Seeing is believing, and for many biologists, what they want to see is fluorescent labels through a microscope. Fluorescence microscopy, which makes it possible to visualize fluorescent proteins or dyes at the cellular and subcellular level, has become a workhorse of modern biology.
The concept of fluorescence, in which a molecule absorbs light of one wavelength and then emits light of a longer wavelength, was first described by British scientist Sir George Stokes in 1852. But the technology to envision that process at high resolution, and to use it for illuminating cellular processes, has come a long way since the 1910s, when German physicists designed early fluorescence microscopes. Scientists started using fluorescent dyes to stain tissues during the 1930s, and fluorescently tagged antibodies came into use in 1942. The discovery in 1961 of the green fluorescent protein from jellyfish helped push the field along, because it gave scientists the ability to label a variety of proteins with fluorescence.
Over past decades, scientists have developed many methods based on fluorescent microscopy to image not only where molecules are, but also how they move and interact.
Scientists have many options to choose from in purchasing a fluorescence microscope, and many factors to consider before they decide. This buyer’s guide discusses how the instruments work, what they can be used for and what to think about when selecting a system for your research needs.
The basics
Cut down to its most basic level, fluorescence microscopy involves illuminating a sample with certain wavelengths of the light spectrum, and then detecting the fluorophores emitted. So it all starts with the light source. The trick is to balance the intensity of the incoming, or excitation light.
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The stronger it is, the more returning, or emission, fluorescence you’ll get from the sample. But intense excitation light can also damage the sample—even killing live cells or tissue—or cause the fluorophores in it to “bleach” and become dimmer with time.
Epifluorescence microscopy
Microscopists have three main choices: a white lamp, LEDs or lasers. Arc lamps and LEDs illuminate the entire sample in what are called epifluorescence microscopes. Lamps, the most common light source, contain gases such as vaporized mercury and discharge white light. The microscope must then include one or more filters that narrow the spectrum down to the desired wavelengths—the blue-green part of the spectrum, for example, to excite green fluorescent protein in the sample. These microscopes bathe the entire sample in light at once, so they can photobleach it. The bulbs take a bit of time to warm up before using the microscope, and they typically last for a couple hundred hours before they fade and burn out. As they fade, they provide different intensity, making it difficult to precisely compare experiments performed even a few weeks apart. The cost of lamps runs $100 or more.
LEDs are another, newer option that is becoming more popular. In this case, each LED produces light in a limited part of the spectrum, so less filtering is necessary than when working with white lamps. Compared to arc lamps, LEDs require less power, last for thousands of hours without fading and don’t require time to warm up or cool off. They also bathe the entire sample in light at once; however, they’re typically not as intense as bulbs and thus have less potential to cause photobleaching. They are cheaper than lamps, costing in the range of a few dollars.
The excitation light from the light source bounces off a dichroic mirror to direct it toward the sample. There it excites fluorophores, which then emit their own, longer wavelength. This light is then collected by the objective lens, which magnifies the image and is crucial for determining resolution and image quality. A given objective will have not only a given magnification (10x, 100x, etc.) but also a number for numerical aperture. The higher the numerical aperture, the higher the resolution and brightness of the resulting image. Some objectives have additional lenses to minimize aberrations in the image.
Another factor that influences the image is the medium the light passes through on its way to that objective.
If the light passes through air first, then hits the glass of the objective lens, some of the light will be scattered at that boundary, because air and glass have different refractive indices. Immersion media, such as oil, have a refractive index to match the objective and minimize light scatter.
The light emitted by the sample’s fluorophores is much fainter than the excitation light, which is also reflected by the sample. That’s why the dichroic mirror, which reflects the excitation light toward the sample, is important. It allows the wavelength of the emitted light through, on its way into the microscope, but blocks the strong excitation wavelengths. The emitted light also passes through an emission filter, which selects for only the output wavelength of the fluorophore under study. These excitation and emission filters must match any fluorophores you wish to use.
The signal can then pass to eyepieces, so you can view the sample. But of course most scientists want to record what they see. The main camera options are an electron-multiplying charge-coupled device (EMCCD) or a complementary metal-oxide semiconductor (CMOS). Both are sensitive digital cameras that convert incoming light into an electrical signal. EMCCDs have long been the standard and are considered more sensitive, particularly in low-light conditions, but CMOS cameras are improving. They may offer faster acquisition of images, larger fields of view and better resolution.
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Confocal microscopy
Hitting the entire sample with incoming light, as epifluorescence microscopes do, means that fluorophores above and below your desired plane of view emit light, limiting resolution. To eliminate that challenge, confocal microscopes use laser light. Using mirrors, they can focus the light on a single point, so fluorophores elsewhere in the sample are not excited. On the emission end, there is also a screen with a pinhole to restrict light from the sample to only the desired focal point.
In laser-scanning confocal microscopy, the machine scans that point of excitation light across a plane and provides a crisper image than epifluorescence typically offers. Varying the level of the plane results in a “stack” of images at different depths, giving one an idea of the three-dimensional arrangement of the sample.
Laser-scanning confocal microscopy is just one version of confocal microscopy. In contrast, spinning disk and programmable array microscopes shine the exciting light—from a laser or epifluorescence source—through multiple pinholes to scan the image. The advantage is that these systems can acquire an image faster, which could be crucial for imaging activities in living cells. However, laser-scanning confocal microscopes typically offer the highest resolution.
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Like lamps, lasers require some warm-up time before imaging, and their intensity eventually fades. But like LEDs, lasers last a long time, perhaps a few thousand hours. Lasers are the most expensive light source, running thousands of dollars.
To detect and amplify the weak emitted light, confocal microscopes use a photomultiplier tube. Incoming photons first hit a light-sensitive photocathode, which absorbs a photon and emits an electron. That electron then interacts with a series of electrodes, each of which emits more electrons than it absorbs. The result is amplification of the signal.
Table: Fluorescence Microscopes
EtalumaLumascope 850 Live Cell Fluorescence MicroscopeNoNoYesEtalumaLumascope 820 Live Cell Fluorescence MicroscopeNoNoYes
Applications
“Almost everybody is using fluorescence now,” says Scott Robinson, manager of the microscopy suite at the Beckman Institute for Advanced Science and Technology at the University of Illinois at Urbana-Champaign. “There’s a really wide range of things that you would want to do.”
“The big one is protein localization,” says Sarah Swanson, a botanist and director of the Newcomb Imaging Center at the University of Wisconsin-Madison. “You can attach your GFP to your favorite protein of interest and look to see where it goes in the living cell.” Or, by linking the GFP to a promoter of interest, one can observe how expression levels vary over time.
Antibodies hooked to fluorophores can also light up a protein of interest. Similarly, fluorescent labels can help scientists see the shapes of structures such as cells and organelles.
Although one can certainly fix tissues and look at them any time, live-cell imaging has also become quite popular. Researchers can follow the proteins or structures of interest over time, enabling them to observe steady-state activity or see what happens when they perturb the system with drugs or other influencers.
For long-term live imaging, a chamber that keeps cells comfortable on the microscope stage, with temperature control and the proper levels of oxygen and carbon dioxide, is crucial.
Microscopy has also found its way into high-throughput screening applications, with automated microscopes that can observe numerous cells over a time course while scientists are engaged elsewhere. This enables researchers to check whether libraries of drugs or diverse treatments affect the location or activities of proteins and to quantify their results. For these types of experiments, the challenge becomes processing and analyzing all the images.
Visit Biocompare's product directory to search for the over 2 million options available for fluorescently-tagged antibodies that can be used in various confocal microscopy systems.
Co-localization
Because light comes in so many colors, scientists can look at multiple proteins or dyes in the same sample, as long as their excitation and emission wavelengths differ. This makes it possible to determine if proteins or membranes of interest are co-localizing—indicating they inhabit the same space and may interact—or not.
Another way to look at the interaction between two labeled molecules in a sample is Förster Resonance Energy Transfer (FRET). When two fluorophores are within nanometers of each other, emission from one can excite fluorescence from the other. The emission wavelengths of the “donor” fluorophore must overlap the excitation spectrum of the “acceptor.” Thus, by exciting the donor with the microscope light, one should be able to observe emission from the acceptor. In this way, scientists can determine that two molecules, or even two parts of the same protein, are near each other.
Investigating ions
Petra Rohrbach, a parasitologist at McGill University in Montreal, says her laser-scanning confocal microscope is integral to nearly all her experiments. She’s interested in how malaria parasites process drugs, pumping them into the acidic digestive vacuole of the parasites and using live-cell imaging to watch the processing. For example, she adds a fluorochrome that the parasite processes as if it were a toxic drug, and she can observe the processing under the microscope.
She also uses dyes that indicate concentrations of calcium, potential across a membrane or pH. For example, by loading the parasites with a fluorochrome that changes color with pH change, she can monitor the acidity of the digestive vacuole. If its pH creeps towards neutral, the digestive enzymes won’t work. That kills the parasite, probably in part because it can’t digest its food, says Rohrbach.
Swanson, too, uses ion-sensitive dyes in her studies of how plants respond to stressors. For example, when a plant is bent, and stressed mechanically, calcium levels spike. Using dyes that change their fluorescence—up, down or to a different color—when bound by calcium, she can observe what’s happening in live plant tissue. Genetically encoded ion indicators can perform the same function.
Laser tricks
The lasers in some confocal microscopes are good for more than just imaging. Researchers also use them to assess how the molecules they’re looking at move around. With fluorescence recovery after photobleaching (FRAP), scientists take advantage of the fact that too intense a light can destroy a fluorophore. They wipe out the fluorophores in one part of their field of view and then watch to see when fluorescence returns. The original fluorophores in that area are gone, so the returning fluorescence must be related to new fluorophores migrating in from elsewhere. This enables researchers to watch the dynamics of diffusion or active trafficking.
A variant of this is FLIP, which stands for fluorescence loss in photobleaching. FLIP helps scientists determine which areas of a sample are connected and allowing materials to diffuse between them. The researchers repeatedly photobleach one spot, destroying any fluorophores as soon as they come in. Throughout the places interconnected to that spot, the overall fluorescence slowly diminishes. In that way, biologists can determine the boundaries of a region.
For many labs, fluorescence imaging is the lifeblood of their research. “It’s everything we do, practically,” says Amy Gladfelter of the University of North Carolina at Chapel Hill. She studies how the cytosol and plasma membrane are organized and uses live-cell imaging, FRAP and other techniques to understand how proteins and organelles are arranged and moving around. She also uses advanced techniques that focus on how quickly proteins move throughout the cell, and at single molecules in cells or in vitro.
Shopping points
There’s a lot to check on when trialing a potential purchase. One concern is ease of use. Microscopes can and do come with lots of bells and whistles, but of course those features are only useful if you need their functionality. Those with fairly basic needs may prefer a cell-imaging system. These simplified microscopes are smaller than their traditional, full-feature counterparts and are easy to use, with a touch-screen interface. Some include a light shield, so you don’t need to be in a darkroom to image your cells. These can be a particular boon for work with students, who aren’t intimidated by the equipment and can focus on the images.
With microscopes simple or fancy, scientists should consider what modes of imaging they’ll need.
In addition to fluorescence in multiple colors, researchers typically want to see the cells with transmitted white light, for example via phase-contrast imaging. That’s always useful, says Rohrbach, to know exactly where the cell’s boundaries are. The number of colors you can use—for example, the number of laser lines the microscope can interface with—could also be an important factor for those using many different fluorophores.
Resolution is a key feature to consider, because it determines what kinds of features you’ll be able to distinguish. Field of view, which is determined by both the microscope and objective in use, also matters. For example, scientists looking at neurons may need a large enough field to see where all the dendrites and axons reach to, but researchers looking at bacteria might be satisfied with a smaller field of view. Another possibility, Robinson adds, is that some microscope software can automatically tile multiple images together to create a large view from several small fields.
Another choice buyers need to make is whether to use an upright microscope—in which the objective faces down at a sample sitting on the stage—or an inverted microscope, in which the objective faces upward. Inverted systems are commonly used for studies of cultured cells, because the broadest part of the cell is that touching the surface of the dish. Of course, if the sample is large—such as an animal for intravital microscopy—only an upright will do, Robinson says.
You’ll also need to decide if you want eyepieces to look through or just a digital image of your sample. Some microscopes lack the eyepieces, Rohrbach points out.
If you can’t afford every feature you want right away, look for a microscope that can be updated after it’s in your lab—for example, by adding an incubator for live-cell imaging—Robinson suggests. At the same time, look for a company with technical support in your area, so you can get help quickly when you need it.
Summary
Although the basics of light microscopy—photons headed into the sample and coming back out, guided by optics—are hundreds of years old, fluorescence microscopy continues to evolve. Scientists and engineers are developing ever-cleverer ways to improve features such as sensitivity and resolution. For example, the emergence of super-resolution techniques means scientists can now distinguish cellular features within fewer than 200 nanometers.
Even as manufacturers update their offerings with the latest bells and whistles, they are also creating simplified, affordable products that help make fluorescence microscopy accessible to labs. Those who simply want to observe a protein or two may be able to get away with a convenient cell-imaging system. For those with more advanced applications in mind—such as live-cell imaging, FRAP or FRET—microscopes such as a confocal system offer more options.
Scientists have plenty of options in buying a microscope. The key question to ask yourself is: “What am I going to be using this for?” That should give you a clear idea of the features you need.
Editor note: We want to acknowledge and thank Ms. Veronika Kortisova-Descamps, global product manager at Bio-Rad Laboratories for her insightful discussions and contributions to this article.
Image: Shutterstock Images