Spatial Biology Services for FFPE and Fresh Frozen Tissue: A Practical Guide

Designing a spatial biology study comes with a lot of decisions, starting with your sample type and quickly expanding into workflows, data outputs, and analysis needs. Whether you’re working with archived clinical samples or collected tissue, the right setup can make the difference between usable data and missed insights.

This guide walks through what to expect from spatial biology services, how to think about FFPE vs fresh frozen tissue, and what to look for in a partner who can support your study end to end.

What Do Spatial Biology Services Include?

At a high level, spatial biology combines molecular profiling with tissue context. You’re not just measuring what’s present, but where it’s happening.

Most projects today fall into a few core categories:

• Spatial transcriptomics: mapping gene expression across a tissue section

• Spatial imaging via fluorescent in situ hybridization (FISH)

• Spatial proteomics: visualizing protein distribution and abundance

• Integrated workflows: combining RNA and protein data for a more complete view

How Does Sample Collection Impact Spatial Biology?

There are many variables that can significantly impact downstream sample quality. It’s essential to prepare thoroughly to ensure optimal outcomes.

Key considerations for sample collection:

• Minimize the time from excision to freezing/fixation (ischemic time)

• Freezing tissue directly in liquid nitrogen can make it unsuitable for most spatial biology platforms

• Ensure that the type of fixative is acceptable for downstream protocols

• Different tissue types have different optimal fixation times

• Plan for global shipping logistics for FFPE vs. frozen samples

For biobanked samples, much of this information is often unavailable thus it is essential to conduct rigorous QC processes before proceeding with any downstream experiments.

Understanding Your Sample Type: FFPE vs Fresh Frozen

The first decisions in planning a spatial biology project hinge on your starting material. Both FFPE and fresh frozen tissue are widely used but each brings its own pros and cons. Depending on which sample type you’re working with, different considerations are necessary during project planning, QC, library prep,and imaging.

FFPE tissue (formalin-fixed, paraffin-embedded)

FFPE is often the default for clinical and archival studies. Formalin-fixed, paraffin-embedded tissue is used for long-term, stable storage, and makes it easier to handle and section the tissue in later analysis.

Why researchers use it:

• Broad availability from biobanks and clinical repositories

• Long-term stability at room temperature

• Global shipping logistics are simplified for FFPE vs. frozen samples

What to plan for:

• RNA fragmentation and degradation. The fixation process is rough on RNA, impacting length and integrity in FFPE samples. Often, researchers need to use low-input RNA sequencing protocols to make the most of remaining RNA content.

• Reliance on optimized QC and library prep workflows. Quality can vary drastically, even throughout the same FFPE sample. Traditional QC metrics like RIN are often not reliable indicators of sample suitability for spatial analysis. A DV200 score <30% is a good indicator that the tissue should not be used for spatial analysis. The DV200 is a measure of the percentage of RNA that is longer than 200bp.

With the right approach, FFPE tissue sample analysis can still deliver high-quality spatial insights. It just requires thorough RNA and morphological QC before attempting any spatial experiments.

Fresh frozen tissue

Fresh frozen samples are typically preferred for methods that require high RNA integrity, such as those using a polyA capture approach. Higher quality preservation of proteins and nucleic acids give you a higher signal for transcriptomic and proteomic detection methods.

Why researchers use it:

• Higher RNA integrity

• Strong performance in spatial transcriptomics workflows

• Can be used for a wide range of other applications that are intolerant of fixation

What to plan for:

• More complex storage and handling requirements. To prepare fresh frozen tissue samples, they should be snap frozen in isopentane immediately post-excision. Failure to use isopentane can lead to ice crystal formation and damage to the morphology, rendering the sample unsuitable for spatial biology analysis by most platforms. Visium 3`HD is a notable exception; their protocol does not require isopentane, just snap freezing. Shipping and storing these samples also present unique challenges. They require deep-freeze storage and rapid, dry-ice shipment to remain frozen. These conditions can be difficult to maintain, particularly if you don’t have the lab infrastructure to support it.

• Less availability for certain clinical cohorts. Many research disciplines can’t rely on large-scale batch collection of fresh frozen tissues. Collecting and shipping frozen tissues across the globe presents a significant set of challenges versus the room temperature stability of FFPE tissues. Retrospective studies often rely on biobanks and repositories, and those resources lean heavily on FFPE sample storage.

What Does an End-to-End Spatial Biology Workflow Look Like?

Once your samples are determined, the next step is execution. In a past blog post, Choosing a Spatial Biology Platform, we went over seven key questions to ask yourself when selecting the instrument, protocol, and other project specs. Here’s what a strong spatial biology workflow typically includes:

Sample QC

• RNA quality assessment. Robust RNA QC is essential before proceeding with any spatial biology project-see above for details.

• Tissue integrity checks. You’re looking for signs that the tissue is in good condition and is likely to yield valuable data. This includes the presence of intact nuclei and distinct tissue regions, clear cell boundaries, and minimal tearing or folding. Tissue with good morphology is more likely to yield robust data and less likely to detach during the often extensive spatial biology workflows.

Histology

The majority of spatial biology platforms have very specific requirements for tissue preparation and placement.

• Slide types. Some platforms require specialty slides to be purchased from the manufacturer.

• Tissue placement. Each platform has guidelines for where tissue can and cannot be placed.

• Tissue staining. Psomagen requires an H&E stained image and/or slide to assess morphology

• Additional slide processing. Visium HD and 3`HD both require an RNase-free H&E slide to enter the workflow

• Scrolls/curls for RNA QC. Scrolls/curls are required by Psomagen for RNA extraction and QC preceding any spatial biology work.

• Post-run staining. Some platforms accommodate post-run H&E or even immunofluorescence staining

Wetlab workflow and data collection

Each type of platform will have a different series of steps in the lab based on the chemistry and the format of the data output. That said, there are some commonalities between all of them given the fundamental goal of spatial biology to collect cell-specific transcript information while retaining each cells’ location within the tissue. Note that the order of these steps may also vary by platform.

• RNA location must be captured. The location of each mRNA transcript must be hybridized to a barcode that denotes its location within the tissue

• Cell segmentation. The boundaries of all cells must be annotated.

• Transcripts must be identified. Individual transcripts must be identified and quantified

• Data overlaid. Transcript identities and locations must be overlaid onto the map of cells within the tissue.

Both imaging and transcriptomic sequencing platforms share the above steps in common. Imagers do not require any downstream library preparation or sequencing. Imagers do all the identification, quantitation, and overlay onto cells within their on-instrument workflows.

Library preparation & assay selection

• FFPE-optimized protocols. This includes steps like additional heat or chemical treatments to reverse crosslinking caused by formaldehyde preservation. Because RNA is fragmented, libraries may be designed for short inserts or probe-based panels.

• Fresh frozen preparations. It is critical that the laboratory make advanced preparations to work with frozen tissue samples. Dry ice will be needed to keep the slides frozen to prevent RNA degradation, and the workflow steps need to be executed without any delay.

• Resolution requirements. Assay choice is heavily influenced by whether the study requires single-cell or subcellular resolution versus region- or spot-level profiling. For assays that require true single-cell resolution,defined as having the x,y, z coordinates for each transcript inside of a single cell, imaging-based technologies are the platforms of choice. Note that many of these platforms are often lower in plex than sequencing-based technologies. If discovery is the goal of the experiment, sequencing-based platforms often deliver whole transcriptome data but lack the ability to provide true single-cell resolution as they often give only x, y coordinates for the transcripts.

Sequencing

This is only applicable for platforms that utilize a sequencing output. Imaging-based platforms do not require any library preparation or sequencing.

• Platform- and assay-specific requirements. Spatial assays come with distinct sequencing recommendations, including read length, paired-end vs single-end reads, and depth per capture area. Aligning sequencing parameters with the assay ensures optimal data quality and avoids under- or over-sequencing.

Bioinformatics & reporting

• Spatial mapping and visualization. Sequencing data is mapped back to its physical location on the tissue, visualizing changes in gene expression within the context of spatial cell arrangement. High-quality imaging alignment is critical to ensure accurate spatial interpretation.

• Clustering and annotation. Computational methods group similar cells based on their expression profiles, followed by identification using known markers and/or reference datasets. This step translates raw data into biologically meaningful insights, identifying cell types and specific tissue sub-regions.

• Data integration and interpretation. Spatial data is often combined with bulk or single-cell RNA-sequencing data insights to strengthen conclusions. Integration helps validate findings by overlaying a deep dataset, which in turn provides a more accurate understanding of the biological system in question.

• Clear, interpretable outputs. Deliverables include raw sequencing data, but are also expandable to include more advanced analyses such as expression, visualizations, cell identification, and summary reports. The goal is to provide results that are accessible and actionable, especially for teams without dedicated bioinformatics expertise.

• Quality control and reporting transparency. Final outputs will include QC metrics (e.g., read depth, gene counts, mapping efficiency) to help assess data reliability and guide downstream interpretation.

What to Look for in a Spatial Biology Service Provider

A few factors tend to separate a smooth project from a frustrating one. When vetting spatial service providers, ask if they have:

• Experience working with both FFPE and fresh frozen tissue

• Clear, usable data deliverables

• Flexibility to adapt to your study design

• Access to scientific support when questions come up

In practice, this is what determines whether you’re only receiving data or actually getting answers.

A well-designed spatial biology project starts with the right sample and succeeds with the right workflow behind it.

That includes:

• Diligent processing and handling of FFPE and fresh frozen tissue

• Integrated QC and optimized library prep

• Thoughtful experimental design

• Clear, actionable data outputs

At Psomagen, our approach is built around that end-to-end experience, supporting researchers from study design through data delivery, with workflows optimized for both archival and fresh-frozen tissue types. If you’re planning a spatial biology study and want to talk through your options, our team is always happy to help you map out the right approach. Reach out here to map your next project with our experts.