Imaging Chromosomal Structure in situ with Vutara

The three dimensional organization of chromosomes plays a key role in chromosomal function. Chromosomes exhibit structural differences on a regional basis, both within and between chromosomes. Understanding these structural differences likely will prove useful in understanding chromosomal function in both normal and pathological states.

Based on the use of OligoSTORM (B. J. Beliveau et al., 2015), the laboratory of Ting Wu at Harvard University, in collaboration with Bruker, has developed a method for imaging and visualizing DNA sequences in specific chromosomal regions at the super resolution level using the Vutara 352 (view webinar). Specially designed oligonucleotides are hybridized to a chromosomal sequence. By sequentially labelling a sample with secondary oligos and then imaging with localization microscopy, a 3-D image of the structure of that lablled region can be generated.

Vutara Maternal and Paternal

14 probes on maternal and paternal homologues

Some of Vutara’s basic capabilities that make it ideal for this application are:

  • 3-D localization with patented biplane technology – each image plane encompasses a 1 um thick slice
  • Extended axial range by Z series – scan through an entire nucleus
  • Imaging depths of up to 30 um in cells
  • Multi-location capture of multiple nuclei
  • Fast acquisition – up to 800 fps for raw images
Both Hom C S01to09 P Cloud60nm Angle2 Box High Res

14 probes on maternal and paternal homologues, view tilted and rotated

SRX software has been optimized to meet the requirements of this demanding application. A critical step was to implement microfluidics control in order to perform sequential labelling steps. The SRX microfluidics control module allows users to create fluidic sequences containing an unlimited number of buffers and reagents per fluidics step, as well as an unlimited number of steps per experiment. SRX assigns a user defined color for localizations in each step, and during Visualization and Analysis, the entire data set is merged. An unlimited number of steps can be visualized and analyzed.

Bruker Microfluidics Accessory

Microfluidics Accessory for Sequential Labelling in Localization Microscopy Applications

SRX fluidics control interface

Cluster Analysis Dialog Box DBSCAN

SRX DBScan interface

The full suite of SRX data filtering and statistical analysis can be utilized, including DBScan for identifying clusters formed by steps and cluster hulls to aid in visualization of clusters.

Semitransparent hulls

Semitransparent view of cluster hulls

Pointsplatting

Point splatting view of clusters

For more methodological details, see https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1007872.

Hear from the authors. WEBINAR: Walking Along Chromosomes: Visualizing in situ Chromosomal Structure with Single Molecule Localization Microscopy.

 

 Contact Us to Learn More