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1. Biocompatible micro tweezers for 3D hydrogel organoid array mechanical characterization

   https://doi.org/10.1371/journal.pone.0262950  

   Alhudaithy, Soliman ; Hoshino, Kazunori ( January 2022 , PLOS ONE)

   Jabbari, Esmaiel (Ed.)

   This study presents novel biocompatible Polydimethylsiloxane (PDMS)-based micromechanical tweezers (μTweezers) capable of the stiffness characterization and manipulation of hydrogel-based organoids. The system showed great potential for complementing established mechanical characterization methods such as Atomic Force Microscopy (AFM), parallel plate compression (PPC), and nanoindentation, while significantly reducing the volume of valuable hydrogels used for testing. We achieved a volume reduction of ~0.22 μl/sample using the μTweezers vs. ~157 μl/sample using the PPC, while targeting high-throughput measurement of widely adopted micro-mesoscale (a few hundred μm-1500 μm) 3D cell cultures. The μTweezers applied and measured nano-millinewton forces through cantilever’ deflection with high linearity and tunability for different applications; the assembly is compatible with typical inverted optical microscopes and fit on standard tissue culture Petri dishes, allowing mechanical compression characterization of arrayed 3D hydrogel-based organoids in a high throughput manner. The average achievable output per group was 40 tests per hour, where 20 organoids and 20 reference images in one 35 mm petri dish were tested, illustrating efficient productivity to match the increasing demand on 3D organoids’ applications. The changes in stiffness of collagen I hydrogel organoids in four conditions were measured, with ovarian cancer cells (SKOV3) or without (control). The Young’s modulus of the control group (Control—day 0, E = 407± 146, n = 4) measured by PPC was used as a reference modulus, where the relative elastic compressive modulus of the other groups based on the stiffness measurements was also calculated (control-day 0, E = 407 Pa), (SKOV3-day 0, E = 318 Pa), (control-day 5, E = 528 Pa), and (SKOV3-day 5, E = 376 Pa). The SKOV3-embedded hydrogel-based organoids had more shrinkage and lowered moduli on day 0 and day 5 than controls, consistently, while SKOV3 embedded organoids increased in stiffness in a similar trend to the collagen I control from day 0 to day 5. The proposed method can contribute to the biomedical, biochemical, and regenerative engineering fields, where bulk mechanical characterization is of interest. The μTweezers will also provide attractive design and application concepts to soft membrane-micro 3D robotics, sensors, and actuators. 

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2. In vivo imaging using surface enhanced spatially offset raman spectroscopy (SESORS): balancing sampling frequency to improve overall image acquisition

   https://doi.org/10.1038/s44303-024-00011-9  

   Nicolson, Fay ; Andreiuk, Bohdan ; Lee, Eunah ; O’Donnell, Bridget ; Whitley, Andrew ; Riepl, Nicole ; Burkhart, Deborah L. ; Cameron, Amy ; Protti, Andrea ; Rudder, Scott ; et al ( April 2024 , npj Imaging)

   In the field of optical imaging, the ability to image tumors at depth with high selectivity and specificity remains a challenge. Surface enhanced resonance Raman scattering (SERRS) nanoparticles (NPs) can be employed as image contrast agents to specifically target cells in vivo; however, this technique typically requires time-intensive point-by-point acquisition of Raman spectra. Here, we combine the use of “spatially offset Raman spectroscopy” (SORS) with that of SERRS in a technique known as “surface enhanced spatially offset resonance Raman spectroscopy” (SESORRS) to image deep-seated tumors in vivo. Additionally, by accounting for the laser spot size, we report an experimental approach for detecting both the bulk tumor, subsequent delineation of tumor margins at high speed, and the identification of a deeper secondary region of interest with fewer measurements than are typically applied. To enhance light collection efficiency, four modifications were made to a previously described custom-built SORS system. Specifically, the following parameters were increased: (i) the numerical aperture (NA) of the lens, from 0.2 to 0.34; (ii) the working distance of the probe, from 9 mm to 40 mm; (iii) the NA of the fiber, from 0.2 to 0.34; and (iv) the fiber diameter, from 100 µm to 400 µm. To calculate the sampling frequency, which refers to the number of data point spectra obtained for each image, we considered the laser spot size of the elliptical beam (6 × 4 mm). Using SERRS contrast agents, we performed in vivo SESORRS imaging on a GL261-Luc mouse model of glioblastoma at four distinct sampling frequencies: par-sampling frequency (12 data points collected), and over-frequency sampling by factors of 2 (35 data points collected), 5 (176 data points collected), and 10 (651 data points collected). In comparison to the previously reported SORS system, the modified SORS instrument showed a 300% improvement in signal-to-noise ratios (SNR). The results demonstrate the ability to acquire distinct Raman spectra from deep-seated glioblastomas in mice through the skull using a low power density (6.5 mW/mm²) and 30-times shorter integration times than a previous report (0.5 s versus 15 s). The ability to map the whole head of the mouse and determine a specific region of interest using as few as 12 spectra (6 s total acquisition time) is achieved. Subsequent use of a higher sampling frequency demonstrates it is possible to delineate the tumor margins in the region of interest with greater certainty. In addition, SESORRS images indicate the emergence of a secondary tumor region deeper within the brain in agreement with MRI and H&E staining. In comparison to traditional Raman imaging approaches, this approach enables improvements in the detection of deep-seated tumors in vivo through depths of several millimeters due to improvements in SNR, spectral resolution, and depth acquisition. This approach offers an opportunity to navigate larger areas of tissues in shorter time frames than previously reported, identify regions of interest, and then image the same area with greater resolution using a higher sampling frequency. Moreover, using a SESORRS approach, we demonstrate that it is possible to detect secondary, deeper-seated lesions through the intact skull.

    

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3. Quantitative 3D refractive index tomography of opaque samples in epi-mode

   https://doi.org/10.1364/OPTICA.410135  

   Ledwig, Patrick ; Robles, Francisco E. ( December 2020 , Optica)

   Three-dimensional (3D) refractive index (RI) tomography has recently become an exciting new tool for biological studies. However, its limitation to (1) thin samples resulting from a need of transmissive illumination and (2) small fields of view (typically$\sim <\#comment/>50\text{µ<\#comment/>}\mathrm{m}\times <\#comment/>50\text{µ<\#comment/>}\mathrm{m}$) has hindered its utility in broader biomedical applications. In this work, we demonstrate 3D RI tomography with a large field of view in opaque, arbitrarily thick scattering samples (unsuitable for imaging with conventional transmissive tomographic techniques) with a penetration depth of ca. one mean free scattering path length ($\sim <\#comment/>100\text{µ<\#comment/>}\mathrm{m}$in tissue) using a simple, low-cost microscope system with epi-illumination. This approach leverages a solution to the inverse scattering problem via the general non-paraxial 3D optical transfer function of our quantitative oblique back-illumination microscopy (qOBM) optical system. A theoretical analysis is presented along with simulations and experimental validations using polystyrene beads, and rat and human thick brain tissues. This work has significant implications for the investigation of optically thick, semi-infinite samples in a non-invasive and label-free manner. This unique 3D qOBM approach can extend the utility of 3D RI tomography for translational and clinical medicine.

    

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4. Mapping optical scattering properties to physical particle information in singly and multiply scattering samples

   https://doi.org/10.1364/BOE.494518  

   Cannon, Taylor M. ; Bouma, Brett E. ; Uribe-Patarroyo, Néstor ( July 2023 , Biomedical Optics Express)

   Optical coherence tomography (OCT) leverages light scattering by biological tissues as endogenous contrast to form structural images. Light scattering behavior is dictated by the optical properties of the tissue, which depend on microstructural details at the cellular or sub-cellular level. Methods to measure these properties from OCT intensity data have been explored in the context of a number of biomedical applications seeking to access this sub-resolution tissue microstructure and thereby increase the diagnostic impact of OCT. Most commonly, the optical attenuation coefficient, an analogue of the scattering coefficient, has been used as a surrogate metric linking OCT intensity to subcellular particle characteristics. To record attenuation coefficient data that is accurately representative of the underlying physical properties of a given sample, it is necessary to account for the impact of the OCT imaging system itself on the distribution of light intensity in the sample, including the numerical aperture (NA) of the system and the location of the focal plane with respect to the sample surface, as well as the potential contribution of multiple scattering to the reconstructed intensity signal. Although these considerations complicate attenuation coefficient measurement and interpretation, a suitably calibrated system may potentiate a powerful strategy for gaining additional information about the scattering behavior and microstructure of samples. In this work, we experimentally show that altering the OCT system geometry minimally impacts measured attenuation coefficients in samples presumed to be singly scattering, but changes these measurements in more highly scattering samples. Using both depth-resolved attenuation coefficient data and layer-resolved backscattering coefficients, we demonstrate the retrieval of scattering particle diameter and concentration in tissue-mimicking phantoms, and the impact of presumed multiple scattering on these calculations. We further extend our approach to characterize a murine brain tissue sample and highlight a tumor-bearing region based on increased scattering particle density. Through these methods, we not only enhance conventional OCT attenuation coefficient analysis by decoupling the independent effects of particle size and concentration, but also discriminate areas of strong multiple scattering through minor changes to system topology to provide a framework for assessing the accuracy of these measurements.

    

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5. NIR-II Nanoprobes: A Review of Components-Based Approaches to Next-Generation Bioimaging Probes

   https://doi.org/10.3390/bioengineering10080954  

   Dunn, Bryce ; Hanafi, Marzieh ; Hummel, John ; Cressman, John R. ; Veneziano, Rémi ; Chitnis, Parag V. ( August 2023 , Bioengineering)

   Fluorescence and photoacoustic imaging techniques offer valuable insights into cell- and tissue-level processes. However, these optical imaging modalities are limited by scattering and absorption in tissue, resulting in the low-depth penetration of imaging. Contrast-enhanced imaging in the near-infrared window improves imaging penetration by taking advantage of reduced autofluorescence and scattering effects. Current contrast agents for fluorescence and photoacoustic imaging face several limitations from photostability and targeting specificity, highlighting the need for a novel imaging probe development. This review covers a broad range of near-infrared fluorescent and photoacoustic contrast agents, including organic dyes, polymers, and metallic nanostructures, focusing on their optical properties and applications in cellular and animal imaging. Similarly, we explore encapsulation and functionalization technologies toward building targeted, nanoscale imaging probes. Bioimaging applications such as angiography, tumor imaging, and the tracking of specific cell types are discussed. This review sheds light on recent advancements in fluorescent and photoacoustic nanoprobes in the near-infrared window. It serves as a valuable resource for researchers working in fields of biomedical imaging and nanotechnology, facilitating the development of innovative nanoprobes for improved diagnostic approaches in preclinical healthcare. 

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