Fluorescence imaging is a broad field of techniques used in biological sciences to visualize and analyze the molecular interactions and dynamics within cells, tissues, and organisms. By utilizing fluorescence, researchers can track cellular processes, quantify biomarkers, and even observe structures that would otherwise be invisible to traditional imaging methods. With its wide range of applications from biological research to medical diagnostics, fluorescence imaging has become an indispensable tool in laboratories around the world. Let’s take a closer look at a few common types of fluorescence imaging techniques used today.
1. Widefield Fluorescence Microscopy
Widefield fluorescence microscopy, or simply widefield microscopy, is one of the most common forms of fluorescence imaging. This basic technique uses fluorescence to visualize and study biological samples with high spatial resolution. It involves exciting fluorophores within a sample to emit light at specific wavelengths. This emitted light is then captured through a microscope objective onto an image sensor (CCD or sCMOS) to create detailed images and quantify fluorescent signals.
One of the main advantages of widefield fluorescence microscopy is its ability to quickly capture large fields of view (hence the name), making it ideal for observing multiple areas of a sample at once. It offers researchers good XY resolution and very fast temporal resolution (imaging speed or acquisition time). Widefield microscopy is also one of the most affordable types of fluorescence imaging, relatively speaking.
Widefield microscopy is commonly used in fields such as cell biology, neuroscience, and pathology for applications like protein localization, cellular dynamics, and tissue imaging. However, it can be limited by background fluorescence and optical artifacts, which may reduce the image quality, especially in thicker samples. Regardless, widefield fluorescence microscopy remains essential in the world of cell and molecular biology.
With widefield microscopy, in general, researchers trade high resolution for a greater field of view (image area). This is typically done by choosing the objective lens magnification, which also requires a major tradeoff in light collection (sensitivity). However, with a scanning system like the Odyssey® M Imaging System, you can still image larger fields of view without sacrificing resolution. In fact, given its imaging surface of 25 x 18 cm, it can quickly and simultaneously scan up to 12 tissue slides or four 96-well plates at high resolution. With over 6 logs of dynamic range, 19 imaging channels, and 5 µm resolution, the Odyssey M Imager delivers exceptional-quality tissue section, whole slide, cell health, and fluorescent protein data in a matter of minutes.
2. Confocal Microscopy
Confocal microscopy is another widely used type of fluorescence imaging. It improves upon traditional fluorescence microscopy by using a laser to scan the sample point by point to produce high-resolution images. The key advantage of this technique is the ability to eliminate out-of-focus light, which enhances the clarity of the images.
In this technique, a laser is focused on a specimen and the emitted fluorescent light is collected through a pinhole aperture. Only light from the focal plane is allowed to pass through, minimizing the interference from out-of-focus fluorescence and significantly improving the image quality.
Confocal microscopy is the most common optical sectioning technique in fluorescence imaging. It’s also exceptionally versatile in terms of its imaging capabilities. Confocal microscopy is used in biological research to study cellular structures, visualize protein localization, and perform multiplex live-cell, fixed-cell, and tissue imaging. It’s useful for detailed surface analysis in materials science and nanotechnology, as well.
While there are numerous noteworthy advantages of confocal microscopy, it can often pose tremendous challenges to researchers. For instance, these microscopes tend to be incredibly expensive, with some high-end models boasting price tags of several million dollars. Their temporal resolution is also relatively low when compared to widefield microscopes and line-scanners like the Odyssey M Imager. Noteworthy exceptions are spinning disk confocal microscopes, which may provide faster imaging speeds than some line-scanners. Finally, confocal microscopes sacrifice sensitivity by rejecting out-of-focus light, and they can carry a risk of photobleaching or phototoxicity given the amount of light energy focused on the sample. However, this presents a unique opportunity to perform preliminary scans of slides on an Odyssey M Imager to save time and reduce the risks of photobleaching and phototoxicity. The best slides of interest can then be taken to a confocal microscope for further analysis.
3. Multiphoton Fluorescence Microscopy
Multiphoton fluorescence microscopy is an advanced laser scanning technique that uses multiple photons to excite a fluorophore simultaneously. This technique utilizes pulsed near-infrared excitation light to enable optical sectioning, rather than a pinhole aperture as in confocal microscopy. Multiphoton microscopy has the distinct advantage of being able to image hundreds of microns into a sample with minimal photodamage and photobleaching outside the focal plane.
In two-photon microscopy, for example, roughly double the amount of energy is absorbed by each fluorescent molecule as would be in single-photon confocal microscopy. However, excitation typically occurs at longer, infrared wavelengths than in widefield and confocal microscopy, which allows for deeper tissue penetration and less potential for sample damage. This is primarily due to the reduced scattering and absorption of infrared wavelengths.
Multiphoton fluorescence microscopy is commonly used in in vivo imaging, particularly in neuroscience, where it allows researchers to visualize brain structures, monitor neuronal activity, and track cellular interactions in living organisms over long periods. Like confocal microscopy, multiphoton microscopes tend to be extremely expensive and fairly slow compared to widefield microscopes and line scanners like the Odyssey M Imager.
Near-infrared fluorescence can be a practical alternative to multiphoton microscopy. While one major advantage of multiphoton microscopy is the penetration depth of its infrared excitation light, one drawback is that its short-wavelength emission light may get trapped within the tissue sample. However, near-infrared fluorescence (like that of the Odyssey M Imager) employs near-infrared wavelengths for both excitation and emission, enabling more emission light to completely exit the sample and be quantified by the image sensor.
4. Total Internal Reflection Florescence Microscopy (TIRF)
Total internal reflection microscopy (TIRF) is a fluorescence imaging technique that enables the visualization of phenomena occurring near a surface. By exploiting the principle of total internal reflection, TIRF microscopy allows for high-resolution imaging of the events at or near the cell membrane, without the interference from the bulk of the sample.
When light is directed at a sample at a certain angle, it reflects off the interface between two media, creating an evanescent wave that penetrates only a few hundred nanometers into the sample. This results in fluorescence from molecules located near the surface, providing excellent contrast for surface-bound molecules.
TIRF is helpful for studying processes like membrane dynamics, protein-protein interactions at the cell surface, and the movement of receptors on cell membranes. It is widely used in cell biology and cancer research because it offers very good z-axis resolution, enables single-molecule insights into real-time mechanisms, and can overcome the background fluorescence common in fluid environments.
With TIRF, its advantage is also its weakness. While this technique can carefully collect light from a very thin volume above a surface, the downside is that you can only look at things within that area. When looking at cells, for instance, the researcher would be limited to molecules touching the surface. In other words, there is no 3D imaging with TIRF.
LICORbio Supports Fluorescence Imaging
Fluorescence imaging has become indispensable in modern biological research. This field offers a range of techniques to explore the molecular and cellular world with high precision. From basic widefield microscopy to cutting-edge confocal imaging, each technique provides unique advantages suited to specific research applications.
Fluorescence is where LICORbio truly shines. We pioneered quantitative near-infrared fluorescence imaging using IRDye® Infrared Dyes with the original Odyssey Imaging System over 20 years ago. Though NIR fluorescence is still fairly uncommon in this field and industry, we’re wholeheartedly committed to educating researchers about the undeniable benefits of NIR fluorescence (reduced autofluorescence, high sensitivity, stable signals, higher SNR, etc.).
We’re determined to be your constant in biological fluorescence imaging. Whether you’re interested in quantifying immunocytochemistry assays, tissue samples, fluorescent proteins, or even Western blots, our instruments quickly deliver exceptional-quality fluorescence data without detector saturation at a fraction of the cost of confocal microscopes. We would love the opportunity to learn more about your lab’s research and unique imaging needs—please don’t hesitate to reach out with any questions, concerns, or feedback today.
References
1. Combs C. A. (2010). Fluorescence microscopy: a concise guide to current imaging methods. Current protocols in neuroscience, Chapter 2, Unit2.1. doi.org/10.1002/0471142301.ns0201s50
2. Jensen, E.C. (2012), Types of Imaging, Part 2: An Overview of Fluorescence Microscopy. Anat Rec, 295: 1621-1627. doi.org/10.1002/ar.22548
3. Axelrod, D. (2001). Total internal reflection fluorescence microscopy. Methods in Cell Biology, 58, 1-23. doi.org/10.1034/j.1600-0854.2001.21104.x