Tips on Purchasing and Using Fluorescence Microscopes

It all began in 1845, when astronomer Sir Frederik William Herschel described the first example of fluorescence. Little did he know, but his discovery started a revolution in microscopy. More than half a century later, physicist Oskar Heimstädt constructed the first working fluorescence microscope. Today, life science and biomedical labs around the world use this technology. Fluorescence microscopes now come in so many versions that it takes a guide to pick the one with the right features for a particular application. Here, we’ll explore how to purchase and use today’s sophisticated fluorescence imaging technology.

Fluorescence features to find

The first step in picking a powerful fluorescence microscope depends on what will be imaged. As Louise Bertrand,product performance manager, widefield, North America—Life Science Division at Leica Microsystems, says, “One of the first things to consider when selecting a fluorescence research microscope is the type of specimen you want to examine.”

Image: Mouse retina was fixed and stained by following reagents: anti-CD31 antibody (green) for endothelia cells; IsoB4 (red) for blood vessels; and microglia anti-GFAP antibody (blue) for astrocytes. Credit: Jeremy Burton, PhD, and Jiyeon Lee, PhD, Genentech, South San Francisco, USA. Imaged by Olga Davydenko, PhD (Leica).

For histological sections or fixed cells mounted on a glass slide, Bertrand says, “an upright microscope will be the choice.” Such a fluorescence scope can also be used for specimens “such as C. elegans maintained on opaque Nematode Growth Medium Agar in petri dishes or for special techniques, such as patch-clamp experiments used to investigate ion channels in detail and recording electrical activity of cells,” she adds.

Scientists turn to inverted fluorescence microscopes for living cells, spheroids, organoid clusters, or whole organs maintained in larger cell-culture vessels filled with culture media. Stereomicroscopes or macroscopes are options for screening, sorting, sample manipulation, and documentation.

The objective plays a crucial role in resolution. A high numerical aperture objective increases resolution to distinguish details. Ultimately, Bertrand says, “The structures to be observed will determine if high resolution is more important than long working distance or vice versa.”

Picking the light

The source of illuminating light also impacts the results. “LED illumination systems have become the light source of choice,” says Kathy Lindsley, an application specialist at Life Sciences, Olympus Scientific Solutions. “However, care must be taken to select a light source that fits the application.”

For routine fluorescence microscopy, most available LED systems serve the purpose. In some cases, such as far-red imaging, a specific LED system will be required.

The LED system also impacts the speed and quality of imaging. For example, Lindsley points out that “broad light sources are typically used with single-channel filter sets,” which produce the best image quality, but the slowest acquisition speeds. Scientists can obtain faster imaging with fast-switching LED illumination, but “the improvement in speed may be at the cost of increased bleed-through,” Lindsley says.

Image: Mouse monoclonal antibodies label endogenous proteins in a section of the hippocampus of a mouse brain. Credit: Imaged with ZEISS Apotome, thank you to Colleen Manning at The Jim Trimmer Lab, UC Davis

Automation can also increase imaging throughput. “If your aim is increased proficiency, consider systems that offer automatic carrier detection, artificial intelligence, label-free sample finding, and hardware autofocus,” says Colleen Manning, NA product marketing manager, widefield and automation at Zeiss Research Microscopy Solutions. “These can reduce your start time to image, while increasing throughput and ease of use.”

Perfecting the process

Even the best fluorescence microscope’s output depends on how it’s used. Let’s explore the top tips from these experts.

“Quantifiable and representative data is key,” says Manning. “Always understand how your data is acquired and processed.” She adds that non-uniform treatment of pixels during acquisition (variable pixel dwell times) or processing with adaptive masks or some deconvolution methods result in non-quantitative data. Plus, opaque processing methodologies or employing adaptive or heterogenous processing of pixels, “yield a non-quantitative manipulated image that misrepresents the science,” Manning notes. “These methods can peddle a pretty picture, but this is both dangerous and misleading.”

Lindsley’s top tips include selecting fluorophores that are spaced as far apart as possible to reduce bleed-through. Plus, she encourages scientists to reduce sources of auto fluorescence if possible. “For example, when conducting live cell–imaging experiments, replace the growth medium with a phenol red–free medium,” Lindsley says. “Immersion oil and mounting medium can also be sources of autofluorescence.” So, immersion oil and mounting medium made for fluorescence applications should be selected.

The technology should also accommodate the samples in fluorescence microscopy. As an example, Lindsley notes: “When imaging thicker specimens, such as tissue sections, matching the refractive index of the sample, mounting medium, and immersion oil will reduce spherical aberration.”

Keeping the image consistent also matters. Otherwise, comparisons cannot be drawn. For instance, Bertrand says, “Ensure that the fluorophore selected is bright and stable.” If some fluorophores are bleaching quicker than others, this decreases the intensity of their fluorescence signal. Also, when performing intensity quantification, scientists should make sure that the parameters, such as light intensity, are constant. Some fluorescence illumination light sources can decrease in intensity over time. This should be considered and accounted for in experiments. It also speaks to the need to update or service the lighting source as needed.

Like all scientific work, researchers should use controls in fluorescence imaging. As Bertrand points out: “In indirect immunofluorescence, a secondary antibody–only control should be performed, following the same staining protocol without the addition of a primary antibody.”

To get the most useful images, scientists must select the best fluorescence microscope, lighting system, fluorophores, and so on. These must all be matched to the sample being imaged. Then, following the top tips for performing the imaging plays a fundamental role in the results. The combination of the technology and how it’s used determines the reproducibility and accuracy of the outcome. Although in 1845 Herschel could not have known the transition in imaging that his work would trigger, it changed the world of what can be seen.