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General Considerations for Labeling and Imaging of Cells
Published in Martin G. Pomper, Juri G. Gelovani, Benjamin Tsui, Kathleen Gabrielson, Richard Wahl, S. Sam Gambhir, Jeff Bulte, Raymond Gibson, William C. Eckelman, Molecular Imaging in Oncology, 2008
In order to perform endogenous cell labeling, gene transfection of a signaling protein into the target cell is necessary. The fluorescence protein family for fluorescence imaging or the luciferase enzyme family for bioluminescence imaging can be employed for optical imaging. In future, nitrogen-rich proteins for chemical exchange saturation transfer (CEST) imaging may become available for in vivo MRI (4,5). Gene transfection methods have been well established in the experimental situation using electroporation, virus vectors, nonviral liposomes or macromolecules, etc. Endogenous cell labeling has technical advantages over exogenous labeling; all cells proliferated from the labeled cell are also labeled, and the dead cell is not visualized because it loses the ability to make signaling proteins. However, gene therapy is yet to be approved in clinical practice because longterm safety for oncogenesis has not been proved. Therefore, although endogenous labeling, despite its inherent disadvantages, is technically a better method, exogenous cell labeling is a more feasible method for use in clinical practice.
Introduction to optical imaging
Published in Ahmad Fadzil Mohamad Hani, Dileep Kumar, Optical Imaging for Biomedical and Clinical Applications, 2017
Dileep Kumar, Ahmad Fadzil Mohamad Hani
Optical imaging techniques can be broadly classified as bioluminescence imaging and fluorescence imaging [9]. Bioluminescence imaging is used for imaging molecules in small animals where the light is emitted into living organisms. The discovery of fluorescence and fluorescence microscopy has been instrumental in imaging the single-cell structures at the microscopic levels with the help of microscopic lenses. Fluorescent images are obtained using a light source of specific wavelength to excite the targeted molecule, which in turn emits light of longer wavelength than absorbed. This emitted light is used to generate fluorescent optical images. Optical imaging techniques being investigated meet the challenges and improvement in molecular imaging in preclinical examination and patient concern. Combination of targeted molecules in vivo and optical contrast agents imaging sensitivity are driven in parts for molecular imaging in order to emphasise an optical imaging systems [10]. By combining optical imaging techniques such as bioluminescence imaging and fluorescence with near-infrared (NIR) spectrum, the signal-to-background ratio for detecting specific molecular signals can be increased and similarly can be achieved with other molecular imaging modalities. In advanced cases, a fundamentally simplified gene expression imaging is produced using bioluminescent and fluorescent proteins that act as synthesised optical active biomarkers. It is also noted that optical imaging with its advancements can now be used in clinical practices for some applications and it is widely investigated for future research directions toward its application in clinics.
Routine and Special Techniques in Toxicologic Pathology
Published in Pritam S. Sahota, James A. Popp, Jerry F. Hardisty, Chirukandath Gopinath, Page R. Bouchard, Toxicologic Pathology, 2018
Daniel J. Patrick, Matthew L. Renninger, Peter C. Mann
In vivo optical imaging includes fluorescence and bioluminescence imaging. Both techniques are highly sensitive (picomolar) at limited depths of a few millimeters; quick and easy to perform (with a high-throughput capability); and in general, do not require costly instrumentation. This makes them particularly suited to the drug development and validation process. Fluorescence imaging uses the ability of traditional or quantum dot fluorochromes (Papagiannaros et al. 2010) to absorb external excitation light of one wavelength and reemit light of a longer wavelength, which can be detected as discussed previously. In bioluminescence imaging, an enzyme (i.e., luciferase from the North American firefly Photinus pyralis or from the sea pansy Renilla) that is capable of generating light in the presence of a substrate (i.e., d-luciferin or coelenterazine, respectively) is used as a reporter to assess the transcriptional activity in cells that are transfected with a genetic construct containing the enzyme’s gene under the control of a promoter of interest. The enzymes can also be used to detect the level of cellular ATP (cell viability or kinase activity assays), tumor growth (Hawes and Reilly 2010), or other enzyme activity (i.e., caspase, cytochrome P450). Thus, the externally detected light is an indicator of biologic/molecular processes. The imaging process involves anesthesia of the animal, injection of the respective substrate, and placement of the animal in a dark chamber with a thermoelectrically cooled CCD camera, which is extremely sensitive to even weak luminescence. The light emitted can then be semiquantitatively analyzed. Disadvantages of optical imaging include low depth of penetration and limited clinical translation (Ying and Monticello 2006).
Targeting cancer homing into the lymph node with a novel anti-CCR7 therapeutic antibody: the paradigm of CLL
Published in mAbs, 2021
Carlos Cuesta-Mateos, Raquel Juárez-Sánchez, Tamara Mateu-Albero, Javier Loscertales, Wim Mol, Fernando Terrón, Cecilia Muñoz-Calleja
The Granta-519-luc+ MCL model was developed through tail vein implantation of 2 × 106 cells/animal into sub-lethally irradiated (2 Gγ), 6–8-weeks-old C.B-17 SCID mice (CB17/Icr-Prkdcscid/IcrIcoCrl, Charles River). The bioluminescent cell line was generated as reported.61 Randomization into study groups was based on both bioluminescence analysis and body weight. Antibodies were dosed intraperitoneally (IP). Further details on bioluminescence imaging tissue analysis can be found in the Supplementary data. For the JVM-3 CLL model, 2 × 107 cells were tail vein injected into sub-lethally irradiated 6–8 weeks old C.B17-SCID mice (Vital River Laboratories, Beijing, China). Randomization was based on body weight and antibodies dosed IP.
Quantification of extracellular vesicles in vitro and in vivo using sensitive bioluminescence imaging
Published in Journal of Extracellular Vesicles, 2020
Dhanu Gupta, Xiuming Liang, Svetlana Pavlova, Oscar P.B Wiklander, Giulia Corso, Ying Zhao, Osama Saher, Jeremy Bost, Antje M. Zickler, Andras Piffko, Cecile L. Maire, Franz L. Ricklefs, Oskar Gustafsson, Virginia Castilla Llorente, Manuela O. Gustafsson, R. Beklem Bostancioglu, Doste R Mamand, Daniel W. Hagey, André Görgens, Joel Z. Nordin, Samir EL Andaloussi
In the past decade, bioluminescence imaging has emerged as a versatile tool with diverse applications in both basic biology and drug delivery research in vitro and in vivo. Because of its great sensitivity, others have tested endogenous bioluminescence labelling of EVs by genetically modifying their source cells. The primary issue with these previous studies has been the luciferase proteins utilised and the strategy used for sorting luciferase proteins into EVs. Most of these studies have simply overexpressed free luciferase or made use of MFGE8 (Lactadherin) fusions in producer cells [13,19,20,22,29], both of which we found to be less efficient than tetraspanin-fusion-based sorting. In addition, a system for sensitive cargo-based labelling of EVs would allow to specifically track different subpopulations of vesicles as compared to passively loaded luciferase enzymes into EVs. Furthermore, these reports have utilised Gaussia or Renilla for EV detection, and while these luciferases are stable, they lack high signal intensity. For instance, Super Rluc8, a Renilla analogue with enhanced activity, showed only modest activity when compared to the ThermoLuc and NanoLuc CD63 fusion proteins used in this study. In addition, we also tested AkaLuc for EV labelling, which was recently described for imaging single cells in vivo [42]. Unfortunately we failed to detect the EVs in vivo using our experimental setup.
Anti-triple-negative breast cancer metastasis efficacy and molecular mechanism of the STING agonist for innate immune pathway
Published in Annals of Medicine, 2023
Xing Lu, Xiang Wang, Hao Cheng, Xiaoqing Wang, Chang Liu, Xiangshi Tan
We created 4T1-Luc cell lines to express firefly luciferase stably, which allowed us to track and quantify cells in vivo. In vivo Biophotonic imaging was used to identify tumour cell spread and proliferation. Mice were weighed and intraperitoneally treated with 150 mg/kg luciferin. After 9 min of luciferin treatment, the animals were pre-anesthetized using an oxygen-isoflurane mixed gas (1%–3%). At 12 min following luciferin administration, the animals were placed into the imaging chamber for bioluminescence measurement assessment using a Xenogen IVIS Lumina XRMS Series III live animal bioluminescence imaging system (Perkin Elmer, USA). On the last day, mice were sacrificed, then their hind limbs and organs were imaged and removed within 10 min.