In collaboration with Dr. Kwasi Kwakwa and Dr. Omer Bayraktar, Wellcome Sanger Institute, Hinxton, Cambridge, UK.
Spatial biology techniques are advancing rapidly as powerful tools to gain an in-depth understanding of tissues and their cellular organization. As a matter of fact, spatially resolved transcriptomics has been selected by Nature Methods as the method of the year in 2021 due to their versatility in transcriptomics profiling (Marx., 2021).
With the primary objective of creating comprehensive gene expression profile maps of entire tissues, spatial biology is able to study the organization and function of an entire tissue with cellular resolution; this is why cancer research, neuroscience, and developmental biology laboratories use this method to quantify mRNAs in a spatial context (Marx, 2021).
A spatially resolved transcriptomics analysis can be performed using in situ sequencing or multiplexed fluorescence in situ hybridization. Following a first step using single-molecule Fluorescence In Situ Hybridization (smFISH), automated high-resolution imaging allows the visualization of gene expression profiles in a specific cellular context or of the entire tissue on a larger scale.
In these studies, the crucial point is the complexity of imaging steps involving large-scale scanning of samples and complex multidimensional acquisitions, including Z-stacks and single-cell resolution multichannel imaging. To support such elaborate acquisitions, a set-up capable of acquiring the sample in its entirety and in a short time is needed, without having to compromise on image resolution.
In these types of experiments, the widefield imaging modality is usually used to increase throughput and prevent long acquisition times. Nevertheless, this type of approach does not allow obtaining such a resolution to properly localize the single RNA molecule expression in cellular compartments or resolve individual RNA molecules in optically crowded tissue areas. Thus, we present in this Application Note a high-speed four-camera set-up, based on CrestOptics confocal technology, used by Dr. Bayraktar’s group in the Wellcome Sanger Institute for spatial transcriptomics analysis.
Dr. Bayraktar and collaborators developed and optimized a large-scale spatial transcriptomics pipeline, consisting of automated multiplexed smFISH, automated high-resolution imaging, single-cell and RNA spot segmentation, single-cell gene expression analysis and mapping. The laboratory uses a top-speed CrestOptics solution consisting of an X-Light V3 spinning disk confocal equipped with four cameras in order to accomplish the complex imaging steps required for this type of research (Figure 1).
Figure 1: High-speed four-camera set-up installed at the Wellcome Sanger Institute for spatial transcriptomics analysis.
This high-end set-up includes an openFrame inverted microscope (Cairn Research) with a Nikon 40X 1.15 NA water immersion objective, an X-Light V3 spinning disk confocal module (CrestOptics), four Kinetix sCMOS cameras (Photometrics) and an LDI laser source (89 North); the entire system is controlled by MicroManager software, which allows cameras synchronization and simultaneous imaging.
In situ mapping of neuronal subtypes is carried out using this high-speed system (Figure 2). By providing homogeneous illumination over the entire 25 mm field of view (FOV), the X-Light V3 confocal spinning disk allows seamless stitching of this very large sample, as well as reducing the number of tiles and increasing the speed. Additionally, the four cameras enable the capture of up to four channels simultaneously on an ultra-large FOV, which further reduces the imaging time.
Furthermore, this installation is further strengthened by the choice of the most appropriate disk pattern for this type of application. The spinning disk box is one of the most versatile components of the CrestOptics X-Light V3 spinning disk confocal and it offers the freedom to choose the most suitable disk geometry depending on the application and imaging requirements. As a matter of fact, our customers have used a disk with slits (HT-Slits), i.e containing continuous spirals instead of pinholes, designed for samples that require high throughput, high speed, and low excitation power. HT-Slits disks are uniquely designed to achieve a balance between throughput and confocality (see our previous application note “Spatial transcriptomics: mapping brain cellular organization”) allowing our customers to obtain very complex images in a short period of time with respect to pinholes disks, thereby speeding up the experiment compared to a standard confocal.
It is shown in Figure 2 how this set-up can be applied to a mouse brain section marked with different species of RNA.
Figure 2: Large image of a mouse brain section marked with different RNA species; nuclei are stained in blue.
In particular, the image was acquired with a 40X 1.15 NA water immersion objective and shows a maximum intensity projection of 15 um (1um Z-step). The entire mouse brain section was covered by 300 tiles acquired over a period of one hour, taking into account both 3D and 2D information.
It is important to note that despite the large sample size, the use of such a high-speed set-up allowed our customers to use high magnification and numerical aperture objectives. Figure 3 illustrates how this approach provides detailed information about tissue compartmentalization, cell quantification, and interaction studies.
Figure 3: Single spots and RNA molecules localization in the hippocampal region. In situ hybridization of 4 different RNA species is shown; nuclei are stained in blue.
In conclusion, this high-speed four-camera set-up, based on CrestOptics X-light V3 confocal, provides a robust solution for spatial transcriptomics analysis. Due to its uniform illumination across a 25 mm field of view, the X-Light V3 is able to stitch images of large samples with ease, collect data without artifacts, and accelerate data collection. HT-Slits disks are particularly suitable for this type of application, as their geometry ensures increased throughput (typically three to four times higher than pinhole disks), while maintaining good axial resolution in samples of no excessive thickness (< 30 um). Furthermore, due to the four-camera setup, the acquisition speed is increased fourfold, making this solution particularly suitable for acquiring tissue at single-cell resolution at rapid speeds.
These features have resulted in this set-up becoming the cutting-edge reference point for spatial biology studies at the Wellcome Sanger Institute.
Acknowledgments:
We thank Dr. Kwakwa and Dr Bayraktar for their support in creating this application note and Cairn Research for the excellent installation and customer support at the Wellcome Sanger Institute.
