCT can distinguish both spatial and spectral features that identify tumor regions in the 9L gliosarcoma rat model. This tool provides fast, label-free, nondestructive, and spatially resolved molecular information that, with future development, can potentially assist in identifying tumor margins in neurosurgery.
A single shot, complete spatiotemporal measurement of the complex electric field E(x,y,z,t) emitted by a high power (>0.1 TW) laser is demonstrated for the first time. We generate movies of the laser's electric field E(x,y,z,t) before and after the chirped pulse amplification chain and examine the temporal, spectral, and spatial field features.
We demonstrate a fiber-based quantitative phase imaging (QPI) system with epi-illumination to acquire tissue and cellar level structure. Our approach is based on quantitative oblique back-illumination microscopy (qOBM), which keeps the advantages of QPI—label-free and non-destructive with nanometer-scale sensitivity—while also delivering tomographic sectioning capabilities in thick scattering samples using epi-illumination. The developed system uses a simple and robust configuration consisting of a flexible fiber bundle and a GRIN lens. Here data are presented with histopathological feature analysis. This technique, with its compact setup and real-time processing algorithm, can lead to in-vivo medical diagnosis, for clinical surgery and endoscopy.
The first-line treatment for brain cancer is surgery, which focuses on maximizing the percentage of the tumor removed during surgery (i.e., extent of resection) while minimizing damage to healthy brain tissue. Data show that extent of resection is one of the most critical factors associated with prolonged survival. However, differentiating between tumor and healthy tissue intraoperatively remains a significant clinical challenge, resulting in an exceedingly low 5-year survival rate of only ~35%. In this work, we show that quantitative oblique back illumination microscopy (qOBM), a novel label-free optical imaging technique that achieves tomographic quantitative phase imaging (QPI) in thick scattering samples, clearly differentiates between tumor and healthy tissue. Using a 9L gliosarcoma rat tumor model, we show that quantitative image features from qOBM provide a robust set of biomarkers for disease. In addition, tumor regions, including diffuse tumor, and healthy brain structures, show excellent structural agreement with H&E stained and sliced brightfield images, the gold standard for cancer detection. The unique attribute of qOBM—low-cost, easy-to-use, label-free, and real-time—make this technology ideally suited to help guide neurosurgery and address this important unmet need. Here we describe our free-space qOBM system and present quantitative results from the 9L gliosarcoma rat tumor model.
Quantitative phase imaging (QPI) provides unique access to cellular and subcellular structures with nanometer-scale sensitivity, making it a valuable tool for non-destructive, label-free imaging of biological samples. However, implementation of QPI typically involves a transmission-based geometry and requires thin samples, preventing use of QPI in many important clinical settings, including endoscopy. In this work we demonstrate a fiber-optic device, with epi-illumination, capable of providing quantitative phase information that is well suited for clinical endoscopy, among other biomedical applications.
Ultrashort pulses emerging from multimode optical fibers are spatiotemporally complex—the multiple fiber modes have different spatial shapes and different propagation velocities and dispersions inside fibers. To measure the complete spatiotemporal field from multimode fibers in real time, we propose and demonstrate a technique for the complete measurement of these pulses using a simple pulse characterization technique, called Spatially and Temporally Resolved Intensity and Phase Evaluation Device: Full Information from a Single Hologram (STRIPED FISH). It yields the complete electric field vs. space and time from multiple digital holograms, simultaneously recorded at different frequencies on a single camera frame.
The general theory of first-order spatiotemporal distortions provides a very helpful framework for understanding beam couplings in ultrashort pulses. The theory describes both real and imaginary coupling terms between 4 pairs of dimensions. The imaginary coupling terms are difficult to understand and visualize because they are difficult to plot in a meaningful way. In general, plotting the spatiotemporal intensity and phase of pulses in in two and three dimensions is a difficult problem. Our work on pulse visualization provides an unprecedented opportunity to study spatiotemporal couplings in ultrashort pulses. We create movies of pulses as they would appear naturally, with all of their evolving spatial, temporal, and spectral structure readily apparent.
We demonstrate ultrashort pulse spatiotemporal field measurement for multimode optical fibers, using a singleframe characterization technique, called Spatially and Temporally Resolved Intensity and Phase Evaluation Device: Full Information from a Single Hologram (STRIPED FISH). We measure STRIPED FISH traces and retrieve the pulse field E(x,y,t) or equivalently E(x,y,ω), to generate movies revealing the field structure induced by propagating modes, due to their differences in field spatial distribution, modal propagation velocity and modal dispersion inside the fiber. We launch femtosecond pulses near 800nm from Ti: Sapphire laser to investigate linearly polarized modes LP01, LP11, LP02 and LP21 in multimode fibers.
Multiple pulsing is a feature of most mode-locked ultrafast laser systems at very high pump powers, and slight variations in the pump power around certain regimes can cause sinusoidally-varying or even chaotic separations among pulses. The impact of this type of unstable multipulsing on modern pulse measurement methods has not been studied. We have performed calculations and simulations and find that allowing only the relative phase of a satellite pulse to vary causes the satellite to wash out of the SPIDER measurement completely. Although techniques like FROG and autocorrelation cannot accurately determine the precise properties of satellite pulses, they do succeed in seeing them.
We demonstrate a simple single-shot device, called Spatially and Temporally Resolved Intensity and Phase Evaluation Device: Full Information from a Single Hologram (STRIPED FISH), for completely characterizing the intensity and phase of an arbitrary ultrashort pulse in space and time (x,y,t). Improvements are made on the measurable bandwidth, aberrations eliminations, and the intensity uniformity of the multiple holograms in our device. To demonstrate the capability, we perform single-camera-frame measurements of spatiotemporally complex subpicosecond crossed and chirped double pulses from a Ti:Sapphire oscillator. To display the resulting four-dimensional intensity-and-phase data, we generate intuitive movies of the measured pulses based on our newly-developed method.
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