Visualizing the Invisible: From Animalcules to Advanced Imaging

By: Inssaf Darraz

In the 17th century, Antoni van Leeuwenhoek, a Dutch merchant, looked through his homemade microscope and discovered a hidden world of “animalcules” in a drop of water. These “little animals”, later identified as bacteria, sparked a revolution in microbiology¹. The simple yet clear terminology of Van Leeuwenhoek laid the foundation for the hidden world of microbes and reshaped our understanding of life. Today, Dutch scientists are still studying infectious diseases, such as COVID-19 and tuberculosis, through the use of microscopy. Here we describe some of the microscopy techniques used to study microorganisms causing disease, highlighting their crucial role in modern biomedical research.

Little animalcules

The tiny organisms, called “animalcules” by van Leeuwenhoek, have been identified as bacteria. These often long-bodied microorganisms serve a crucial role in our existence, playing key roles in our gut microbiota and offering protection². However, there are also pathogenic bacteria that, when they invade our cells, can lead to various diseases and even death³. 

Microscopy is essential for visualizing bacteria and other microorganisms to facilitate microbiological research of host-pathogen interactions. Among various microscopic techniques, the light microscope (LM) stands out as one of the most important methods⁴. It emerged in the late 19th century and uses visible light to magnify tiny objects, including bacteria⁵. Here’s how it works: when light passes through the sample, different glass lenses work together to focus and amplify the image. Through the objective lens and eyepiece, a total magnification of 400 to 1,000 times can be achieved, sufficient to visualize bacteria and cellular components^4,5.

One specific LM method provides a fascinating way to visualize specific proteins or components in bacteria and cells more clearly. Fluorescence microscopy (FM) uses special molecules, like antibodies or genetically modified proteins, that glow when they are hit with a certain wavelength of light. They resemble “glow-in-the-dark”, attached to the cells or proteins of interest. FM enables scientists to localize and quantify cells, as demonstrated in a Dutch university study that used FM to locate pathogenic mycobacteria expressing a specific enzyme in zebrafish models⁶. The findings shed new light on how this enzyme aids mycobacterium survival.

Highlighting the practical importance of this technique, imagine the challenge of finding a single cell from a lung biopsy. With FM, it is like searching for a tiny green-pea seed in a water tank of 1×1 m. This would be the comparison when both a cell and a 1×1 mm tissue block are magnified 1000 times. Just as a pea seed is challenging to find in a water tank, the cell remains hidden until brought to light through FM (Figure 1). 

But what about viruses and the bacterial interior?

A light microscope cannot resolve the finer details of microbial components, such as the intricate structures of the bacterial interior, or most viruses smaller than 0.2 μm. Super-resolution FM and transmission electron microscopy (TEM) provide solutions to this limitation^7,8,9. TEM, which emerged in the early 20th century, uses an electron beam that facilitates high resolution down to 0.1 nm and magnifications up to 1,500,000 times^5,9.

To illustrate this magnification, imagine the search for a virus as the search for an orange in a crowded city of 1×1 km (Figure 2). A complicating factor of TEM is that it requires sections as thin as 60 nm, even thinner than a single virus particle, to minimize electron interference. This makes searching for a virus similar to looking for an orange in a section of the city, while finding a cross-section of a mycobacterium is like searching for a trash can within that same section (Figure 2). This highlights how challenging research on these small microorganisms could be. However, it also shows the potential of EM to overcome these challenges.

TEM research covers ultrastructural analysis, including internal structures, such as DNA, and external components, such as bacterial cell walls. A Dutch study focused on examining ultrastructural changes in mycobacteria after antibiotic treatment. Interestingly, DNA usually appears as chains of small segments within the bacteria, but after antibiotic treatment it appeared in a single enlarged cluster¹⁰. This physiological response observed with TEM highlights its capability to reveal detailed ultrastructural changes. Furthermore, insights gained from this study have recently contributed to the development of a potential new drug targeting mycobacteria¹¹.

But how does TEM make DNA condensation visible? The detailed strucures observed in TEM images are a result of careful sample preparation techniques. Contrast in electron micrographs is achieved through electron scattering using heavy metal staining. In this process, electrons interact with the atomic nuclei of the sample, with higher atomic numbers leading to more electron scattering and thus higher contrast⁵. However, most biological elements, such as nitrogen and oxygen, have relatively low atomic numbers, making it easy for electrons to pass through them.  Using heavy metals that selectively bind to proteins enhances the contrast in the images. Lipids do not absorb these metals, so they appear as white areas on electron micrographs (see cell wall of M. marinum in Figure 2) – a feature known as electron-lucent.

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Look, the pathogen has escaped!

Preparative techniques have been developed to improve the visibility of microstructures, such as microbial components. Among these is Immuno Electron Microscopy (immuno-EM) which uses, like in FM, antibodies to detect specific proteins but is now tagged to small gold particles (Figure 3)⁹. With this approach, viruses and bacteria can be located in infected samples and smaller proteins can be visualized. 

Recent research on Mycobacterium tuberculosis, the bacterium that causes tuberculosis and is known to escape from macrophages, used immuno-EM. By using gold-labeled antibodies directed against Lysosomal-associated membrane protein 1 (LAMP-1), a key membrane protein on lysosomes, the scientists were able to visualize the precise locations of these waste-disposing organelles¹². This allowed researchers to determine whether the bacteria were inside or outside these organelles. This method effectively revealed the subcellular localization of bacteria in macrophages.

Illustrative images of Mycobacterium marinum, genetically similar to M. tuberculosis, show this escape mechanism. In these cross sections, gold particles appear as black dots, membranes as white lines, and the cytosol as a gray area surrounding the bacteria. Figure 3A shows a bacterium marked by gold particles at the membrane, located within the compartment where engulfed particles are digested (phagolysosome). In contrast, figure 3B shows four free bacteria in the cytosol, indicating these have escaped from the phagolysosome. These images show the dynamic interactions between bacteria and the immune system and provide valuable insights into disease processes.

Functional and structural information

Can we combine the strengths of both light and electron microscopy? Efforts in the late 20th century led to correlative light and electron microscopy (CLEM), an innovative technique that integrates the capabilities of both imaging methods¹³. It works as follows: first, LM is used to capture areas of interest in the sample. Then, the same sample is imaged with EM, focusing on the regions previously identified. Using specialized software, the images from both techniques are merged, resulting in an overlayed image that combines information from both methods¹⁴.

An illustrative example of this method can be seen in a Dutch study that investigated the localization of proteins in SARS-CoV-2 infected lung tissue¹⁵. Figure 4 shows an EM image of an infected cell with small (white) electron-lucent circles outside the nucleus. At the same time, the same cell image is displayed in FM with the nucleus in blue, lipids labeled in red and viral proteins in green. By overlaying these two images, a CLEM image is created, highlighting the red-labeled lipids within the cell’s translucent compartments. This technique revealed a viral-induced structure that may be associated with the extensive immune response in COVID-19. Thus, CLEM combines the structural details obtained from EM with the functional insights from FM, offering a more comprehensive perspective.

Pitfalls of EM

EM is valuable for studying infectious diseases, but it has its limitations. With TEM, inaccurate sample preparation can cause clumps or artifacts visible in the images, requiring expert operators for accurate sample processing. Additionally, EM requires a high vacuum to prevent electron scattering with air molecules, which also damages or destroys live cells. Therefore, samples must be fixed before imaging. Another disadvantage is the inability to capture 3D images, as it only provides 2D projections of 3D objects. To address this, 3D electron tomography can be used, which reconstructs detailed 3D images from multiple 2D TEM images taken from different angles¹⁶.

A lasting legacy

Microscopy has played an important role in scientific progress, from Antoni van Leeuwenhoek’s observations of “animalcules” to the development of more advanced techniques FM, TEM, and CLEM. This method of observing biological components invisible to the naked eye has led to a better understanding of pathogenic mechanisms and the intricate details of infected cells, providing insights into their structure as well as their function. Looking ahead, emerging microscopy techniques hold promise for further discoveries and will continue to be essential for Dutch research and global scientific progress.

About the author

Further reading

1. Davis IM. Antoni van Leeuwenhoek and measuring the invisible: The context of 16th and 17th century micrometry. Studies in History and Philosophy of Science Part A. 2020;83. ↩︎
2. Zhang YJ, Li S, Gan RY, Zhou T, Xu DP, Li H Bin. Impacts of gut bacteria on human health and diseases. Vol. 16, International Journal of Molecular Sciences. 2015. ↩︎
3. Ikuta KS, Swetschinski LR, Aguilar GR, Sharara F, Mestrovic T, Gray AP, et al. Global mortality associated with 33 bacterial pathogens in 2019: a systematic analysis for the Global Burden of Disease Study 2019. The Lancet. 2022;400(10369). ↩︎
4. Murphy DB, Davidson MW. Fundamentals of Light Microscopy and Electronic Imaging: Second Edition. Fundamentals of Light Microscopy and Electronic Imaging: Second Edition. 2012. ↩︎
5. Rohde M. Microscopy. In: Methods in Microbiology. 2011. p. 61–100. ↩︎
6. Boot M, Jim KK, Liu T, Commandeur S, Lu P, Verboom T, et al. A fluorescence-based reporter for monitoring expression of mycobacterial cytochrome bd in response to antibacterials and during infection. Sci Rep. 2017;7(1). ↩︎
7. Robb NC. Virus morphology: Insights from super-resolution fluorescence microscopy. Biochim Biophys Acta Mol Basis Dis. 2022;1868(4). ↩︎
8. Carsten A, Wolters M, Aepfelbacher M. Super-resolution fluorescence microscopy for investigating bacterial cell biology. Molecular Microbiology. John Wiley and Sons Inc; 2023. ↩︎
9. Curry A, Appleton H, Dowsett B. Application of transmission electron microscopy to the clinical study of viral and bacterial infections: Present and future. Vol. 37, Micron. 2006. ↩︎
10. Scutigliani EM, Scholl ER, Grootemaat AE, Khanal S, Kochan JA, Krawczyk PM, et al. Interfering with DNA decondensation as a strategy against mycobacteria. Front Microbiol. 2018;9(SEP). ↩︎
11. van der Niet S, Green KD, Schimmel IM, de Bakker J, Lodder B, Reits EA, et al. Zafirlukast induces DNA condensation and has bactericidal effect on replicating Mycobacterium abscessus. Antimicrob Agents Chemother. 2024;68(8). ↩︎
12. van der Wel N, Hava D, Houben D, Fluitsma D, van Zon M, Pierson J, et al. M. tuberculosis and M. leprae Translocate from the Phagolysosome to the Cytosol in Myeloid Cells. Cell. 2007;129(7). ↩︎
13. van den Dries K, Fransen J, Cambi A. Fluorescence CLEM in biology: historic developments and current super-resolution applications. Vol. 596, FEBS Letters. 2022. ↩︎
14. Santarella-Mellwig R, Haselmann U, Schieber NL, Walther P, Schwab Y, Antony C, et al. Correlative light electron microscopy (CLEM) for tracking and imaging viral protein associated structures in cryo-immobilized cells. Journal of Visualized Experiments. 2018;2018(139). ↩︎
15. Grootemaat AE, van der Niet S, Scholl ER, Roos E, Schurink B, Bugiani M, et al. Lipid and Nucleocapsid N-Protein Accumulation in COVID-19 Patient Lung and Infected Cells. Microbiol Spectr. 2022;10(1). ↩︎
16. Ercius P, Alaidi O, Rames MJ, Ren G. Electron Tomography: A Three-Dimensional Analytic Tool for Hard and Soft Materials Research. Adv Mater. 2015;27(38). ↩︎