Manual Spatial Proteomics: Fusing Cycles into OME-TIF was Never Easier

Spatial Proteomics was named the Nature Method of the Year 2024. One predominant method is Cyclic Immunofluorescence (CycIF). Here, a tissue sample is stained and scanned during a first cycle. Next, the stain is bleached away, and the sample is restained for other antigens before being scanned again. This process can be repeated in multiple cycles. Typically, a nuclear stain like DAPI is used in each cycle to facilitate high-accuracy alignment of the cycles. Ultimately, the resulting low-plex scans — one per cycle — are aligned (aka co-registered) and fused into a single high-plex scan, e.g., in OME-TIFF format.

This process is automated by instruments such as PhenoCycler by Akoya Biosciences, Comet by Lunaphore, MACSima by Miltenyi Biotec, and others (see free download of List of Commercial Spatial Biology Instruments, Assays & Softwares). These instruments come with software that carries out the alignment automatically. The resulting slides can be directly opened and analyzed using MIKAIA^®. For more, refer to our Overview of MIKAIA’s Spatial Analysis Apps for Multiplexed Immunofluorescence Slides.

However, if no such instruments are available, the process can also be carried out manually by using a standard low-plex fluorescent scanner like the Zeiss AxioScan or the Olympus VS100. In this case, the last step of fusing the low-plexes into a single high-plex has been a technical challenge, but it is now easily possible with MIKAIA’s new Slide Align module, powered by the HistokatFusion technology developed by Fraunhofer MEVIS.

Here is a step-by-step guide on how to align 16 markers scanned in three cycles.

Load slides and start Slide Align Wizard

Load the slides into MIKAIA^® by either dragging them into the “Slides” workspace or by adding the enclosing folder as a workspace folder to include all contained slides. In the toolbar of the “Slides” panel, click the “Align…” button to start the alignment wizard:

Structure of image sources

Grouping slides into cases: Composing alignment jobs

Each cycle is represented as a single multi-channel file in the job compositor page, which is designed to group files into cases. Here, all three files belong to the same case. Since the case name is not coded into the file names, the easiest approach is to click the “All to same case” button and enter a name (e.g., “case A”). If the case name is included in the file path, it can be automatically extracted by entering delimiters that inform how to extract the Case ID (and, in the case of brightfield scans, also the stain name), from the source paths.

The three rows need to be included by checking the box in the first column (in case you have further slides in the workspace that are not part of any alignment job). You also need to select a reference cycle needs. The DAPI channel is automatically pre-selected as the alignment channel. Optionally, you can select multiple channels. The resulting transform and deformation field will then be applied to the remaining channels accordingly.

Click the “Proceed” button to move to the next wizard step.

Output channel configuration when creating high-plexes

It is recognized that at least one case contains fluorescence data, which brings up the “Channel Setup” page. (When aligning brightfield sections, this page is skipped.) On this page, you have the option to edit marker names, colors, and order. Here, we deselected all but the first cycle’s DAPI channel, ensuring that only this channel will be included in the generated output OME-TIF high-plex file.

Export configuration

Next comes the general configuration page, where the output folder, naming scheme, and resolution can be configured. In the case of CycIF, the “<Stain>” placeholder is ignored in the output naming scheme, since all channels result in the same output file.

Reviewing alignment result before exporting

Once the alignment has been computed, BEFORE (“original”) and AFTER (“aligned”) thumbnails are generated and can be inspected by clicking on the “Open” button with the lens-icon. This will bring up the “Inspect alignment results” dialog . On the left half, it shows the channels/stains in carousel mode (one at a time), whereas on the right half, all images are displayed in a gallery, side by side. When hovering an image with the mouse pointer, press the SPACE key to zoom in to the maximum available preview resolution. Press SPACE again to zoom out again.

If the automatic alignment has failed,you can use this dialog to manually place three landmarks in both the reference image and in the channel that did not auto-align. After both sets of three landmarks have been placed, click the “Re-align” button to repeat the alignment. If the alignment still does not yield satisfactory results, uncheck the “Include this stain” checkbox to skip this alignment pair during the following alignment.

Exporting aligned & fused OME-TIF

Viewing and analyzing generated High-plex OME-TIF in MIKAIA^®

High-plex OME-TIF generated in the previous steps can now be viewed and analyzed.

For analysis options, please visit Overview of MIKAIA’s Spatial Analysis Apps for Multiplexed Immunofluorescence Slides.

MIKAIA^® Slide Align is powered by award-winning science: HistokatFusion by Fraunhofer MEVIS

Fore more details, visit the Fraunhofer MEVIS’s HistokatFusion website.

Trying out MIKAIA Slide Align

In the free MIKAIA^® lite edition, the Slide Align can be tried out, but the maximum output resolution is limited to 5 microns/px. Get in touch (mikaia@iis.fraunhofer.de) if you want to try out an eval version of MIKAIA studio, incl. Slide Align export at full resolution.

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Data source

The scans are kindly provided by DAKEWE Medical with permission to use them for this article.

Funding

This MIKAIA^® extension has been kindly made possible thanks to venture capital provided by the Fraunhofer Future Foundation (Fraunhofer Zukunfsstiftung). Project: “Histology AI Author”, consortium: Fraunhofer IIS & Fraunhofer MEVIS