Merge pull request #9 from tidyomics/technology-description

update technology description

Author: stemangiola

inst/images/cosmx.png

@@ -39,18 +39,24 @@ By the end of this session, participants will be able to:
 
 ## Experimental technologies
 
+Here we briefly describe some of the major technologies. This section is contributed by Dr Luciano Martellotto.
+
 **Spatial-omics** encompasses a suite of powerful methods that reveal not only which genes are active in a tissue but also exactly where those genes are switched on. One widely used strategy involves laying a thin slice of tissue onto a specially prepared glass slide that carries an array of microscopic “spots,” each spot marked with its own unique molecular barcode. As the tissue is gently broken down, the messenger RNA molecules released from each cell adhere to the underlying spots and pick up that spot’s barcode. By sequencing the barcodes together with the captured RNA, researchers can reconstruct a two-dimensional map of gene expression. For example, the Visium platform from 10x Genomics uses this barcoded-surface approach to chart gene activity across tumour biopsies, helping oncologists to identify pockets of treatment-resistant cells within a cancerous mass.
 
 An alternative method, known as **combinatorial FISH** (fluorescence in situ hybridisation), skips the need for physical barcodes by using fluorescent probes that bind directly to RNA molecules within intact tissue. Each probe is tagged with a small coloured label, and by carrying out multiple rounds of staining, imaging and probe removal, a unique sequence of coloured dots is generated for each target gene. It’s akin to reading a barcode of coloured spots: once the entire sequence of images has been captured, computational decoding reveals which gene each pattern corresponds to and pinpoints its exact location. This technique underlies MERFISH (Multiplexed Error-Robust FISH), which neuroscientists often employ to map hundreds of genes simultaneously in brain sections, illuminating the molecular identities of different neuronal subtypes.
 
 **In-situ sequencing** offers yet another route to spatially resolved transcriptomics by performing the sequencing reactions directly within fixed tissue sections. Rather than relying on pre-made probes, this approach uses a series of enzymatic ligation or polymerisation steps to read out the RNA sequence base by base. At each cycle, fluorescently labelled reagents indicate which nucleotide (A, C, G or T) has been incorporated, and repeated imaging across multiple cycles yields short sequence reads in situ. Once these reads are matched to a reference genome, they reveal precisely where specific transcripts lie. Developmental biologists have harnessed this method—pioneered by technologies such as Fluorescent In Situ Sequencing (FISSEQ)—to follow gene expression patterns during embryo formation, tracking how cells differentiate according to their spatial context.
 
+
+
 ```{r, echo=FALSE, out.width="700px"}
 library(here)
 
 knitr::include_graphics(here("inst/images/three_technologies.png"))
 ```
 
+### Visium
+
 The **Visium CytAssist** platform from 10x Genomics brings the power of spatial transcriptomics into a streamlined, sequencing-based workflow. At its heart lies a standard glass slide bearing an 11 mm by 11 mm capture area patterned with roughly 14 000 microscopic spots (or 5 000 spots on a smaller 6.5 mm by 6.5 mm format). Each spot is densely coated with millions of identical oligonucleotides, each bearing a unique spatial barcode, a unique molecular identifier (UMI) and a poly(dT) tail designed to bind the polyadenylated tails of mRNA. When a fresh‐frozen or FFPE tissue section is mounted onto this slide, RNA molecules released during permeabilisation will hybridise to these oligos, effectively “stamping” each transcript with its precise tissue coordinates.
 
 The CytAssist instrument automates the critical steps of permeabilisation, RNA digestion and probe release. Rather than capturing native transcripts directly, Visium employs probe hybridisation: a comprehensive set of probes tiles the entire transcriptome (v2 chemistry covers some 18 000 human or 19 000 mouse genes), binding selectively to their target RNAs. Once the tissue has been permeabilised, these probes are enzymatically released and immediately recaptured by the underlying barcoded array. A short extension reaction then attaches the probe insert to the spatial barcode and UMI, before a denaturation step frees the complete construct for library preparation.
@@ -59,21 +65,27 @@ Sequencing libraries are configured so that Read 1 decodes the slide’s spatial
 
 In practice, Visium CytAssist has found widespread use across many fields. Cancer researchers have applied it to map immune cell infiltration and stromal niches within melanoma or breast carcinoma biopsies. Developmental biologists use it to chart gene expression gradients in embryonic tissues, revealing how cells acquire distinct identities in different locations. Even neuroscientists have begun to dissect the molecular architecture of brain regions, linking spatial patterns of gene activity with anatomy and function. By combining a turnkey instrument with a comprehensive probe set and high‐throughput sequencing, Visium offers an accessible route to the spatial “geography” of gene expression in virtually any tissue.
 
+
+
 ```{r, echo=FALSE, out.width="700px"}
 library(here)
 
 knitr::include_graphics(here("inst/images/visium.png"))
 ```
+### Visum HD
 
 The **Visium HD** system represents a next-generation leap in spatial transcriptomics, offering subcellular resolution on a standard CytAssist instrument. Instead of discrete 55 µm spots, the Visium HD slide presents a continuous lawn of capture oligonucleotides across a 6.5 mm × 6.5 mm area, each oligo bearing a unique spatial barcode and UMI. These barcodes are patterned in a fine grid of 2 µm × 2 µm squares, which are digitally binned into 8 µm × 8 µm “pixels” for data analysis. In practice, this means that gene expression can be mapped at roughly one-cell or even subcellular scale—more than a six-fold improvement in resolution compared with the original Visium array.
 
 As with the standard Visium workflow, fresh-frozen or FFPE tissue sections are first stained (H&E or immunofluorescence, if desired) and imaged for morphological context. The CytAssist then automates permeabilisation, RNA digestion and probe‐release steps: a comprehensive probe set tiles the entire transcriptome, binding each target mRNA; released probes are recaptured by the underlying barcoded oligo lawn; and a short extension reaction fuses the probe insert to its spatial barcode and UMI. After denaturation frees these constructs, they undergo library preparation and high-throughput sequencing. Read 1 decodes the spatial barcode and UMI, while Read 2 reads into the probe insert to identify the gene. To cover the full 6.5 mm capture area at HD resolution, Visium HD recommends approximately 275 million read-pairs per run.
 
+
+
 ```{r, echo=FALSE, out.width="700px"}
 library(here)
 
 knitr::include_graphics(here("inst/images/visiumhd.png"))
 ```
+### STOmics 
 
 **BGI’s STOmics** system brings spatial transcriptomics onto DNA nanoball (DNB) patterned chips that can cover areas from a few square millimetres right up to an entire microscope slide, offering both enormous scale and subcellular resolution. The process begins with the creation of a dense array of molecular “nanoballs,” each just 220 nm across and stamped onto the chip in a precise grid. During chip manufacture, each nanoball is endowed with three key elements: a poly-T tail for capturing polyadenylated mRNA, a unique molecular identifier (UMI) to count individual transcripts, and a coordinate identifier (CID) that records its exact X–Y position on the array.
 

inst/images/merfish.png

@@ -26,6 +26,51 @@ In this session we will learn the basics of imaging-derived spatial transcriptom
 
 We will maintain the use of `tidyomics` that we learned in `Session 2`. The programming style, in contrast of `Session 1` will make use of the `|>` (pipe) operator.
 
+## Experimental technologies
+
+Here we briefly describe some of the major technologies. This section is contributed by Dr Luciano Martellotto.
+
+### Xenium
+
+The Xenium platform from 10x Genomics translates the principles of padlock-probe chemistry and rolling-circle amplification into a fully automated, imaging-based workflow that delivers subcellular maps of RNA within tissue sections. Each sample is secured in a proprietary glass cassette that holds up to several serial sections in a rigid fiducial frame, ensuring precise registration between fluorescent images and subsequent histological stains. Once loaded, the instrument carries out probe hybridisation and ligation steps in situ, converting each target transcript into a circular DNA molecule that serves as the template for highly localised rolling-circle amplification.
+
+As amplification proceeds, each RNA-derived padlock probe generates a dense bundle of amplified DNA—known as an amplification product dot—at the original site of the transcript. These dots are then illuminated in successive rounds of fluorescent detection and cleavage, producing a unique spatial barcode for each gene target. By capturing high-resolution images after every cycle, Xenium reads out the identity and localisation of hundreds of transcripts at true subcellular resolution (often down to 200 nm), while preserving tissue morphology throughout the experiment.
+
+Following completion of the imaging sequence, sections can undergo immunofluorescence or standard haematoxylin & eosin staining without loss of register, enabling seamless integration of protein, RNA and anatomical data. The accompanying Xenium Explorer software performs automated cell segmentation—typically using DAPI-stained nuclei—and assigns each amplification dot to its host cell, yielding single-cell, spatially resolved gene expression matrices ready for downstream analysis.
+
+```{r, echo=FALSE, out.width="700px"}
+library(here)
+
+knitr::include_graphics(here("inst/images/xenium.png"))
+```
+
+### CoxMx
+
+The CosMx Single Molecular Imager, commercialised by NanoString, brings truly single-molecule mapping of RNA—and even proteins—into intact tissue sections with subcellular precision. In this approach, each target transcript is first recognised by a bespoke in situ hybridisation probe carrying a unique “readout domain” of oligonucleotides. These readout domains each host multiple photocleavable sites. During the experiment, a series of up to sixteen fluorescent reporter sets are sequentially hybridised to the readout domains, imaged in high-resolution three-dimensional fields, then cleaved away and washed off. The presence or absence of fluorescence across the sixteen cycles produces a binary barcode that identifies each individual molecule, while the precise x, y and z coordinates captured by the microscope pin down its location within or between cells.
+
+CosMx is compatible with both fresh-frozen and formalin-fixed paraffin-embedded samples and can scan as much as 300 mm² of tissue by stitching together up to 384 fields of view. Beyond RNA, the same cyclic detection chemistry can be applied to barcoded antibodies for multiplexed protein measurement—in some panels up to 68 different targets in the same section. After imaging, the AtoMx software platform integrates DAPI or membrane-marker images to segment individual cells and assign each molecular dot to its host cell, yielding spatially resolved count matrices at single-cell, subcellular resolution.
+
+```{r, echo=FALSE, out.width="700px"}
+library(here)
+
+knitr::include_graphics(here("inst/images/cosmx.png"))
+```
+
+### MERFISH
+
+The Vizgen MERSCOPE system brings the power of MERFISH (Multiplexed Error-Robust Fluorescence In Situ Hybridisation) into a user-friendly, end-to-end instrument for high-plex, subcellular mapping of RNA within intact tissue sections. In practice, a fixed tissue slice—whether fresh-frozen or formalin-fixed paraffin-embedded—is first permeabilised and then incubated with a library of “encoding” probes. Each encoding probe carries a short, target‐specific sequence that binds to its RNA of interest, followed by a readout sequence that serves as the scaffold for later detection.
+
+Once the probes are hybridised, the MERSCOPE instrument carries out a series of imaging rounds that reveal the unique binary barcode of each transcript. In each cycle, a set of fluorescent “readout” probes is flowed over the sample and allowed to bind to their complementary readout sequences. A high-resolution image is captured, the fluorophores are chemically cleaved and washed away, and the next readout cycle begins. By repeating this process—typically over a dozen or more rounds—each RNA species is assigned a distinct pattern of “on” and “off” signals across the images. Sophisticated error-robust encoding ensures that even if a spot is missed or a signal fluctuates, the correct gene identity can still be recovered.
+
+Following image acquisition, the built-in MERSCOPE Visualizer software aligns the hundreds of image stacks, decodes each fluorescent barcode into a specific RNA identity, and maps thousands of individual molecules back to their precise positions within cells. Researchers can then overlay the decoded RNA map with standard histology or immunofluorescence stains, revealing how gene expression patterns relate to tissue morphology.
+
+```{r, echo=FALSE, out.width="700px"}
+library(here)
+
+knitr::include_graphics(here("inst/images/merfish.png"))
+```
+
+
 ### 1. Working with imaging-based data in Bioconductor with MoleculeExperiment
 
 ```{r, message=FALSE, warning=FALSE}
@@ -392,7 +437,7 @@ tx_spe =
 
 We then visualise what is the relationship between variance and total expression across cells.
 
-```{r, fig.width=3, fig.height=2}
+```{r, fig.width=5, fig.height=5}
 tx_spe |> 
   
   # Gene variance