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Journal articles on the topic 'Multiplexed fluorescence imaging'

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Author: Grafiati

Published: 29 April 2023

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1

Cappella, Paolo, and Fabio Gasparri. "Highly Multiplexed Phenotypic Imaging for Cell Proliferation Studies." Journal of Biomolecular Screening 19, no. 1 (July 29, 2013): 145–57. http://dx.doi.org/10.1177/1087057113495712.

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The application of multiplexed imaging technologies in phenotypic drug discovery (PDD) enables profiling of complex cellular perturbations in response to drug treatment. High-content analysis (HCA) is among the most pursued approaches in PDD, with a proven capability to identify compounds with a given cellular mechanism of action (MOA), as well as to unveil unexpected drug cellular activities. The ability of fluorescent image-based cytometric techniques to dissect the phenotypic heterogeneity of cell populations depends on the degree of multiplexing achievable. At present, most high-content assays employ up to four cellular markers separately detected in distinct fluorescence channels. We explored the possibility to increase HCA multiplexing through analysis of multiple proliferation markers in the same fluorescence channel by taking advantage of the different timing of antigen appearance during the cell cycle, or differential intracellular localization. Simultaneous analysis of DAPI staining and five immunofluorescence markers (BrdU incorporation, active caspase-3, phospho-histone H3, phospho-S6, and Ki-67) resulted in the first six-marker high-content assay readily applicable to compound MOA studies. This approach allows detection of rare cell subpopulations, unveiling a high degree of phenotypic heterogeneity in exponentially growing cell cultures and variability in the individual cell response to antiproliferative drugs.

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Liu, Hsiou-Yuan, Jingshan Zhong, and Laura Waller. "Multiplexed phase-space imaging for 3D fluorescence microscopy." Optics Express 25, no. 13 (June 21, 2017): 14986. http://dx.doi.org/10.1364/oe.25.014986.

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3

Luo, Teng, Ting Zhou, Yihua Zhao, Liwei Liu, and Junle Qu. "Multiplexed fluorescence lifetime imaging by concentration-dependent quenching." Journal of Materials Chemistry B 6, no. 13 (2018): 1912–19. http://dx.doi.org/10.1039/c8tb00095f.

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4

Ko, Jina, Juhyun Oh, Maaz S. Ahmed, Jonathan C. T. Carlson, and Ralph Weissleder. "Ultra‐fast Cycling for Multiplexed Cellular Fluorescence Imaging." Angewandte Chemie 132, no. 17 (April 20, 2020): 6906–13. http://dx.doi.org/10.1002/ange.201915153.

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5

Ko, Jina, Juhyun Oh, Maaz S. Ahmed, Jonathan C. T. Carlson, and Ralph Weissleder. "Ultra‐fast Cycling for Multiplexed Cellular Fluorescence Imaging." Angewandte Chemie International Edition 59, no. 17 (March 6, 2020): 6839–46. http://dx.doi.org/10.1002/anie.201915153.

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6

Gamber, Kevin, Arne Christians, Spencer Schwarz, Adam Northcutt, Jason Forys, Thomas Campbell, and Crystal Winkeler. "Quantitative Immune Profiling of Human Tumor Tissues with Multiplexed ChipCytometry." Journal of Immunology 206, no. 1_Supplement (May 1, 2021): 68.05. http://dx.doi.org/10.4049/jimmunol.206.supp.68.05.

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Abstract Cancer therapies that rely on manipulating or engineering immune cells have shown much promise in recent years for effectively combating a broad array of cancer types. In order to determine the effectiveness of such therapies, it is necessary to accurately profile the complement of immune cell types present in the tumor microenvironment. Methods to reliably obtain immune cell signatures require the combination of powerful resolution to discriminate cell boundaries, broad dynamic range to capture markers of varying expression levels, and accurate cell segmentation across a wide range of cell sizes and morphologies. Many methods for quantitative immune cell profiling have demonstrated shortcomings when it comes to the aforementioned attributes. To remedy these shortcomings, we present here ChipCytometry, an imaging technology for immune profiling of cells and sectioned tissues. ChipCytometry is a fluorescence-based imaging system that utilizes multiplexed immuno-fluorescence staining in combination with high-dynamic-range (HDR) imaging to facilitate quantitative phenotyping of individual cells within tissue samples. Employing this technology to profile metastatic and primary tumors, multiple biomarkers were stained in iterative multiplex assays to profile single cells with an AI-powered cell segmentation algorithm. The HDR imaging allows for both strong and weak fluorescent signals to be simultaneously obtained without loss of sensitivity. This immune profiling was completed on 6 different cryosectioned human tumor tissues (head & neck, liver, lung, breast, colon, and pancreas).

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7

Mizuno, T., E. Hase, T. Minamikawa, Y. Tokizane, R. Oe, H. Koresawa, H. Yamamoto, and T. Yasui. "Full-field fluorescence lifetime dual-comb microscopy using spectral mapping and frequency multiplexing of dual-comb optical beats." Science Advances 7, no. 1 (January 2021): eabd2102. http://dx.doi.org/10.1126/sciadv.abd2102.

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Fluorescence lifetime imaging microscopy (FLIM) is a powerful tool for quantitative fluorescence imaging because fluorescence lifetime is independent of concentration of fluorescent molecules or excitation/detection efficiency and is robust to photobleaching. However, since most FLIMs are based on point-to-point measurements, mechanical scanning of a focal spot is needed for forming an image, which hampers rapid imaging. Here, we demonstrate scan-less full-field FLIM based on a one-to-one correspondence between two-dimensional (2D) image pixels and frequency-multiplexed radio frequency (RF) signals. A vast number of dual-comb optical beats between dual optical frequency combs are effectively adopted for 2D spectral mapping and high-density frequency multiplexing in the RF region. Bimodal images of fluorescence amplitude and lifetime are obtained with high quantitativeness from amplitude and phase spectra of fluorescence RF comb modes without the need for mechanical scanning. The parallelized FLIM will be useful for rapid quantitative fluorescence imaging in life science.

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8

Xu, Jian, Tianyue Zhang, Shenyu Yang, Ziwei Feng, Haoying Li, Dejiao Hu, Fei Qin, et al. "Plasmonic Nanoprobes for Multiplexed Fluorescence-Free Super-Resolution Imaging." Advanced Optical Materials 6, no. 20 (August 5, 2018): 1800432. http://dx.doi.org/10.1002/adom.201800432.

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9

Lv, Yanlu, Jiulou Zhang, Dong Zhang, Wenjuan Cai, Nanguang Chen, and Jianwen Luo. "In vivosimultaneous multispectral fluorescence imaging with spectral multiplexed volume holographic imaging system." Journal of Biomedical Optics 21, no. 6 (June 3, 2016): 060502. http://dx.doi.org/10.1117/1.jbo.21.6.060502.

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10

Keshri, Puspam, Bin Zhao, Tianfa Xie, Yousef Bagheri, James Chambers, Yubing Sun, and Mingxu You. "Quantitative and Multiplexed Fluorescence Lifetime Imaging of Intercellular Tensile Forces." Angewandte Chemie 133, no. 28 (June 10, 2021): 15676–83. http://dx.doi.org/10.1002/ange.202103986.

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11

Keshri, Puspam, Bin Zhao, Tianfa Xie, Yousef Bagheri, James Chambers, Yubing Sun, and Mingxu You. "Quantitative and Multiplexed Fluorescence Lifetime Imaging of Intercellular Tensile Forces." Angewandte Chemie International Edition 60, no. 28 (June 10, 2021): 15548–55. http://dx.doi.org/10.1002/anie.202103986.

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12

Barash, Eugene, Sean Dinn, Christopher Sevinsky, and Fiona Ginty. "Multiplexed Analysis of Proteins in Tissue Using Multispectral Fluorescence Imaging." IEEE Transactions on Medical Imaging 29, no. 8 (August 2010): 1457–62. http://dx.doi.org/10.1109/tmi.2010.2045005.

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13

Bhagavatula, Sharath, Devon Thompson, Sebastian W. Ahn, Kunj Upadhyaya, Alex Lammers, Kyle Deans, Christine Dominas, et al. "A Miniaturized Platform for Multiplexed Drug Response Imaging in Live Tumors." Cancers 13, no. 4 (February 6, 2021): 653. http://dx.doi.org/10.3390/cancers13040653.

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By observing the activity of anti-cancer agents directly in tumors, there is potential to greatly expand our understanding of drug response and develop more personalized cancer treatments. Implantable microdevices (IMD) have been recently developed to deliver microdoses of chemotherapeutic agents locally into confined regions of live tumors; the tissue can be subsequently removed and analyzed to evaluate drug response. This method has the potential to rapidly screen multiple drugs, but requires surgical tissue removal and only evaluates drug response at a single timepoint when the tissue is excised. Here, we describe a “lab-in-a-tumor” implantable microdevice (LIT-IMD) platform to image cell-death drug response within a live tumor, without requiring surgical resection or tissue processing. The LIT-IMD is inserted into a live tumor and delivers multiple drug microdoses into spatially discrete locations. In parallel, it locally delivers microdose levels of a fluorescent cell-death assay, which diffuses into drug-exposed tissues and accumulates at sites of cell death. An integrated miniaturized fluorescence imaging probe images each region to evaluate drug-induced cell death. We demonstrate ability to evaluate multi-drug response over 8 h using murine tumor models and show correlation with gold-standard conventional fluorescence microscopy and histopathology. This is the first demonstration of a fully integrated platform for evaluating multiple chemotherapy responses in situ. This approach could enable a more complete understanding of drug activity in live tumors, and could expand the utility of drug-response measurements to a wide range of settings where surgery is not feasible.

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Xiao, Lu, Joshua Labaer, and Jia Guo. "Highly Sensitive and Multiplexed In Situ RNA Profiling with Cleavable Fluorescent Tyramide." Cells 10, no. 6 (May 21, 2021): 1277. http://dx.doi.org/10.3390/cells10061277.

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Understanding the composition, regulation, and function of complex biological systems requires tools that quantify multiple transcripts at their native cellular locations. However, the current multiplexed RNA imaging technologies are limited by their relatively low sensitivity or specificity, which hinders their applications in studying highly autofluorescent tissues, such as formalin-fixed paraffin-embedded (FFPE) tissues. To address this issue, here we develop a multiplexed in situ RNA profiling approach with a high sensitivity and specificity. In this approach, transcripts are first hybridized by target-specific oligonucleotide probes in pairs. Only when these two independent probes hybridize to the target in tandem will the subsequent signal amplification by oligonucleotide hybridization occur. Afterwards, horseradish peroxidase (HRP) is applied to further amplify the signal and stain the target with cleavable fluorescent tyramide (CFT). After imaging, the fluorophores are chemically cleaved and the hybridized probes are stripped by DNase and formamide. Through cycles of RNA staining, fluorescence imaging, signal cleavage, and probe stripping, many different RNA species can be profiled at the optical resolution. In applying this approach, we demonstrated that multiplexed in situ RNA analysis can be successfully achieved in both fixed, frozen, and FFPE tissues.

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15

Wegner, K. David, and Niko Hildebrandt. "Quantum dots: bright and versatile in vitro and in vivo fluorescence imaging biosensors." Chemical Society Reviews 44, no. 14 (2015): 4792–834. http://dx.doi.org/10.1039/c4cs00532e.

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16

Kuang, Yu, Guillem Pratx, Magdalena Bazalova, Bowen Meng, Jianguo Qian, and Lei Xing. "First Demonstration of Multiplexed X-Ray Fluorescence Computed Tomography (XFCT) Imaging." IEEE Transactions on Medical Imaging 32, no. 2 (February 2013): 262–67. http://dx.doi.org/10.1109/tmi.2012.2223709.

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17

Jin, Zhao-Hui, Atsushi B. Tsuji, Mélissa Degardin, Pascal Dumy, Didier Boturyn, and Tatsuya Higashi. "Multiplexed Imaging Reveals the Spatial Relationship of the Extracellular Acidity-Targeting pHLIP with Necrosis, Hypoxia, and the Integrin-Targeting cRGD Peptide." Cells 11, no. 21 (November 4, 2022): 3499. http://dx.doi.org/10.3390/cells11213499.

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pH (low) insertion peptides (pHLIPs) have been developed for cancer imaging and therapy targeting the acidic extracellular microenvironment. However, the characteristics of intratumoral distribution (ITD) of pHLIPs are not yet fully understood. This study aimed to reveal the details of the ITD of pHLIPs and their spatial relationship with other tumor features of concern. The fluorescent dye-labeled pHLIPs were intravenously administered to subcutaneous xenograft mouse models of U87MG and IGR-OV1 expressing αVβ3 integrins (using large necrotic tumors). The αVβ3 integrin-targeting Cy5.5-RAFT-c(-RGDfK-)4 was used as a reference. In vivo and ex vivo fluorescence imaging, whole-tumor section imaging, fluorescence microscopy, and multiplexed fluorescence colocalization analysis were performed. The ITD of fluorescent dye-labeled pHLIPs was heterogeneous, having a high degree of colocalization with necrosis. A direct one-to-one comparison of highly magnified images revealed the cellular localization of pHLIP in pyknotic, karyorrhexis, and karyolytic necrotic cells. pHLIP and hypoxia were spatially contiguous but not overlapping cellularly. The hypoxic region was found between the ITDs of pHLIP and the cRGD peptide and the Ki-67 proliferative activity remained detectable in the pHLIP-accumulated regions. The results provide a better understanding of the characteristics of ITD of pHLIPs, leading to new insights into the theranostic applications of pHLIPs.

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18

Moffitt, Jeffrey R., Junjie Hao, Guiping Wang, Kok Hao Chen, Hazen P. Babcock, and Xiaowei Zhuang. "High-throughput single-cell gene-expression profiling with multiplexed error-robust fluorescence in situ hybridization." Proceedings of the National Academy of Sciences 113, no. 39 (September 13, 2016): 11046–51. http://dx.doi.org/10.1073/pnas.1612826113.

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Image-based approaches to single-cell transcriptomics, in which RNA species are identified and counted in situ via imaging, have emerged as a powerful complement to single-cell methods based on RNA sequencing of dissociated cells. These image-based approaches naturally preserve the native spatial context of RNAs within a cell and the organization of cells within tissue, which are important for addressing many biological questions. However, the throughput of these image-based approaches is relatively low. Here we report advances that lead to a drastic increase in the measurement throughput of multiplexed error-robust fluorescence in situ hybridization (MERFISH), an image-based approach to single-cell transcriptomics. In MERFISH, RNAs are identified via a combinatorial labeling approach that encodes RNA species with error-robust barcodes followed by sequential rounds of single-molecule fluorescence in situ hybridization (smFISH) to read out these barcodes. Here we increase the throughput of MERFISH by two orders of magnitude through a combination of improvements, including using chemical cleavage instead of photobleaching to remove fluorescent signals between consecutive rounds of smFISH imaging, increasing the imaging field of view, and using multicolor imaging. With these improvements, we performed RNA profiling in more than 100,000 human cells, with as many as 40,000 cells measured in a single 18-h measurement. This throughput should substantially extend the range of biological questions that can be addressed by MERFISH.

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19

Luo, Teng, Danying Lin, Ting Zhou, Yuan Lu, Shaoxiong Liu, and Junle Qu. "Identification and characterization of different tissues in blood vessel by multiplexed fluorescence lifetimes." Analyst 143, no. 10 (2018): 2243–48. http://dx.doi.org/10.1039/c8an00392k.

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Herein, fluorescence lifetime imaging microscopy (FLIM) was used to directly measure eosin fluorescence lifetimes from H&E-stained umbilical artery, and a further utilization of eosin for high-content and multi-target analysis was proposed for the first time.

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20

Xiao, Lu, and Jia Guo. "Multiplexed single-cell in situ RNA analysis by reiterative hybridization." Analytical Methods 7, no. 17 (2015): 7290–95. http://dx.doi.org/10.1039/c5ay00500k.

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A novel method to quantify the identities, positions, and copy numbers of a large number of different RNA species in single cells has been developed by reiterative cycles of target hybridization, fluorescence imaging and photobleaching.

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21

Sograte-Idrissi, Shama, Nazar Oleksiievets, Sebastian Isbaner, Mariana Eggert-Martinez, Jörg Enderlein, Roman Tsukanov, and Felipe Opazo. "Nanobody Detection of Standard Fluorescent Proteins Enables Multi-Target DNA-PAINT with High Resolution and Minimal Displacement Errors." Cells 8, no. 1 (January 14, 2019): 48. http://dx.doi.org/10.3390/cells8010048.

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DNA point accumulation for imaging in nanoscale topography (PAINT) is a rapidly developing fluorescence super-resolution technique, which allows for reaching spatial resolutions below 10 nm. It also enables the imaging of multiple targets in the same sample. However, using DNA-PAINT to observe cellular structures at such resolution remains challenging. Antibodies, which are commonly used for this purpose, lead to a displacement between the target protein and the reporting fluorophore of 20–25 nm, thus limiting the resolving power. Here, we used nanobodies to minimize this linkage error to ~4 nm. We demonstrate multiplexed imaging by using three nanobodies, each able to bind to a different family of fluorescent proteins. We couple the nanobodies with single DNA strands via a straight forward and stoichiometric chemical conjugation. Additionally, we built a versatile computer-controlled microfluidic setup to enable multiplexed DNA-PAINT in an efficient manner. As a proof of principle, we labeled and imaged proteins on mitochondria, the Golgi apparatus, and chromatin. We obtained super-resolved images of the three targets with 20 nm resolution, and within only 35 minutes acquisition time.

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Tewson, Paul H., Anne Marie Quinn, and Thomas E. Hughes. "A Multiplexed Fluorescent Assay for Independent Second-Messenger Systems." Journal of Biomolecular Screening 18, no. 7 (April 11, 2013): 797–806. http://dx.doi.org/10.1177/1087057113485427.

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There is a growing need in drug discovery and basic research to measure multiple second-messenger components of cell signaling pathways in real time and in relevant tissues and cell types. Many G-protein–coupled receptors activate the heterotrimeric protein, Gq, which in turn activates phospholipase C (PLC). PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to produce two second messengers: diacylglycerol (DAG), which remains in the plasma membrane, and inositol triphosphate (IP3), which diffuses through the cytosol to release stores of intracellular calcium ions (Ca2+). Our goal was to create a series of multiplex sensors that would make it possible to simultaneously measure two different components of the Gq pathway in living cells. Here we describe new fluorescent sensors for DAG and PIP2 that produce robust changes in green or red fluorescence and can be combined with one another, or with existing Ca2+ sensors, in a live-cell assay. These assays can detect multiple components of Gq signaling, simultaneously in real time, on standard fluorescent plate readers or live-cell imaging systems.

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23

Zhao, Ming, Yu Li, and Leilei Peng. "Parallel excitation-emission multiplexed fluorescence lifetime confocal microscopy for live cell imaging." Optics Express 22, no. 9 (April 21, 2014): 10221. http://dx.doi.org/10.1364/oe.22.010221.

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24

Maryu, Gembu, Michiyuki Matsuda, and Kazuhiro Aoki. "Multiplexed Fluorescence Imaging of ERK and Akt Activities and Cell-cycle Progression." Cell Structure and Function 41, no. 2 (2016): 81–92. http://dx.doi.org/10.1247/csf.16007.

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25

Northcutt, Adam J., Arne Christians, Jason T. Forys, Thomas D. Campbell, and Crystal L. Winkeler. "Quantitative immune profiling of human tumor tissues with multiplexed ChipCytometry." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 159.10. http://dx.doi.org/10.4049/jimmunol.204.supp.159.10.

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Abstract Objective Many immune cell profiling methods have demonstrated limits on the number of biomarkers that can be reliably assayed from the same cell; to remedy this, we present here ChipCytometry, an imaging technology for quantitative immune profiling of cells and sectioned tissues. Methods ChipCytometry is a fluorescence-based imaging system that utilizes multiplexed immuno-fluorescence staining in combination with high-dynamic-range (HDR) imaging to facilitate quantitative phenotyping of individual cells within tissue samples. Employing this technology to profile metastatic and primary tumors, multiple biomarkers were stained in iterative multiplex assays to profile single cells with an AI-powered cell segmentation algorithm. This immune profiling was completed on 6 different cryosectioned human tumor tissues (head & neck, liver, lung, breast, colon, and pancreas). Results ChipCytometry allowed for the quantification of both tumor and immune cells in all 6 of the human tumor tissues that were analyzed, revealing the diversity of T cells (helper, cytotoxic, regulatory, naïve/effector/memory), B-cells, granulocytes, monocytes, dendritic cells, and natural killer cells present in the tumor microenvironment. The resolution, dynamic range, and AI-powered cell segmentation offered by ChipCytometry allowed for quantification of 15 immune cell types at the single-cell level. The data show exact counts of each specific cell type, relative percentages within each tissue, and the precise spatial distribution of each cell. Conclusion These results demonstrate the potential of ChipCytometry to broadly probe a diverse set of human tumor tissue types and acquire a deep immunological cell type profile.

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Potier, Guillaume, Aurélie Doméné, and Perrine Paul-Gilloteaux. "A flexible open-source processing workflow for multiplexed fluorescence imaging based on cycles." F1000Research 11 (September 29, 2022): 1121. http://dx.doi.org/10.12688/f1000research.124990.1.

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Background Multiplexing tissue imaging is developing as a complement for single cell analysis, bringing the spatial information of cells in tissue in addition to multiple parameters measurements. More and more commercial or home-made systems are available. These techniques allow the imaging of tens of fluorescent reporters, where the spectral overlap is solved by imaging by cycles the fluorophores using microfluidics to change the reporters between each cycle. Methods For several systems, the acquisition system coupled to the microfluidic system is a wide field microscope, and the acquisition process is done by mosaicking to cover a large field of view, relying on image processing to obtain the data set to be analysed in intensity. The processed data set allows the identification of different populations, quite similarly to cytometry analysis, but with spatial information in addition. To obtain the final image for analysis from the raw acquisitions, several preprocessing steps are needed for inter-cycle registration, tissue autofluorescence correction or mosaicking. We propose a workflow for this preprocessing, implemented as an open source software (as a library, command line tool and standalone). Results We exemplify the workflow on the commercial system PhenoCycler® (formerly named CODEX®) and provide a reduced size data set for testing. Conclusions We compare our processor with the commercially provided processor and show that we solve some problems also reported by other users.

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WANG Da-hui, 王大辉, 钱航 QIAN Hang, 赵学庆 ZHAO Xue-qing, 马连英 MA Lian-ying, 张永生 ZHANG Yong-sheng, 邵碧波 SHAO Bi-bo, 冯刚 FENG Gang, 易爱平 YI Ai-ping, and 赵军 ZHAO Jun. "Automatic alignment of multiplexed beams of excimer laser system based on fluorescence imaging." Optics and Precision Engineering 23, no. 4 (2015): 949–55. http://dx.doi.org/10.3788/ope.20152304.0949.

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28

Harmon, Jeffrey, Hideharu Mikami, Hiroshi Kanno, Takuro Ito, and Keisuke Goda. "Accurate classification of microalgae by intelligent frequency-division-multiplexed fluorescence imaging flow cytometry." OSA Continuum 3, no. 3 (February 19, 2020): 430. http://dx.doi.org/10.1364/osac.387523.

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29

Haedicke, Katja, Susanna Gräfe, Frank Lehmann, and Ingrid Hilger. "Multiplexed in vivo fluorescence optical imaging of the therapeutic efficacy of photodynamic therapy." Biomaterials 34, no. 38 (December 2013): 10075–83. http://dx.doi.org/10.1016/j.biomaterials.2013.08.087.

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Theodorou, Ioannis G., Pakatip Ruenraroengsak, Daniel A. Gonzalez-Carter, Qianfan Jiang, Ernesto Yagüe, Eric O. Aboagye, R. Charles Coombes, Alexandra E. Porter, Mary P. Ryan, and Fang Xie. "Towards multiplexed near-infrared cellular imaging using gold nanostar arrays with tunable fluorescence enhancement." Nanoscale 11, no. 4 (2019): 2079–88. http://dx.doi.org/10.1039/c8nr09409h.

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Wegner, Kyle A., Adib Keikhosravi, Kevin W. Eliceiri, and Chad M. Vezina. "Fluorescence of Picrosirius Red Multiplexed With Immunohistochemistry for the Quantitative Assessment of Collagen in Tissue Sections." Journal of Histochemistry & Cytochemistry 65, no. 8 (July 10, 2017): 479–90. http://dx.doi.org/10.1369/0022155417718541.

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The low cost and simplicity of picrosirius red (PSR) staining have driven its popularity for collagen detection in tissue sections. We extended the versatility of this method by using fluorescent imaging to detect the PSR signal and applying automated quantification tools. We also developed the first PSR protocol that is fully compatible with multiplex immunostaining, making it possible to test whether collagen structure differs across immunohistochemically labeled regions of the tissue landscape. We compared our imaging method with two gold standards in collagen imaging, linear polarized light microscopy and second harmonic generation imaging, and found that it is at least as sensitive and robust to changes in sample orientation. As proof of principle, we used a genetic approach to overexpress beta catenin in a patchy subset of mouse prostate epithelial cells distinguished only by immunolabeling. We showed that collagen fiber length is significantly greater near beta catenin overexpressing cells than near control cells. Our fluorescent PSR imaging method is sensitive, reproducible, and offers a new way to guide region of interest selection for quantifying collagen in tissue sections.

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Zhao, Ming, Yu Li, and Leilei Peng. "FPGA-based multi-channel fluorescence lifetime analysis of Fourier multiplexed frequency-sweeping lifetime imaging." Optics Express 22, no. 19 (September 15, 2014): 23073. http://dx.doi.org/10.1364/oe.22.023073.

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Margineanu, Anca, Romain Laine, Sunil Kumar, Clifford Talbot, Sean Warren, Christopher Kimberley, James McGinty, et al. "Multiplexed Time Lapse Fluorescence Lifetime Readouts in an Optically Sectioning Time-Gated Imaging Microscope." Biophysical Journal 100, no. 3 (February 2011): 183a. http://dx.doi.org/10.1016/j.bpj.2010.12.1221.

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Matsuda, Takahiko, and Izumi Oinuma. "Imaging endogenous synaptic proteins in primary neurons at single-cell resolution using CRISPR/Cas9." Molecular Biology of the Cell 30, no. 22 (October 15, 2019): 2838–55. http://dx.doi.org/10.1091/mbc.e19-04-0223.

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Fluorescence imaging at single-cell resolution is a crucial approach to analyzing the spatiotemporal regulation of proteins within individual cells of complex neural networks. Here we present a nonviral strategy that enables the tagging of endogenous loci by CRISPR/Cas9-mediated genome editing combined with a nucleofection technique. The method allowed expression of fluorescently tagged proteins at endogenous levels, and we successfully achieved tagging of a presynaptic protein, synaptophysin (Syp), and a postsynaptic protein, PSD-95, in cultured postmitotic neurons. Superresolution fluorescence microscopy of fixed neurons confirmed the identical localization patterns of the tagged proteins to those of endogenous ones verified by immunohistochemistry. The system is also applicable for multiplexed labeling and live-cell imaging. Live imaging with total internal reflection fluorescence microscopy of a single dendritic process of a neuron double-labeled with Syp-mCherry and PSD-95-EGFP revealed the previously undescribed dynamic localization of the proteins synchronously moving along dendritic shafts. Our convenient and versatile strategy is potent for analysis of proteins whose ectopic expressions perturb cellular functions.

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Nissler, Robert, and Sebastian Kruss. "Towards Monochiral Chemical Sensing with Near Infrared Fluorescent Carbon Nanotubes." ECS Meeting Abstracts MA2022-01, no. 9 (July 7, 2022): 722. http://dx.doi.org/10.1149/ma2022-019722mtgabs.

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Semiconducting single wall carbon nanotubes (SWCNTs) fluoresce in the near infrared (NIR) and the emission wavelength depends on their chirality (n,m). Interactions with the environment affect the fluorescence and can be tailored by functionalizing SWCNTs with biopolymers such as DNA, which is the basis for fluorescent biosensors. So far, such biosensors were mainly assembled from mixtures of SWCNT chiralities with large spectral overlap, which affects sensitivity as well as selectivity and prevents multiplexed sensing. The main challenge to gain chirality pure sensors has been to combine approaches to isolate specific SWCNTs and generic (bio)functionalization approaches. Here, we report several approaches to assemble chirality pure SWCNT-based NIR biosensors, with a major focus on aqueous two-phase extraction (ATPE) assisted purification and novel surface exchange protocols. We study the impact of SWCNT chirality/handedness on chemical sensing and highlight possible applications for detection of neurotransmitters, ROS, bacterial motives of plant metabolites. Finally, we use multiple monochiral/single-color SWCNTs as combined ratiometric/multiplexed nanosensors for NIR detection and imaging.

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CHAI, JINGWEN, and QING SONG. "MULTISPECTRAL CONCURRENT DETECTION OF MULTIPLE PROTEINS." Nano LIFE 02, no. 03 (September 2012): 1241004. http://dx.doi.org/10.1142/s1793984412410048.

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Proteins constitutively function within networks. Concurrent detection of multiple proteins is crucial to clinical diagnoses and multidimensional drug profiling. Fluorescence microscopy is capable of multicolor imaging, and has the capability to quantify essentially any physiological changes that occur at the single-cell level and in the context of live single cells, and thus provides an alternative to flow cytometry for multiplexed live single-cell assay. The staining of cells with multiple labels is still a technical challenge while multiplexed assays are complicated by spectral emission overlaps and measurement errors. In this study, we applied emission fingerprinting technique provided by Zeiss LSM 510 META detector, and achieved concurrent detection of ten proteins expressed on the same endothelial cell sample. This approach can be further applied to real-time measurement of multiple proteins expressed on live single cell surface, and therefore will enable a novel approach of multiplexed live single cell detection.

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Paulson, Bjorn, Saeed Bohlooli Darian, Youngkyu Kim, Jeongmin Oh, Marjan Ghasemi, Kwanhee Lee, and Jun Ki Kim. "Spectral Multiplexing of Fluorescent Endoscopy for Simultaneous Imaging with Multiple Fluorophores and Multiple Fields of View." Biosensors 13, no. 1 (December 27, 2022): 33. http://dx.doi.org/10.3390/bios13010033.

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Complex clinical procedures and small-animal research procedures can benefit from dual-site imaging provided by multiple endoscopic devices. Here, an endoscopic system is proposed which enables multiple fluorescence microendoscopes to be spectrally multiplexed on a single microscope base, enabling light sources and optical relays to be shared between endoscopes. The presented system is characterized for resolution using USAF-1951 resolution test charts and for modulation transfer function using the slanted edge method. Imaging is demonstrated both directly and with microendoscopes attached. Imaging of phantoms was demonstrated by targeting USAF charts and fiber tissues dyed for FITC and Texas Red fluorescence. Afterwards, simultaneous liver and kidney imaging was demonstrated in mice expressing mitochondrial Dendra2 and injected with Texas Red-dextran. The results indicate that the system achieves high channel isolation and submicron and subcellular resolution, with resolution limited by the endoscopic probe and by physiological movement during endoscopic imaging. Multi-channel microendoscopy provides a potentially low-cost means of simultaneous multiple endoscopic imaging during biological experiments, resulting in reduced animal harm and potentially increasing insight into temporal connections between connected biological systems.

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Xu, Jian, Tianyue Zhang, Shenyu Yang, Ziwei Feng, Haoying Li, Dejiao Hu, Fei Qin, et al. "Super-Resolution Optical Imaging: Plasmonic Nanoprobes for Multiplexed Fluorescence-Free Super-Resolution Imaging (Advanced Optical Materials 20/2018)." Advanced Optical Materials 6, no. 20 (October 2018): 1870077. http://dx.doi.org/10.1002/adom.201870077.

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Pham, Thai, Renjie Liao, Joshua Labaer, and Jia Guo. "Multiplexed In Situ Protein Profiling with High-Performance Cleavable Fluorescent Tyramide." Molecules 26, no. 8 (April 12, 2021): 2206. http://dx.doi.org/10.3390/molecules26082206.

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Understanding the composition, function and regulation of complex cellular systems requires tools that quantify the expression of multiple proteins at their native cellular context. Here, we report a highly sensitive and accurate protein in situ profiling approach using off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this method, protein targets are stained with horseradish peroxidase (HRP) conjugated antibodies and CFT. Subsequently, the fluorophores are efficiently cleaved by mild chemical reagents, which simultaneously deactivate HRP. Through reiterative cycles of protein staining, fluorescence imaging, fluorophore cleavage, and HRP deactivation, multiplexed protein quantification in single cells in situ can be achieved. We designed and synthesized the high-performance CFT, and demonstrated that over 95% of the staining signals can be erased by mild chemical reagents while preserving the integrity of the epitopes on protein targets. Applying this method, we explored the protein expression heterogeneity and correlation in a group of genetically identical cells. With the high signal removal efficiency, this approach also enables us to accurately profile proteins in formalin-fixed paraffin-embedded (FFPE) tissues in the order of low to high and also high to low expression levels.

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Mooney, Nancie. "Abstract 5631: Expanding tools for multiplex mRNA imaging in spatialomics through the ViewRNA tissue assay kits." Cancer Research 83, no. 7_Supplement (April 4, 2023): 5631. http://dx.doi.org/10.1158/1538-7445.am2023-5631.

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Abstract Spatialomics is a rapidly growing field as it allows researchers to gain a deeper understanding of transcriptomes and corresponding protein expression profiles in cells within complex tissue microenvironments. One critical technology for spatialomics is in-situ hybridization (ISH) technology, which enables direct visualization and quantitation of nucleic acid in cells with single molecule resolution. The Invitrogen ViewRNA ISH assays incorporate branched DNA (bDNA) technology provides tools for interrogating multiple RNA transcripts at the same time, paving the way for improved spatialomics research. This technology is a powerful tool for spatialomics, giving insight into important mechanisms within cells and tissue. With researchers increasingly using spatialomic data in fields like neuroscience, immuno-oncology & single-cell analysis, these scientists will need the rapid and efficient detection of mRNA co-expression profiles that ViewRNA portfolio provides. In our newest offering, ViewRNA fluorescence tissue kits we extended our branched DNA technology, proprietary probe set design and signal amplification technology with Alexa Fluor probes offering a unique, robust, and sensitive in situ hybridization assay for RNA localization in fixed tissues. With fluorescence detection using Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 594, Alexa Fluor 647, and Alexa Fluor 750, these kits allow multiplexed detection and imaging paving the way for improved spatialomics. This technology lays the foundation for expanded fluorescent profiles to provide scientists additional tools to probe multiple transcripts per sample, allow broader spatialomics research. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Nancie Mooney. Expanding tools for multiplex mRNA imaging in spatialomics through the ViewRNA tissue assay kits. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5631.

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McMahon, Nathan P., Jocelyn A. Jones, Ashley N. Anderson, Matthew S. Dietz, Melissa H. Wong, and Summer L. Gibbs. "Flexible Cyclic Immunofluorescence (cyCIF) Using Oligonucleotide Barcoded Antibodies." Cancers 15, no. 3 (January 29, 2023): 827. http://dx.doi.org/10.3390/cancers15030827.

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Advances in our understanding of the complex, multifaceted interactions between tumor epithelia, immune infiltrate, and tumor microenvironmental cells have been driven by highly multiplexed imaging technologies. These techniques are capable of labeling many more biomarkers than conventional immunostaining methods. However, multiplexed imaging techniques suffer from low detection sensitivity, cell loss—particularly in fragile samples—, and challenges with antibody labeling. Herein, we developed and optimized an oligonucleotide antibody barcoding strategy for cyclic immunofluorescence (cyCIF) that can be amplified to increase the detection efficiency of low-abundance antigens. Stained fluorescence signals can be readily removed using ultraviolet light treatment, preserving tissue and fragile cell sample integrity. We also extended the oligonucleotide barcoding strategy to secondary antibodies to enable the inclusion of difficult-to-label primary antibodies in a cyCIF panel. Using both the amplification oligonucleotides to label DNA barcoded antibodies and in situ hybridization of multiple fluorescently labeled oligonucleotides resulted in signal amplification and increased signal-to-background ratios. This procedure was optimized through the examination of staining parameters including staining oligonucleotide concentration, staining temperature, and oligonucleotide sequence design, resulting in a robust amplification technique. As a proof-of-concept, we demonstrate the flexibility of our cyCIF strategy by simultaneously imaging with the original oligonucleotide conjugated antibody (Ab-oligo) cyCIF strategy, the novel Ab-oligo cyCIF amplification strategy, as well as direct and indirect immunofluorescence to generate highly multiplexed images.

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Li, Qingbo, and Edward S. Yeung. "Evaluation of the Potential of a Charge-Injection Device for DNA Sequencing by Multiplexed Capillary Electrophoresis." Applied Spectroscopy 49, no. 6 (June 1995): 825–33. http://dx.doi.org/10.1366/0003702953964598.

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Despite the rapid growth in the use of imaging detectors in spectroscopy, the charge-injection device (CID) has unique features that have not been fully exploited. The advantages of the CID as a two-dimensional array detector for laser-induced fluorescence detection in highly multiplexed capillary electrophoresis are evaluated. In such a system, the CID maintains both high sensitivity and high sampling rate, which are usually difficult to achieve simultaneously with other array detectors. Applying the electronic windowing function significantly improves the scan rate and greatly reduces the volume of data generated. With 1-s exposure time and 488-nm excitation, the detection limit of the system is 10−12 M fluorescein with the device cryogenically cooled and 10−11 M fluorescein at ambient temperature. The low dark current of the CID imager allows operation at room temperature without significantly affecting sensitivity when combined with moderate laser powers. We demonstrate that the CID is well suited for high-speed, high-throughput DNA sequencing based on multiplexed capillary electrophoresis with on-column laser-induced fluorescence detection.

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Chiodi, Elisa, George G. Daaboul, Allison M. Marn, and M. Selim Ünlü. "Multiplexed Affinity Measurements of Extracellular Vesicles Binding Kinetics." Sensors 21, no. 8 (April 9, 2021): 2634. http://dx.doi.org/10.3390/s21082634.

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Extracellular vesicles (EVs) have attracted significant attention as impactful diagnostic biomarkers, since their properties are closely related to specific clinical conditions. However, designing experiments that involve EVs phenotyping is usually highly challenging and time-consuming, due to laborious optimization steps that require very long or even overnight incubation durations. In this work, we demonstrate label-free, real-time detection, and phenotyping of extracellular vesicles binding to a multiplexed surface. With the ability for label-free kinetic binding measurements using the Interferometric Reflectance Imaging Sensor (IRIS) in a microfluidic chamber, we successfully optimize the capture reaction by tuning various assay conditions (incubation time, flow conditions, surface probe density, and specificity). A single (less than 1 h) experiment allows for characterization of binding affinities of the EVs to multiplexed probes. We demonstrate kinetic characterization of 18 different probe conditions, namely three different antibodies, each spotted at six different concentrations, simultaneously. The affinity characterization is then analyzed through a model that considers the complexity of multivalent binding of large structures to a carpet of probes and therefore introduces a combination of fast and slow association and dissociation parameters. Additionally, our results confirm higher affinity of EVs to aCD81 with respect to aCD9 and aCD63. Single-vesicle imaging measurements corroborate our findings, as well as confirming the EVs nature of the captured particles through fluorescence staining of the EVs membrane and cargo.

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Xiao, Lu, Renjie Liao, and Jia Guo. "Highly Multiplexed Single-Cell In Situ RNA and DNA Analysis by Consecutive Hybridization." Molecules 25, no. 21 (October 23, 2020): 4900. http://dx.doi.org/10.3390/molecules25214900.

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The ability to comprehensively profile nucleic acids in individual cells in their natural spatial contexts is essential to advance our understanding of biology and medicine. Here, we report a novel method for spatial transcriptomics and genomics analysis. In this method, every nucleic acid molecule is detected as a fluorescent spot at its natural cellular location throughout the cycles of consecutive fluorescence in situ hybridization (C-FISH). In each C-FISH cycle, fluorescent oligonucleotide probes hybridize to the probes applied in the previous cycle, and also introduce the binding sites for the next cycle probes. With reiterative cycles of hybridization, imaging and photobleaching, the identities of the varied nucleic acids are determined by their unique color sequences. To demonstrate the feasibility of this method, we show that transcripts or genomic loci in single cells can be unambiguously quantified with 2 fluorophores and 16 C-FISH cycles or with 3 fluorophores and 9 C-FISH cycles. Without any error correction, the error rates obtained using the raw data are close to zero. These results indicate that C-FISH potentially enables tens of thousands (216 = 65,536 or 39 = 19,683) of different transcripts or genomic loci to be precisely profiled in individual cells in situ.

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Tu, Siyu, David Rioux, Josée Perreault, Danny Brouard, and Michel Meunier. "Fluorescence and Scattering Dual-Mode Multiplexed Imaging with Gold–Silver Alloy Core Silica Shell Nanoparticles." Journal of Physical Chemistry C 121, no. 16 (April 13, 2017): 8944–51. http://dx.doi.org/10.1021/acs.jpcc.6b11954.

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Braselmann, Esther, and Nadia Sarfraz. "Abstract 1289: Multiplexed illumination of RNAs in live mammalian cells by fluorescence lifetime imaging microscopy." Journal of Biological Chemistry 299, no. 3 (2023): S70. http://dx.doi.org/10.1016/j.jbc.2023.103200.

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Afsari, Hamid Samareh, Marcelina Cardoso Dos Santos, Stina Lindén, Ting Chen, Xue Qiu, Paul M. P. van Bergen en Henegouwen, Travis L. Jennings, et al. "Time-gated FRET nanoassemblies for rapid and sensitive intra- and extracellular fluorescence imaging." Science Advances 2, no. 6 (June 2016): e1600265. http://dx.doi.org/10.1126/sciadv.1600265.

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Time-gated Förster resonance energy transfer (FRET) using the unique material combination of long-lifetime terbium complexes (Tb) and semiconductor quantum dots (QDs) provides many advantages for highly sensitive and multiplexed biosensing. Although time-gated detection can efficiently suppress sample autofluorescence and background fluorescence from directly excited FRET acceptors, Tb-to-QD FRET has rarely been exploited for biomolecular imaging. We demonstrate Tb-to-QD time-gated FRET nanoassemblies that can be applied for intra- and extracellular imaging. Immunostaining of different epitopes of the epidermal growth factor receptor (EGFR) with Tb- and QD-conjugated antibodies and nanobodies allowed for efficient Tb-to-QD FRET on A431 cell membranes. The broad usability of Tb-to-QD FRET was further demonstrated by intracellular Tb-to-QD FRET and Tb-to-QD-to-dye FRET using microinjection as well as cell-penetrating peptide–mediated endocytosis with HeLa cells. Effective brightness enhancement by FRET from several Tb to the same QD, the use of low nanomolar concentrations, and the quick and sensitive detection void of FRET acceptor background fluorescence are important advantages for advanced intra- and extracellular imaging of biomolecular interactions.

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Hanley, Quentin S., Peter J. Verveer, and Thomas M. Jovin. "Optical Sectioning Fluorescence Spectroscopy in a Programmable Array Microscope." Applied Spectroscopy 52, no. 6 (June 1998): 783–89. http://dx.doi.org/10.1366/0003702981944364.

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We report the use of a programmable array microscope (PAM) for the acquisition of spectrally resolved and high-throughput optical sections. The microscope is based on the use of a spatial light modulator for defining patterns of excitation and/or detection of fluorescence. For obtaining optically sectioned spectral images, the entrance slit of an imaging spectrograph and a line illumination pattern defined with a spatial light modulator are placed in conjugate optical positions. Compared to wide-field illumination, optical sectioning led to greater than 3× improvement in the rejection of out-of-focus fluorescence emission and nearly 6× greater peak-to-background ratios in biological specimens, yielding better contrast and spectral characterization. These effects resulted from a reduction in the artifacts arising from spectral contributions of structures outside the region of interest. We used the programmable illumination capability of the spectroscopic system to explore a variety of excitation/detection patterns for increasing the throughput of optical sectioning microscopes. A Sylvester-type Hadamard construction was particularly efficient, performing optical sectioning while maintaining a 50% optical throughput. These results demonstrate the feasibility of full-field highly multiplexed confocal spectral imaging.

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Gómez-García, Pablo A., Erik T. Garbacik, Jason J. Otterstrom, Maria F. Garcia-Parajo, and Melike Lakadamyali. "Excitation-multiplexed multicolor superresolution imaging with fm-STORM and fm-DNA-PAINT." Proceedings of the National Academy of Sciences 115, no. 51 (December 3, 2018): 12991–96. http://dx.doi.org/10.1073/pnas.1804725115.

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Recent advancements in single-molecule-based superresolution microscopy have made it possible to visualize biological structures with unprecedented spatial resolution. Determining the spatial coorganization of these structures within cells under physiological and pathological conditions is an important biological goal. This goal has been stymied by the current limitations of carrying out superresolution microscopy in multiple colors. Here, we develop an approach for simultaneous multicolor superresolution imaging which relies solely on fluorophore excitation, rather than fluorescence emission properties. By modulating the intensity of the excitation lasers at different frequencies, we show that the color channel can be determined based on the fluorophore’s response to the modulated excitation. We use this frequency multiplexing to reduce the image acquisition time of multicolor superresolution DNA-PAINT while maintaining all its advantages: minimal color cross-talk, minimal photobleaching, maximal signal throughput, ability to maintain the fluorophore density per imaged color, and ability to use the full camera field of view. We refer to this imaging modality as “frequency multiplexed DNA-PAINT,” or fm-DNA-PAINT for short. We also show that frequency multiplexing is fully compatible with STORM superresolution imaging, which we term fm-STORM. Unlike fm-DNA-PAINT, fm-STORM is prone to color cross-talk. To overcome this caveat, we further develop a machine-learning algorithm to correct for color cross-talk with more than 95% accuracy, without the need for prior information about the imaged structure.

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Cooper, Justin T., and Joel M. Harris. "Spatially Multiplexed Imaging: Fluorescence Correlation Spectroscopy for Efficient Measurement of Molecular Diffusion at Solid–Liquid Interfaces." Applied Spectroscopy 70, no. 4 (February 17, 2016): 695–701. http://dx.doi.org/10.1177/0003702816631312.

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