Imaging the Genome
rate, flow time, volume, and wait times. Te acquisition setup also features a multi-location capture option that
is useful for reducing
cost and increasing throughput for experiments with long hybridization steps. Aſter running an experiment, data can be visualized and analyzed according to probe number directly within SRX, which offers unlimited probe support. Initial genomic imaging data
can appear very noisy. SRX soſtware offers built-in analysis tools that use the data associated with every local- ization to isolate signal from noise via cluster analysis. Tere are sev- eral algorithm options available for performing cluster analysis, includ- ing density-based spatial clustering applications with noise (DBSCAN) [29], ordering points to identify the clustering structure (OPTICS) [30], Delaunay analysis [31], and image- based clustering algorithms
[32].
DBSCAN has been used to gener- ate OligoSTORM data, and this algorithm uses
particle
Figure 6: Vutara ecosystem. (A) Left: Fully integrated fluidics unit that has been designed with single-molecule multiplexed and genomic imaging in mind. The unit contains four large washing reservoirs and fifteen small reser- voirs for labeling reagents. The small reservoirs are swappable during an experiment, making imaging of greater than fifteen targets possible. Middle: The Vutara microscope for single-molecule super-resolution imaging. Right: An isosurface rendering of a topologically associated domain (blue) and compartment (magenta) visualized using SRX software. (B) Schematic of density-based spatial clustering of applications with noise (DBSCAN). Top left: Radius term is defined by the circles around the points. The minimum points for determining a cluster in this example is three, meaning each point must contain two additional points within its circle to be considered a core point of the cluster (blue dots). Dots reachable from a core point but not containing enough points to be a core point are also considered part of the cluster (red dots). Black dots represent points that are not reachable by any core points and are considered noise and are filtered from the final image. Top right: Cartoon representing all data points from an acquisition. Bottom right: DBSCAN is applied to the data, three clusters are identified; core points are represented by blue, grey, and dark blue dots. Red dots are reachable from a cluster and included in the final image on the bottom left.
applications like OligoSTORM feasible for the large number of targets spatial genomics studies require. Te microscope uses biplane detection making each acquisition 3D, allowing for imaging of the entire depth of the nucleus if necessary. Te incorporation of the fluidics unit is critical for these types of experiments due to the large number of targets to be imaged. Te fluidics unit has been carefully designed with multiplexing and single-molecule imaging in mind. Each unit has four large reservoirs for high-volume reagents, such as imaging and wash buffers, along with fiſteen smaller reservoirs for more costly labeling reagents. Tese smaller reservoirs can be swapped out during an experiment, making imaging greater than fiſteen targets possible (Figure 6). Te fluidics component is fully integrated into Vutara’s
SRX soſtware at every step from acquisition to analysis. Begin- ning with the experimental setup, the user interface allows for full control over sequences and parameters, including flow
24 distances
and densities to assign clusters ( Figure 6). Once localizations have been assigned to clusters, SRX offers a suite of statistical analysis options to calculate metrics. Some of these metrics include the number of parti- cles in a cluster, volume, surface area, sphericity ratio, density of particles, and radius of gyration. Bruker’s Vutara SMLM imag-
ing platform with SRX soſtware provides a powerful system for 3D SMLM of the genome along with a wide range of other sample types.
Te ability to integrate fluidics with the system makes multi- plexed imaging applications much more accessible, and SRX provides an all-in-one solution for data acquisition, visualiza- tion, and analysis.
Future Directions Currently, progress
is being made on integrating a
workflow into SRX called ORCA (Optical Reconstruction of Chromatin Architecture) [34]. ORCA uses oligopaints like Oli- goSTORM does but, rather than imaging at the single-mole- cule level, it takes sub-diffraction images at the wide-field level (Figure 7). All the fluorophores in a single genomic region are imaged as an ensemble rather than stochastically. Te center of mass is determined from the image, and an ORCA walk is generated by connecting the center of masses. Te technique uses small probe step sizes (2–10 kb) and is ideal for looking at smaller regions or single genes.
www.microscopy-today.com • 2020 November
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