Imaging Flashcards
What is imaging?
Visualising organisms, tissues, cells or cellular components.
Why is imaging used?
PCR, sequencing and western blotting requires lysing to extract the information so they lack spatial context:
Which cell type the DNA or RNA protein originated from.
Where were the cells located in the tissue.
What structures the cells were nearby.
Imaging provides spatial context.
What is the exception to the lack of spatial context?
Single-cell sequencing can give information about which cells nucleic acids came from but can’t say where the cells were located or structures nearby, so imaging is still required.
What can be imaged?
Thin section of preserved tissue.
Cell monolayer
Tissue slice and organoids for live cell imaging.
Whole animal - zebrafish, mouse.
Humans.
What is preserved tissue?
Either formalin-fixed paraffin-embedded (FFPE) tissue, or fresh-frozen tissue.
What is a cell monolayer?
Cell suspension - cytospin or dot cells onto slide.
Or adherent cells - the culture on coverslip.
What is FFPE tissue?
The tissue is fixed in formalin and embedded in paraffin, which provides support to cut very thin sections using a microtome - thinner than a cell.
The tissue sections are then separated in a water bath to smooth them, then mounted on glass slides and charged so they are fixed on.
What are the approaches for imaging?
H&E staining
Immunohistochemistry staining
Immunofluorescence staining
In situ hybridisation / RNAscope
Multiplex spatial proteomics or transcriptomics.
Single cell spatial multi-omics.
What is multiplex spatial proteomics and transcriptomics?
Uses:
COMET
Phenocycler
GeoMx digital spatial profiling
What is H&E staining?
Most widely used stain.
Very reproducible
Haematoxylin stains acidic structures e.g. DNA a purple colour
Eosin stains basic structures e.g. ribosomes, pink cytoplasm.
How are the H&E structures analysed by pathologists?
Identify different types of cells and tissues.
Help diagnose diseases such as cancer.
Use of AI and deep learning models are being explored for analysis.
What is immunohistochemistry staining?
IHC is used to visualise proteins within tissues or cells.
How is the tissue prepared for IHC staining?
Fixing to preserve cell morphology and tissue architecture.
Antigen retrieval to reverse effects of harsh fixation - formalin that causes cross-linking of proteins, masks epitopes and changes structures of proteins.
For intracellular antigens, permeabilise so antibodies can enter cells to get to antigens.
Then block using serum or BSA to reduce background or non-specific staining.
What is the process of adding antibodies for IHC staining?
Add primary antibody specific for the antigen, will bind, incubate at optimum time for best staining. Then wash the excess antibody off.
Then stain with enzyme e.g. peroxidase or alkaline phosphate conjugated secondary antibody, species specific (e.g. anti-rabbit antibody). Incubate, and wash off.
How is the sample analysed in IHC staining?
Then add substrate for colour development to form coloured precipitate around the site of where the primary antibody bound. e.g. DAB forms brown precipitate.
Wash, counterstain using Haematoxylin, stains nuclei so can visualise all cells regardless of antibody, and mount - preservative glue on a coverslip, so can be imaged.
What are the controls for IHC?
Positive - add sample known to include antigen of interest - will definitely stain so know acid works.
Negative - sample that does not include the antigen of interest - shouldn’t stain, shows no non-specific or background staining.
Isotype control - antibody of same isotype of primary antibody but not specific to antigen of interest. Doesn’t bind, so secondary antibody doesn’t bind, so should have no substrate deposition.
What are the advantages of IHC staining?
Simple workflow, can view tissue architecture, standard brightfield microscopy, permanent.
What is the disadvantage of IHC staining?
Difficult to multiplex - stain for more than one antigen.
Could stain with 2 different primary antibodies and 2 secondary antibodies, but hard to distinguish staining.
What is the uses of IHC in medicine?
Diagnosis of neurodegenerative diseases e.g. Parkinson’s and Alzheimer’s.
Diagnosis and staging of cancer, and prediction of treatment response.
How is IHC used to predict treatment response?
PD-L1 expressed on tumour cells.
Binds to PD-1, suppresses anti-tumour immune responses (Switches them off).
These immune responses could recognise and kill tumour cells.
Antibodies that block the interaction to switch immune responses back on - immunotherapy.
PD-L1 IHC is an approved test to select patients for immunotherapy.
What do you notice about this image and the distribution of CLEC14A staining?
There is red staining around blood vessels.
Only the tumour tissue is stained, which supports the notion that it is a tumour endothelial marker.
What further controls could be used in IHC?
Use something that stains all blood vessels - CD31, and stain the next section.
Alternatively could use immunofluorescence with multiple colours.
What is immunofluorescence staining?
IF is similar to IHC but uses fluorescence instead of enzymatic detection.
Same key treatment steps:
Primary antibody, wash incubate, secondary antibody with fluorescent label, incubate wash, then use fluorescent microscope to visualise.
Labels are excited by light at the appropriate wavelength and emit light that is then detected.
What is the advantage of IF staining?
Allows multiplexing using primary antibodies raised in different species and secondary antibodies conjugated to different fluorochromes.
What is specificity in IF and IHC staining?
Specificity - degree to which antibody only binds to protein of interest.
Determined mainly by the primary antibody - only bind to antigen nothing else.
Tissue quality will also effect specificity.
What is sensitivity in IHC and IF staining?
Lower limit of detection, the minimum amount of antigen that can be detected.
Determined by primary antibody and tissue quality, and the detection system used.
What are the types of detection in IHC and IF staining?
Indirect detection - primary antibody added then fluorescent labelled secondary antibody binds, multiple binding so amplification of signal.
Direct - fluorochrome is conjugated to primary antibody. More simple but less sensitive.
Can be made more sensitive by amplification by detection system.
What is UEA1?
A lectin that binds to a-linked fucose residues in glycoproteins and glycolipids in blood.
This is a marker for endothelial cells.
It is not an antibody but is directly conjugated to a fluorophore, so is easy to combine with a range of different antibodies, without worrying about species specificities of secondary antibodies.
What is DAPI?
A fluorescent stain that binds strongly to DNA in the nucleus.
So it is a good positive control as it shows where the cells are.
What is DNA in situ hybridisation?
Uses a labelled nucleotide probe complementary to the target DNA instead of primary antibody.
Once bound, can detect using chromogenic detection (ISH) or fluorescent detection (FISH).
What is the process of DNA-ISH and FISH?
DNA is denatured to give single strand that the complementary labelled probe can bind to.
Then add primary antibody specific to the label.
Then secondary antibody with enzyme attached to convert chromogen to visual precipitate or fluorescent label.
What are the uses of DNA-ISH/FISH?
Assess chromosome integrity
Detect viral infection
DNA-FISH can be multiplexed.
What is RNA-FISH?
Detect gene expression
Very useful in short-lived or secreted proteins (hard to visualise).
What is an RNAscope?
A more advanced method of RNA fluorescent in situ hybridisation.
Can multiplex, has high specificity and sensitivity - as 2 probes are bound and it is amplified.
What is the process of using an RNAscope?
Complementary probe binds, then second complementary probe binds adjacent to first.
When the two probes are bound together, then pre-amplifier can bind, then amplifier, then labelled probe.
What are the ranges of FISH and ISH?
IHC/ISH can be used to visualised 1-3 proteins or genes.
IF/FISH can be used to visualise 4-6 proteins or genes.
It is limited by the number of different species for primary and secondary antibodies.
Also limited to the number of different fluorochromes that can be visualised together.
What is the COMET platform?
Sequential immunofluorescence (seqIF).
Has successive cycles of staining, imagining, then elution.
Two antigens are imaged per cycle.
It is fast, uniform and reproducible staining.
How can COMET be multi-omics?
Can combine with RNAscope to analyse both protein and RNA, spatial multi-omics
Analyse 40+ targets.
What is the phenocycler?
Use primary antibodies with unique oligonucleotide barcode attached
Add complementary oligonucleotide sequences to the barcodes, have fluorescent dyes, allows specific detection of each antibody.
Iterative imagining cycles - stain, reveal 3 antibodies, image, remove, repeat, until all proteins in section are imaged.
What is GeoMx digital spatial profiling?
Stain up to 3 proteins using fluorescent labelled antibodies which are morphology Markers - used to decide which parts of the tissue to look at - the regions of interest.
It is then DNA stained to identify it.
To examine RNA, add probes specific to the RNA wanted to analyse.
What are the probes in GeoMx digital spatial profiling?
Probes have UV-photocleavable oligonucleotide barcode attached, they’re cleaved by UV light.
For proteins, add antibodies specific to the proteins, have oligonucleotide barcode attached that is UV photocleavable.
Only those with the probes or antibodies attached will be cleaved.
How is GeoMx analysed?
UV light is shone onto the regions of interest.
Only the oligonucleotide barcodes from antibodies that are bound by proteins are cleaved.
The barcodes are collected and dispensed into a 96-well plate, then counted using NGS or an integrated system.
What is the results of GeoMx digital spatial profiling?
It provides spatial context - where the region is from, and quantitative expression data - the level of gene/protein expression in the region.
What is single cell spatial multi-omics?
Quantify gene expression in regions but there are multiple cell types
Want to look at specific cells, so can use Xenium platform, CosMx, phenocycler-fusion.
What is sample preparation for CosMx?
Hybridisation of probes
Assembling of flow cell
Can then be analysed by interactive data analysis at a single cell level.
What is the complexity and cost of the imaging techniques in DNA and RNA?
DNA/RNA-ISH /FISH are least complex and expensive.
Then RNAscope, then spatial transcriptomics then single cell transcriptomics.
What is the complexity and cost of the imaging techniques in proteins?
Least:
IHC
IF
Multiplex IF
Spatial proteomics
Single cell proteomics.
What is the process of live cell imaging using tissue slice culture models?
Tissue or tumour is embedded in agarose gel for support.
Sliced used vibratome, thicker sections of live tissue.
Produces tumour slice for analysis and imaging
Can be cultured and kept alive for a week.
How can the live tissue slice culture models be used?
Treated with drugs or antibodies for checkpoint inhibitors
Culture under different conditions e.g. low oxygen and see effects
Add immune cells to see behaviour
Killing assay if tumour cells, see if immune cells can kill tumour cells.
How can the tissue slice culture be used further?
Can stain using limited numbers of markers.
Takes layers and stacks together to produce 3d live image.
Then can fix and embed cultures for higher plex imaging.
