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DNA Methods in Veterinary Medicine
Published in Rebecca A. Krimins, Learning from Disease in Pets, 2020
An orthogonal method to identify larger scale variation that complements sequencing is optical mapping. In this approach, long DNA molecules are isolated, tagged by incorporating fluorescent nucleotides at specific DNA sequences and then electrophoresed as single DNA molecules through nanochannels where the patterns of marked sites are imaged. The single molecule images are then reassembled into maps which can be aligned to a reference sequence for the species. This method, and variants of it, are being used to aid de novo gene assembly and to look at changes in genomes that can occur in cancer and certain inherited diseases. A commercial supplier of optical mapping technology is Bionano Genomics (www.bionanogenomics.com) and optical maps have been used as an adjunct to the assembly of many genomes (e.g., Miga et al., 2019; Kronenberg et al., 2018) or characterizing regions with repetitive sequences that are hard to assemble with most DNA sequencing methods (e.g., Demaerel et al., 2019) (Figure 8.5). As with other long DNA methods, optical mapping also requires long DNA (ideally greater than 300 kb) which is best obtained from nucleated cells (~1–2 × 106).
Experimental models and measurements to study cardiovascular physiology
Published in Neil Herring, David J. Paterson, Levick's Introduction to Cardiovascular Physiology, 2018
Neil Herring, David J. Paterson
Fluorescence imaging as described in Section 19.3 with regard to single cells, can also be used in multicellular preparations using optical mapping techniques as pioneered by the Salama group. However, several limitations are particularly relevant to this approach in multicellular tissue. The first arises from the method of tissue perfusion, which may cause uneven loading of the dye throughout the tissue. The second arises in the mechanical restraint required to focus on a large area of curved tissue that may be spontaneously beating. This can be done via mechanical restraint for example, compressing a glass slide over a beating heart, or pharmacologically with a mechanical uncoupling agent such as blebbistatin, a small molecule inhibitor of myosin II. Blebbistatin will remove any mechano-electric feedback from the heart and may also directly interfere with cardiac electrophysiolog y, although this is controversial. Tissues, such as the epicardial surface of the heart, can also be loaded with more than one dye so that several parameters (membrane voltage, cytoplasmic calcium or SR Ca2+) can be monitored with high temporal and spatial resolution across the tissue. This is particularly useful for the study of cardiac arrhythmias where intracellular Ca2+ handling events can be related to the initiation and propagation of electrical waves of excitation.
Optical Coherence Tomography and Quantitative Optical Imaging of Brain Cancer
Published in Yu Chen, Babak Kateb, Neurophotonics and Brain Mapping, 2017
Carmen Kut, Jordina Rincon-Torroella, Alfredo Quinones-Hinojosa, Xingde Li
OCT can bring tremendous value to brain cancer management when used in conjunction with existing imaging technologies. OCT adds unique advantages as it provides real-time, continuous intra-operative guidance and is able to image volumetric tissue samples quickly and efficiently at micron-level resolution. Furthermore, quantitative optical mapping techniques using OCT data can provide surgeons with color-coded visual cues in the detection of cancer versus brain cancer surgery at high sensitivity and specificity. However, OCT cannot provide molecular characteristics of brain cancer and, as a result, can benefit from complementary surface imaging techniques such as 5-ALA and Raman spectroscopy. In addition, OCT does not provide a whole-brain field of view (unlike MRI or CT); as a result, it should be integrated into a surgical guidance system (e.g., Polaris or Brainlab) that can map the 3D position of the OCT probe, relative to the brain resection cavity, in real time.
3D bioprinting for organ and organoid models and disease modeling
Published in Expert Opinion on Drug Discovery, 2023
Amanda C. Juraski, Sonali Sharma, Sydney Sparanese, Victor A. da Silva, Julie Wong, Zachary Laksman, Ryan Flannigan, Leili Rohani, Stephanie M. Willerth
A gold-standard read-out for functional assessment of 3D printed cardiac tissues following their response to drugs is high-speed high-resolution optical mapping [89]. Optical mapping is a widely used noninvasive assessment tool to visualize action potential and its wave propagation to study cardiac electrophysiology. Voltage-sensitive dyes (e.g. Di-4-ANEPPS) or Ca2+ sensitive dyes (e.g. X-Rhod, Fluo-4) are used to monitor transmembrane potential changes or Ca2+ transient propagation, respectively [90,91]. Measuring transient Ca2+ provides quantitative information about Ca2+ handling properties of the cardiac tissue, such as conduction velocity and signal intensity, which helps investigate the clinical phenotype of inherited heart diseases in vitro [7]. For example, it can be used to recapitulate hallmark features of catecholaminergic polymorphic ventricular tachycardia by showing ectopic Ca2+ propagation from patient-derived cardiac tissues with rapid electrical pacing or adrenergic stimulation [92].
The clinical implementation of copy number detection in the age of next-generation sequencing
Published in Expert Review of Molecular Diagnostics, 2018
Jayne Y. Hehir-Kwa, Bastiaan B. J. Tops, Patrick Kemmeren
Short-read technologies are limited in structural variation detection [93,94]. The presence of numerous repetitive elements that are longer than the sequencing library insert size makes it close to impossible to detect medium length SVs and resolve large CNVs with base pair accuracy [95]. Long-read (LR) sequencing preserves the long-range genome architecture and can provide breakpoint resolution of SVs often inaccessible to shorter reads [96–98]. The first sequences produced using the single-molecule, real-time (SMRT) long-read sequencing platform achieved on average reads longer than 7 kb, which some reads exceeding 40 kb [90,99]. Alternative methods to capture long-range information have also been introduced, such as BioNano optical mapping [93] and 10× Genomics linked-read technology [100] (Table 2). While short-read next-generation sequencing data rely on multiple (often) indirect sources of information in order to identify SVs, these can be directly derived in long-read data [51]. However, the ability to meet sample requirements and high molecular weight is a challenge in many settings. In addition, the benefits of longer reads come at a cost as the per-base error rates of such technologies are higher than current short-read sequencing technologies [101].
The Visible Heart® project and methodologies: novel use for studying cardiac monophasic action potentials and evaluating their underlying mechanisms
Published in Expert Review of Medical Devices, 2018
Megan M. Schmidt, Paul A. Iaizzo
As previously mentioned, due to their ability to represent the underlying TAPs, MAP recordings in novel groups of patients may provide unique insights relative to abnormal myocyte repolarizations (also known as repolarization syndromes). While other mapping technologies, such as optical mapping, may be able to record repolarization patterns from more points at any given time, the undetected morphology focal waveforms can be altered due to the use of paralytic drugs (thus the underutilization of stretch-activated channels) required for such techniques [42–45]. For example, Blana et al. conducted studies with mice genetically modified to express long QT syndrome type 3; they concluded that this model for long QT demonstrated both structural and electrophysiologic changes in atrial substrates [46]. Similarly, Shimizu et al. conducted some early studies on long QT syndrome and, through the study of afterdepolarizations, concluded that both verapamil and propranolol could help improve these abnormalities [47]. Interestingly, verapamil and propranolol have also been studied by other researchers, focusing on the ability to recreate a Brugada-like action potential [48–51]. Recently, using Visible Heart methodologies, we have initiated similar studies to generate Brugada-like action potentials in the right ventricular outflow tract of reanimated large mammalian hearts. This model allows us to monitor the focal and global applications of a variety of current and novel therapies for the treatment of early repolarization diseases.