FLUORESCENCE IMAGING/ NANOPARTICLE TRACKING ANALYSIS: Fluorescent labeling 101

BOB CARR

Fluorescent labeling can be used in any situation where it is necessary to distinguish a particular subset of particles within a complex background. For instance, it may be useful for labeling drug-delivery nanoparticles; small biological particles such as exosomes and microvesicles; or virus and virus-like particles (VLPs). Fluorescence is an important tool for life scientists, a critical support for microscopy techniques from confocal to multiphoton, and also for techniques such as flow cytometry and nanoparticle tracking analysis (see sidebar, "NTA and fluorescence").

Choosing a fluorophore

The appropriate fluorescence labeling that a scientist uses depends on what he or she wishes to label and the goals of the research. In the case of nanoparticles for drug delivery, it is possible to fluorescently tag or load particles and distinguish them within a complex medium. For instance, mucin is highly complex, making it impossible to see or track drug-delivery nanoparticles against this high background. By fluorescently tagging them, it is possible to use a specially selected filter to remove the scattered light from the mucin and allow only the longer wavelength emission from the fluorescently labeled particles to be imaged and tracked.

Fluorescent labeling can be very specific, or rather general. For example, antibody labeling can specifically target a known marker on a particle of interest within a mixed sample. This type of labeling is often useful when studying small biological particles such as exosomes or microvesicles. For labeling of more general constituents, it is possible to use a dye with a specific affinity—for lipids, proteins, or sugars, for instance—and thus target these within a sample. Sometimes the dye is "switched on" only when it becomes attached to the molecule of interest; for example, some lipid membrane dyes exhibit substantially enhanced fluorescence within a lipid environment when compared to an aqueous environment.

Choosing an appropriate fluorophore means evaluating a number of criteria, including excitation maximum, emission maximum, Stokes shift, and fluorescence emission. For instance, the excitation maximum of the fluorophore should be close to the wavelength of the laser (excitation source) fitted to the instrument. Also, the fluorophore you choose should have an emission maximum that is longer than the wavelength cutoff point of the filter you are using (see Table 1 for laser wavelengths and standard filters for NTA). The greater the difference between a fluorophore's excitation maximum and emission maximum—that is, its Stokes shift—the better the results will be. Finally, the fluorophore you choose should have fluorescence emission as bright and photo-stable as possible. Table 2 shows rates and number of fluorophores using these criteria, and lists the applications for which they are best suited.

Tips for success

Achieving the best possible results with fluorescent labeling sometimes requires finessing. These guidelines may help:

1. Try varying the concentration of fluorescent label to sample particles until the best signal-to-noise ratio is achieved. If using primary and secondary antibodies rather than directly labeling, try varying the ratios of these, too.

2. Adding large quantities of fluorescent label will not necessarily result in better labeling. Unbound label in free solution becomes a significant problem, raising the background intensity and drowning out the signal from labeled particles. Sometimes too much fluorescent label can result in quenching of the fluorescent signal.

3. Rather than beginning at the optimal concentration range, it is better to label particles at a high concentration and dilute immediately prior to analysis/imaging. This approach may also help to dilute out any unbound fluorescent label that is left in the sample, and to increase the likelihood (rate) of interaction between fluorophore and target. For NTA, try overnight incubation of the sample with fluorescent label; a 30-minute staining protocol that works for flow cytometry will not necessarily work for NTA.

Bob Carr, Ph.D., is the inventor of nanoparticle tracking analysis (NTA) technology and founder of NanoSight Ltd., Amesbury, England; e-mail: [email protected]; www.nanosight.com.