Since its initial development in the early 20th century (1), immunofluorescence has now become a quintessential visual tool for research scientists to convey their discoveries. Immunofluorescence images are arguably the most gorgeous images in science but creating them is a daunting task. Few scientists and research technicians are trained in how to create a publication quality image for immunofluorescence – or in what constitutes an excellent one. This guide is by no means a comprehensive guide on fluorescence microscopy, and it is impossible to cover everything in a blog post. However, I want to tell you about some beginner tips that I learned when I was a graduate research student working in a research laboratory specializing in widefield and confocal fluorescence microscopy.
Tip 1: Focus on the most important information that you are trying to convey.
When I was a research student, I was frustrated by how some journal publications have very poor immunofluorescence images. There are many reasons for a poor fluorescence image: incorrect focus, incorrect exposure, high autofluorescence, weak signals, inappropriate microscopy method, and many others. When one of these problems is sufficiently severe, this can negatively affect the signal and the information that the image is attempting to convey to the reader. Even very good journals sometimes have publications that are full of poor images. For example, 16S rRNA Fluorescence in-situ Hybridization (FISH) is frequently employed to visualize and identify bacterial cells in the lumen or mucosa of the gastrointestinal tissues. High quality 16S rRNA FISH images should clearly demonstrate individual bacterial cells that have hybridized with the probe as well as morphology of the bacterial cells. For superb 16S rRNA FISH images, the reader is encouraged to review the Microbiology Society article, Fluorescence In Situ Hybridisation: Making Microbes Glow to See How They Organise.(2) In contrast, notice the difference in quality of the 16S rRNA FISH images when compared to figure 6 in the article, Helicobacter bilis Infection Alters Mucosal Bacteria and Modulates Colitis Development in Defined Microbiota Mice(3). Here, the signal appears blurry, and no individual cells can be seen. The morphology of the bacteria is completely lost.
My advice is to use the best method that will convey what you are trying to show in the image to convince your readers. For the best 16S rRNA FISH images, you should consider using confocal microscopy. In my opinion, when the poor quality of fluorescence images is not addressed, it certainly affects the credibility of the publication. If confocal microscopy will give you a more convincing image and if it is the best tool to convey the most important information that you are trying to show in a publication, then use it to acquire your image. Why would you settle for less and compromise the quality and credibility of the publication? To determine the most suitable microscopy method for your needs, the beginner is encouraged to read the publication, Fluorescence microscopy (4).
Tip 2: Trash in. Trash out. Make sure your assay quality is as good as possible.
This is perhaps the most self-explanatory tip. This idea is true for many aspects of histotechnology, and it is also true for creating a publication quality fluorescence image. Taking a good image depends on whether the original quality of the assay is good or bad. If the quality of the assay is bad, no amount of processing or tricks will improve the quality of the image. For example, when we have a very weak signal intensity located in an area of very high autofluorescence, it is incredibly difficult if not impossible to adjust during image processing. There are many factors that we should consider for the best assay outcome. As technicians, we are constantly troubleshooting these assays. While troubleshooting immunofluorescence can be a little laborious, it is always important that we have the best assay quality to facilitate our image acquisition and processing steps.
Tip 3: Be cognizant of nuances during image acquisition and processing.
Generally, when you are trying to show differences in protein levels between samples, it is best practice to use the same capture parameters. This is to ensure that the signal intensity is true, which is important for image analysis. This is not that critical if you are just taking representative images, and adjustments can be made to improve the quality of the images depending on the signal strength of each sample.
Commercially available photo-editing programs, such as Adobe Photoshop, are great for image processing. However, using Adobe Photoshop requires a steep learning curve. It is also expensive if your institution does not subscribe to Adobe Creative Cloud. Alternatively, you can use a free software for image processing like FIJI, an enhanced version of ImageJ2. Finally, the majority of immunofluorescence images have primarily utilized two colors: red and green. My advice is to change the red color into magenta to accommodate readers with color blindness. Some reputable journals are starting to make this a requirement. For a more comprehensive guide on creating a publication quality immunofluorescence image, I really like this article from Nature, Best bractices and tools for reporting reproducible fluorescence microscopy methods (5). Always aim for the best quality! Nature’s guidelines are as stringent as far as scientific publications go.
Tip 4. Always learn from the world’s greatest fluorescent images.
In my opinion, the Nikon Small World competition is the world’s most prestigious immunofluorescence competition. Here, you can find an extensive list of some of the world’s most beautiful fluorescent images. I think in order to improve our own skills, we should analyze these images and figure out why they are high quality. For example color choices, level of exposure, background, the level of autofluorescence and intensity, are all factors that we should keep in mind when acquiring an image.
Written by: Ka Lam Nguyen, MS, BS, HTL(ASCP)
References:
(1). Coons AH. The beginnings of immunofluorescence. The Journal of Immunology. 1961 Nov 1;87(5):499-503.
(2). https://microbiologysociety.org/publication/past-issues/imaging/article/fluorescence-in-situ-hybridisation-making-microbes-glow-to-see-how-they-organise.html
(3). Atherly T, Mosher C, Wang C, Hostetter J, Proctor A, Brand MW, Phillips GJ, Wannemuehler M, Jergens AE. Helicobacter bilis infection alters mucosal bacteria and modulates colitis development in defined microbiota mice. Inflammatory bowel diseases. 2016 Nov 1;22(11):2571-81.
(4). Sanderson MJ, Smith I, Parker I, Bootman MD. Fluorescence microscopy. Cold Spring Harbor Protocols. 2014 Oct;2014(10):pdb-top071795.
(5). Montero Llopis P, Senft RA, Ross-Elliott TJ, Stephansky R, Keeley DP, Koshar P, Marqués G, Gao YS, Carlson BR, Pengo T, Sanders MA. Best practices and tools for reporting reproducible fluorescence microscopy methods. Nature Methods. 2021 Dec;18(12):1463-76.
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