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1. Phase imaging with computational specificity (PICS) for measuring dry mass changes in sub-cellular compartments

   https://doi.org/10.1038/s41467-020-20062-x  

   Kandel, Mikhail E. ; He, Yuchen R. ; Lee, Young Jae ; Chen, Taylor Hsuan-Yu ; Sullivan, Kathryn Michele ; Aydin, Onur ; Saif, M. Taher A. ; Kong, Hyunjoon ; Sobh, Nahil ; Popescu, Gabriel ( December 2020 , Nature Communications)

   Due to its specificity, fluorescence microscopy has become a quintessential imaging tool in cell biology. However, photobleaching, phototoxicity, and related artifacts continue to limit fluorescence microscopy’s utility. Recently, it has been shown that artificial intelligence (AI) can transform one form of contrast into another. We present phase imaging with computational specificity (PICS), a combination of quantitative phase imaging and AI, which provides information about unlabeled live cells with high specificity. Our imaging system allows for automatic training, while inference is built into the acquisition software and runs in real-time. Applying the computed fluorescence maps back to the quantitative phase imaging (QPI) data, we measured the growth of both nuclei and cytoplasm independently, over many days, without loss of viability. Using a QPI method that suppresses multiple scattering, we measured the dry mass content of individual cell nuclei within spheroids. In its current implementation, PICS offers a versatile quantitative technique for continuous simultaneous monitoring of individual cellular components in biological applications where long-term label-free imaging is desirable.

    

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2. A drug‐selectable acoustic reporter gene system for human cell ultrasound imaging

   https://doi.org/10.1002/btm2.10584  

   Howells, Alessandro R. ; Welch, Phoebe J. ; Kim, John ; Forest, Craig R. ; Shi, Chengzhi ; Lian, Xiaojun Lance ( August 2023 , Bioengineering & Translational Medicine)

   A promising new field of genetically encoded ultrasound contrast agents in the form of gas vesicles has recently emerged, which could extend the specificity of medical ultrasound imaging. However, given the delicate genetic nature of how these genes are integrated and expressed, current methods of producing gas vesicle‐expressing mammalian cell lines requires significant cell processing time to establish a clonal/polyclonal line that robustly expresses the gas vesicles sufficiently enough for ultrasound contrast. Here, we describe an inducible and drug‐selectable acoustic reporter gene system that can enable gas vesicle expression in mammalian cell lines, which we demonstrate using HEK293T cells. Our drug‐selectable construct design increases the stability and proportion of cells that successfully integrate all plasmids into their genome, thus reducing the amount of cell processing time required. Additionally, we demonstrate that our drug‐selectable strategy forgoes the need for single‐cell cloning and fluorescence‐activated cell sorting, and that a drug‐selected mixed population is sufficient to generate robust ultrasound contrast. Successful gas vesicle expression was optically and ultrasonically verified, with cells expressing gas vesicles exhibiting an 80% greater signal‐to‐noise ratio compared to negative controls and a 500% greater signal‐to‐noise ratio compared to wild‐type HEK293T cells. This technology presents a new reporter gene paradigm by which ultrasound can be harnessed to visualize specific cell types for applications including cellular reporting and cell therapies.

    

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3. Overexpression of caveolin‐1 in inflammatory breast cancer cells enables IBC‐specific gene delivery and prodrug conversion using histone‐targeted polyplexes

   https://doi.org/10.1002/bit.26022  

   Ross, Nikki L. ; Sullivan, Millicent O. ( June 2016 , Biotechnology and Bioengineering)

   Gene therapy platforms offer a variety of potentially effective solutions for development of targeted agents that can be exploited for cancer treatment. The physicochemical properties of nanocarriers can be tuned to enhance their localization in tumors, and cell specificity can also be increased by appropriate selection of gene targets. A relatively underexploited approach to enhance therapeutic selectivity in cancer tissues is the use of nanocarriers whose nuclear targeting and uptake are triggered by the altered expression of specific endomembrane trafficking proteins in cancer cells. Previously, we showed that histone 3 (H3) peptide‐targeted DNA polyplexes traffic to the nucleus efficiently through caveolar endocytosis followed by transfer through the Golgi and endoplasmic reticulum (ER). We hypothesized that these polyplexes would exhibit enhanced activity in inflammatory breast cancer (IBC) cells, which overexpress caveolin‐1 as part of their invasive phenotype, and we also posited that this targeting effect could be exploited to facilitate IBC‐specific transfection and prodrug conversion in the presence of normal breast epithelial cells. Using cellular transfection experiments, function‐blocking assays, and confocal imaging in both IBC SUM149 cell monocultures and IBC SUM149 co‐cultures with MCF10A normal breast epithelial cells, we found that our H3‐targeted polyplexes selectively transfected IBC SUM149 cells at a 4‐fold higher level than normal breast epithelial cells. This selectivity and increased transfection were caused by a 2.2‐fold overexpression of caveolin‐1 in IBC SUM149 cells, which led to increased polyplex trafficking to the nucleus through the Golgi and ER. We also saw similar enhancements in cell selectivity and transfection when cells were transfected with a suicide gene/prodrug combination, as the increased expression of the suicide gene in IBC SUM149 cells led to a 55% decrease in viability in IBC SUM149 cells as compared to a 25% decrease in MCF10A cells. These findings demonstrate that differences in the expression of the endocytic membrane protein caveolin‐1 can be exploited for cell‐selective gene delivery, and ultimately, these gene‐based targeting approaches may be useful in potential treatments for aggressive cancer types. Biotechnol. Bioeng. 2016;113: 2686–2697. © 2016 Wiley Periodicals, Inc.

    

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4. Optical coherence tomography detects necrotic regions and volumetrically quantifies multicellular tumor spheroids

   Huang, Yongyang ; Wang, Shunqiang ; Guo, Qiongyu ; Kessel, Sarah ; Rubinoff, Ian ; Chan, Leo Li-Ying ; Li, Peter ; Liu, Yaling ; Qiu, Jean ; Zhou, Chao ( October 2017 , Cancer research)

   Three-dimensional (3D) tumor spheroid models have gained increased recognition as important tools in cancer research and anti-cancer drug development. However, currently available imaging approaches employed in high-throughput screening drug discovery platforms e.g. bright field, phase contrast, and fluorescence microscopies, are unable to resolve 3D structures deep inside (>50 μm) tumor spheroids. In this study, we established a label-free, non-invasive optical coherence tomography (OCT) imaging platform to characterize 3D morphological and physiological information of multicellular tumor spheroids (MCTS) growing from ~250 μm up to ~600 μm in height over 21 days. In particular, tumor spheroids of two cell lines glioblastoma (U-87 MG) and colorectal carcinoma (HCT 116) exhibited distinctive evolutions in their geometric shapes at late growth stages. Volumes of MCTS were accurately quantified using a voxel-based approach without presumptions of their geometries. In contrast, conventional diameter-based volume calculations assuming perfect spherical shape resulted in large quantification errors. Furthermore, we successfully detected necrotic regions within these tumor spheroids based on increased intrinsic optical attenuation, suggesting a promising alternative of label-free viability tests in tumor spheroids. Therefore, OCT can serve as a promising imaging modality to characterize morphological and physiological features of MCTS, showing great potential for high-throughput drug screening. 

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5. 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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