Beauty through the scope

What do you enjoy about microscopy? 

Microscopy has always been very cool to me; the human brain is amazing at finding patterns. Finding animals in the shapes of clouds, faces of celebrities in your morning toast, constellations in the sky, etc., it's a phenomenon called pareidolia. Molecular images offer the same kind of opportunity, I see stained glass windows in meiotic spindles, spider webs in lung tissue, and rivers in thymus sections.

Why is the image notable to your work?

The image I submitted is a section of chicken thymus that was later used for spatial transcriptomic analysis. To our knowledge, this is the first time that anyone has spatially analyzed the transcriptome of avian thymus. It is shaping up to be a really interesting project. Our hope is that by fully characterizing the avian immune system we can develop better tools and treatments to mitigate the damage done by these outbreaks. 

From a technical aspect, I imagine there’s a lot of choices that go into deciding what scope to use, what staining to employ and how to prepare a sample to examine. Can you walk me through what was involved in obtaining this image? 

Interestingly enough, some of the difficulty in obtaining this image comes from the fact that there are not very many choices when it comes to processing for the spatial transcriptomic platform. The protocol dictates the quality and thickness of the tissue, the reagents you can use, the imaging resolution, and more. We're lucky that the Advanced Light Microscopy Core and the Genomics Technology Core have the equipment and instrumentation necessary to perform these analyses. Tissues for this analysis get frozen into a block of what's called O.C.T. Compound at -80 degrees Celsius, then we cut a thin slice of the tissue in a machine called a cryostat which keeps the tissue at approximately -20 degrees Celsius. Once a thin section is obtained without rolling or tearing, the tissue is placed on a slide and stored back at -80 degrees Celsius. 

The rest of the analysis protocol occurs within a 48-hour period: the slide gets thawed, fixed, stained, and imaged all within approximately 2 hours. The Ti2 Eclipse microscope at the ALMC has a widefield scanning color camera and allows you to quickly scan the entire tissue and then select the region that you'd like to image at a higher resolution. Because we need a high-resolution image for this analysis, the microscope takes many images with a small field of view and then 'stitches' them together to create one large image at high resolution. This type of imaging used to take days but now takes 15 to 20 minutes per tissue. 

What did you find most challenging in creating the image? 

I think that any histopathologist, which I certainly am not, would tell you that the thymus and lymph nodes are a difficult tissue to section. They have a very uniquely high cellular density and fat content which makes it especially difficult to find the correct cutting temperature. More than that, it's very difficult to preserve the biological structure of the tissue while taking such a 'thick' section of a very dense tissue. While a histopathologist might take a 5- or 7-micron section of this tissue, the spatial platform we're using specifies a 10-micron thick section. However, this tissue doesn't just need to be a pretty picture; it needs to contain enough RNA to capture and sequence later!

Getting an image that’s scientifically relevant/useful and one that’s beautiful seem like separate challenges. How did you approach getting the most interesting image while balancing its usefulness?

The analysis of spatial transcriptomics allows us to get an idea of the specific RNA transcripts being expressed by individual cells at a specific moment in time; part of the nuance of this analysis is that in order to have a nice spatial analysis of the RNA, you need to have a really nice looking tissue. The margin for error is very narrow. I submitted this image specifically not because it was difficult, but because to me, it looked very much like a topographic map. It was like I was seeing the larger reality reflected in the molecular reality of something like the topography of the amazon within the avian thymus, much like the Helix Nebula looks like a human eye.  

What do you enjoy about microscopy? What did you learn from the process?

I enjoy microscopy because it lets me directly see the biology I study. It turns complex cellular interactions into something visible and meaningful. I learned that a strong microscopy image needs both technical precision and biological insight. The most effective images are the ones that are beautiful because they are scientifically true.

Why is the image notable to your work?

Staining with neuronal and mitochondrial markers allows us to visualize the expression patterns of these structures, compare them with healthy mouse optic nerve, and assess neurodegenerative changes and mitochondrial accumulation alongside our other datasets.

From a technical aspect, I imagine there’s a lot of choices that go into deciding what scope to use, what staining to employ and how to prepare a sample to examine. Can you walk me through what was involved in obtaining this image? 

To obtain this image, we prepared cross-sections of mouse optic nerve tissue and stained them with DAPI, neurofilament, GFAP, and COX IV to label nuclei, axons, astrocytes, and mitochondria, respectively. We then used confocal fluorescence microscopy at about 20X to capture a field that balanced tissue detail with overall nerve organization, allowing us to visualize expression patterns and compare them with healthy optic nerve tissue

What did you find most challenging in creating the image? 

The hardest part was balancing all four stains, so the image was both clear and informative. In a dense tissue like the optic nerve, it takes careful preparation and imaging to avoid weak signal or overlap.

Getting an image that’s scientifically relevant/useful and one that’s beautiful seems like separate challenges. How did you approach getting the most interesting image while balancing its usefulness?

I focused first on making the image scientifically accurate and representative. Its beauty came naturally from the organization of the tissue and the contrast between the markers.

What do you enjoy about microscopy?

I enjoy being able to directly visualize the tumor microenvironment-seeing how immune cells and structures are spatially organized makes the biology much more tangible and insightful than abstract data alone.

Why is the image notable to your work?

It reveals the spatial organization of immune infiltration in the tumor, helping us determine how SPIKE alters the microenvironment to promote effective anti-tumor responses. 

This image is a multiplex immunofluorescence of a mouse ovarian tumor cryosection, where specific markers highlight dendritic cell networks (CD21/CD35, CD11c) and vascular structures (Meca-79), allowing us to visualize immune architecture in situ and highlighting immune cell networks that might show some mechanism underlying the effect of SPIKE

What did you learn from the process?

I learned how critical proper sample preparation and staining are for preserving tissue architecture and getting reliable signals. It also taught me how to interpret spatial patterns in context, which is essential for understanding how treatments like SPIKE impact immune infiltration and tumor structure.

Working with ALMC’s Senior Research Specialist Katherine Rodriguez-Lukey, we determined the best approach. Here Rodriguez-Lukey describes the process. 

Sample Preparation:
Fresh frozen tumor tissue embedded in Optimal Cutting Temperature compound (OCT) was selected to preserve native tissue morphology while enabling rapid processing. This approach is particularly advantageous for tumor samples, as it minimizes degradation and maintains antigen integrity for downstream immunofluorescence analysis. Tissue sections were cut at a thickness of 10 µm, which is standard for immunofluorescence and provides an optimal balance between structural preservation and sufficient resolution to visualize individual cells of interest.

Staining Strategy:
A multiplex immunofluorescence approach was employed using three markers: MECA-79 (rat IgM), CD21/35 (rat IgG2a), and CD11c (Armenian hamster). A critical consideration in the staining design was determining whether each target antigen was intracellular or membrane-bound, as this dictates permeabilization requirements and antibody accessibility. Additionally, the presence of two primary antibodies derived from rat required careful optimization to prevent cross-reactivity and ensure specific binding of secondary antibodies to their intended targets. This added complexity necessitated thoughtful selection of antibody combinations and blocking strategies.

Imaging and Microscope Selection:
The choice of microscope was driven by the need to balance spatial resolution with the ability to image large tissue areas. A Nikon fluorescence microscope platform was selected due to its high-quality air objectives, which provide sufficient resolution for cellular-level analysis without the constraints of immersion media. Furthermore, the system is equipped with a highly sensitive camera and supports tile-scanning (image stitching), enabling the acquisition of large, high-resolution composite images across extended regions of the tissue.

Image Processing and Visualization:
For the final image, lookup tables (pseudo-colors) were adjusted to enhance visual contrast and interpretability. Color assignments were selected not only to create a more visually impactful representation of the data, but also to ensure accessibility, including compatibility with common forms of color vision deficiency. This step is important for accurately conveying multiplex information while maintaining clarity for a broad audience.

What did you find most challenging in creating the image? 

The most challenging aspect of generating this image was the optimization of the multiplex immunofluorescence staining protocol. The combination of markers introduced complexity, particularly due to the use of primary antibodies derived from the same host species, which increased the risk of cross-reactivity and non-specific binding. As a result, significant effort was dedicated to optimizing key parameters, including permeabilization conditions, blocking strategies, fixation times, and washing steps.

A major focus was achieving an optimal signal-to-noise ratio, ensuring that specific staining was both strong and clearly distinguishable from background fluorescence. Additionally, careful coordination was required to ensure compatibility among antibodies so that each marker could be reliably detected without interference. This iterative optimization process was essential to obtain high-quality, interpretable multiplex imaging data.

Getting an image that’s scientifically relevant/useful and one that’s beautiful seem like separate challenges. How did you approach getting the most interesting image while balancing its usefulness?

Balancing scientific relevance with visual impact was an intentional part of the workflow rather than a separate step. The primary focus throughout was to ensure that the image accurately represented the underlying biology. This began with careful sample preparation and optimization of the multiplex immunofluorescence protocol to achieve high specificity and a strong signal-to-noise ratio, ensuring that each marker reliably reflected its target cell population.

During image acquisition, parameters were strictly controlled to preserve data integrity and ensure compatibility with downstream processing, including deconvolution and quantitative analysis. Exposure settings were carefully optimized to avoid signal saturation, as overexposure can introduce artifacts and compromise the accuracy of the data. Maintaining consistent and conservative acquisition settings was critical to generating reproducible and reliable images.

At the same time, imaging parameters were selected to capture both detail and context. The use of a widefield system with tile-scanning capabilities allowed us to image large tissue regions while maintaining sufficient resolution to resolve individual cellular features. This approach ensured that the image was not only informative at the cellular level but also provided spatial insight into tissue organization.

During post-processing, care was taken to apply minimal and controlled adjustments. Overprocessing was deliberately avoided, as excessive manipulation (including aggressive deconvolution or background subtraction) can introduce artifacts that do not reflect the true biological signal. The final image therefore represents a faithful enhancement of the raw data rather than a reconstruction.

Finally, visual presentation was refined through thoughtful selection of lookup tables (pseudo-colors). The color scheme was adjusted from a traditional green–orange–red palette to a cyan–magenta–yellow combination on a black background. This change improved contrast between channels, enhanced interpretability, and increased accessibility for viewers with color vision deficiencies. Overall, aesthetic adjustments were applied in a way that supports clarity while preserving scientific accuracy.

What do you enjoy about microscopy? What did you learn from the process?

For me, microscopy or photomicrography is a skill set that require a lot of patience and persistence. Although it can be challenging at times, but I find it very rewarding when it helps answer complex scientific questions. At the same time, it probably also satisfies my artistic inclinations, as I enjoy choosing color combinations and look for meaningful patterns within the data. Altogether, the process is both intellectually and visually engaging. 

Why is the image notable to your work?

We have developed a three-dimensional co-culture system, an assembloid model of endometriosis associated pain, that provides a unique platform to investigate pain signaling. The image demonstrates that this assembloid model recapitulates a neuronal-epithelial-stromal crosstalk. 

From a technical aspect, I imagine there’s a lot of choices that go into deciding what scope to use, what staining to employ and how to prepare a sample to examine. Can you walk me through what was involved in obtaining this image?

These endometriosis assembloids can typically grow up to ~300 µm in size. A whole mount immunostaining protocol was optimized with optical clearing to visualize tissues labeled with cytokeratin-8 (endometrial epithelial cells marker), beta-tubulin III (neuronal marker), and synaptotagmin-1 (a calcium sensor marker associated with neuronal activity). High resolution 3D optical sectioning was performed using a diode white laser Leica TCS SP8 confocal microscope at the Advanced Light Microscopy Core at Mizzou, allowing minimal crosstalk and phototoxicity. Acquired Images were processed and reconstructed in 3D using IMARIS, an Image processing software for publication-ready visualization. 

What did you find most challenging in creating the image? 

While technological advancements in staining protocols and microscopy techniques have improved imaging capabilities, each experiment presents a unique challenge, from sample preparation to the acquisition of scientifically meaningful image and these steps are highly interdependent. I found it challenging to maintain the tissue integrity during the staining process, yet it was essential for generating images that have the most scientific value and aesthetically compelling.

Getting an image that’s scientifically relevant and one that’s beautiful seem like separate challenges. How did you approach getting the most interesting image while balancing its usefulness?

My approach begins with a clear understanding of hypothesis and scientific question that is being addressed. While the image should represent a specific data point, it should also be aesthetically appealing, first to me and then to the reviewers and broader audience to enhance engagement. I remain extremely careful while adjusting contrast, 3D rendering parameters to avoid introducing artifacts and to ensure that representation does not compromise data integrity.

What do you enjoy about microscopy? What did you learn from the process?

What I enjoy most about microscopy is the ability to directly visualize complex biological processes that are otherwise abstract. It transforms molecular and cellular mechanisms into something tangible and intuitive. From this process, I learned the importance of patience and precision—small technical details can significantly influence the final outcome. I also gained a deeper appreciation for how cellular architecture and spatial organization contribute to function, particularly in early embryonic development where subtle structural differences can have profound biological consequences.

Why is the image notable to your work?

The image is notable because it visually represents how structural organization within the embryo supports accurate cell division and developmental progression, which are fundamental determinants of embryo quality and fertility.

From a technical aspect… can you walk me through what was involved in obtaining this image?

Obtaining this image required careful optimization at multiple steps. Embryos were fixed using 4% paraformaldehyde to preserve cellular architecture, followed by permeabilization with Triton X-100 to allow antibody access. Blocking conditions were optimized to minimize background while preserving signal specificity. Microtubules were labeled using an anti–α-tubulin antibody conjugated to Alexa Fluor 488, while nuclei were stained with DAPI. Imaging was performed using high-resolution confocal microscopy, with careful adjustment of laser intensity, z-stack acquisition, and optical sectioning to capture the three-dimensional organization of the embryo. The image was further processed using Huygens deconvolution to improve resolution and signal clarity. Post-acquisition processing involved minimal additional adjustments to enhance visualization while preserving biological accuracy.

What did you find most challenging in creating the image?

One of the main challenges was preserving the delicate three-dimensional structure of the embryo during fixation and staining. Early embryos are highly sensitive, and even slight variations in handling can disrupt cytoskeletal organization. Additionally, achieving a balance between signal intensity and background noise required multiple rounds of optimization, particularly for uniform staining across all blastomeres. Capturing the full spatial complexity of microtubule networks in a relatively large, multicellular embryo also required careful imaging settings and reconstruction.

Balancing scientific usefulness and visual appeal—how did you approach this?

I approached this by prioritizing biological accuracy first—ensuring that the staining faithfully represents microtubule organization and nuclear positioning. Once that was achieved, I optimized imaging parameters such as contrast, depth, and color separation to enhance clarity and visual impact. The inherent symmetry and organization of the embryo naturally lend themselves to visually striking patterns, so the goal was to highlight these features without introducing artifacts or over-processing. In this way, the image remains both scientifically informative and visually engaging.