Multiplex imaging of tissues broadly refers to techniques that allow for the capture of 3+ targets of interest from a single sample. For example, a standard Masson’s Trichrome Stain allows the visualization of collagen, muscle tissue, cytoplasm, and nuclei. However, most standard bright field stains do not allow the simultaneous visualization of more than 4 targets in a tissue sample. This is due to a few reasons: 1) lack of stain specificity; 2) steric hindrance (e.g., limited space for dye molecules to penetrate into the sample and interference from dye molecules with other targets); and 3) limited spectrum space to visualize more targets, making it difficult to distinguish between different co-localizations of targets.
To work around these limitations, most highplex (>3 targets) imaging techniques are fluorescence-based. Fluorescent dyes are typically excited and imaged one at a time, which means that the signals are more specific and easily distinguishable. It is also possible to turn on and off individual channels, making it easier to focus on the relevant signals of interest. With fluorescence microscopy, an arbitrarily large number of channels (i.e., markers) can be added to an image (though it is only recommended to capture and view 3-4 markers simultaneously to avoid the necessity of signal de-mixing).
At Enable Medicine, we use the Akoya PhenoCycler (formerly CODEX) system to acquire multiplex images of tissue samples. The PhenoCycler system allows highplex (50+ biomarkers) whole slide images (WSI) to be generated using a combination of immunohistochemistry (IHC), DNA barcoding, and fluorescence imaging (read on for more details). The PhenoCycler platform is used only for protein markers with a nuclear counterstain.
Image from Akoya Biosciences.
Tissue samples are collected, sectioned, and mounted on poly-L-lysine coated coverslips. The poly-L-lysine coating facilitates the adhesion of the tissue sample to the coverslip due to the electrostatic interactions between the negatively charged cell membrane and the positively charged surface coating.
Because images are typically taken at 10-20x with objectives that have a depth of field of as little as 1 μm, tissue samples would ideally be sectioned into 1 μm or thinner slices so that the entirety of the sample would be in focus. However, many prepared tissue samples are thicker. Thus, given the depth of field, there will be features which are out of focus in these thicker samples. Our workflow usually involves acquiring images at various focal planes throughout the thickness of the sample and then choosing the focal plane that has the best image quality for each FOV to stitch into the final image.
After the sample is mounted onto the coverslip, the PhenoCycler processing begins.
The sample is treated with a solution containing antibodies for all the biomarkers of interest. The antibodies are all conjugated to oligonucleotides (oligos), which are short single-stranded DNA sequences that act as a barcode or unique identifier for each of the markers. For example, all CD3 antibodies could be conjugated to a ATTCG DNA molecule sequence, while CD20 antibodies might be conjugated to CTACG, and CD67 antibodies are conjugated to ACGAC oligos. The DNA-labeled antibodies bind to the all targets of interest in a single staining step.
Next, the sample is washed with a solution containing the complementary sequence for 3 oligo sequences, each of which is conjugated to a different fluorescent molecule. In our example, we might have a solution containing the complementary strands for CD3 (complementary strand TAAGC), CD20 (complementary strand CATGC), and CD67 (complementary strand TGCTG), which are bound to red, yellow, and green fluorescent tags, respectively. The red, yellow, and green fluorescent molecules are excited and fluoresce, allowing the camera to detect the local expression of CD3, CD20, and CD67.
After detection, the complementary strands are detached from the sample (i.e., stripped), and then the sample is washed with another solution containing another set of three complementary strands that are conjugated to fluorescent molecules. This next set of complementary strands will attach to 3 other targets of interest and allow for their imaging.
The cycle of stripping, washing, and detection will continue until all targets have been imaged. The final file will be an image of the entire tissue section with separate channels for each biomarker.
Multiplex imaging is a spatial biology technique, which means that the biomarker localization is maintained within the tissue section. The spatial information allows us to gain a more detailed understanding of cell-cell and cell-environment interactions, since it is possible to determine where various cell phenotypes are located within the tissue and their spatial relationship to other cell phenotypes and tissue structures. These local interactions play a key role in the progression of many diseases, and have implications for treatment course and disease prognosis.
In contrast, many other tissue analysis techniques, such as flow cytometry, Western blotting, ELISA, and gene sequencing (including single-cell sequencing technologies), require tissue digestion (i.e., breakdown of the tissue structure into smaller parts) to perform the analysis. This allows for whole samples to be analyzed as a “bulk” and generates a lot of information about the tissue sample. These methods can even provide information down to the single-cell level, e.g., in the case of single-cell sequencing, making it possible to detect rare phenotypes. However, because the spatial structure is not preserved, it is difficult to delve deeply into the mechanisms and interactions which define tissue function.
The PhenoCycler platform is unique amongst IHC (immunohistochemistry) and IF (immunofluorescence) methods in that it allows researchers to stain for over 50 biomarkers simultaneously. (Theoretically, the platform can capture the spatial data of hundreds of biomarkers, but we currently only use 51 readily available validated markers for our standard panel.) Other IHC and IF techniques typically allow researchers to stain for only a few biomarkers at once due to steric hindrance of the dyes and/or a limited ability for the human eye to perceive individual stain colors and differentiate between colocalization of stains and another color. With PhenoCycler, while our perception still limits the number of stains that we can see by eye at once, because the individual biomarkers are acquired separately and the image data for each marker is stored in a separate channel, it is easy to view a variety of biomarker combinations on a single tissue slide. Thus, unlike a typical staining method, researchers are not locked into viewing a specific tissue region with just the 4 biomarkers that they stained for initially.