Choosing a Tracer—Section 14.1
To serve as an effective tracer of cell morphology, a fluorescent probe or other detectable molecule must have the capacity for localized introduction into a cell or organelle, as well as for long-term retention within that structure. If used with live cells and tissues, then the tracer should also be biologically inert and nontoxic. When these conditions are satisfied, the fluorescence or other detectable properties of the tracer can be used to track the position of the tracer over time. Fluorescent tracers can be employed to investigate flow in capillaries, to define neuronal cell connectivity and to study dye translocation through gap junctions, as well as to follow cell division, cell lysis or liposome fusion. Furthermore, they can be used to track the movements of labeled cells in culture, tissues or intact organisms. The review of techniques for tracing neuronal pathways by Bohland and co-workers is particularly recommended.
Although the predominant tracers have been fluorescent, not all of the useful tracers are intrinsically detectable. For example, biotin derivatives are widely used as polar tracers, especially in neurons. However, when a biotinylated or haptenylated tracer is used in live cells, detection requires cell fixation and permeabilization to allow access to fluorescent dye– or enzyme-labeled conjugates of avidin and streptavidin (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6) or of antibodies (Anti-Dye and Anti-Hapten Antibodies—Section 7.4).
In many of these tracing applications, the physical dimensions of the tracer molecule are an important consideration. We offer fluorescent tracers ranging in size from small molecules about 1 nm in diameter to polystyrene microspheres up to 15 µm in diameter. This chapter discusses our diverse selection of fluorescent tracers, as well as biotin derivatives and other tracers:
- Cell-permeant cytoplasmic labels (Membrane-Permeant Reactive Tracers—Section 14.2). We have developed several thiol-reactive CellTracker probes (), which yield fluorescent products that are retained in many live cells through several generations and are not transferred to adjacent cells in a population, except possibly by transport through gap junctions. These probes represent a significant breakthrough in the cellular retention of fluorescent dyes and are ideal long-term tracers for transplanted cells or tissues.
- Microinjectable cytoplasmic labels (Polar Tracers—Section 14.3). Polar tracers such as lucifer yellow CH, Cascade Blue hydrazide, the Alexa Fluor hydrazides and biocytin are membrane-impermeant probes that are usually introduced into cells by whole-cell patch clamping, iontophoresis, osmotic lysis of pinocytic vesicles or comparable methods (Techniques for loading molecules into the cytoplasm—Table 14.1). These tracers are commonly used to investigate cell–cell and cell–liposome fusion, as well as membrane permeability and transport through gap junctions or cell uptake during pinocytosis (Probes for Following Receptor Binding and Phagocytosis—Section 16.1).
- Nissl stains for retrograde tracing in neurons (Polar Tracers—Section 14.3). We have developed five fluorescent Nissl stains that not only provide a wide spectrum of fluorescent colors for staining neurons, but are also more sensitive than the conventional dyes.
- Membrane tracers—DiI, DiO, DiD, DiR, DiA, R18, FM 1-43, FM 4-64 and their analogs (Tracers for Membrane Labeling—Section 14.4). Lipophilic carbocyanine, aminostyryl and rhodamine dyes can be introduced into membranes by direct application of a dye crystal onto a cell, by bulk loading from aqueous dispersions prepared from our Vybrant DiI, DiO and DiD cell-labeling solutions or by application of the NeuroTrace DiI, DiO and DiD tissue-labeling pastes. Lateral diffusion of the dye within the membrane eventually stains the entire cell. These probes are widely used for neuroanatomical tracing and long-term assays of cell–cell association. Some of our DiI and DiO analogs exhibit superior solubility and brightness and, in some cases, produce a cell-staining pattern that persists through fixation by aldehyde-based reagents and through acetone permeabilization ().
- Fluorescent and biotinylated dextran conjugates (Fluorescent and Biotinylated Dextrans—Section 14.5). Dextran conjugates are ideal cell-lineage tracers because they are relatively inert, exhibit low toxicity and are retained in cells for long periods. These membrane-impermeant probes are usually loaded into cells by invasive techniques such as microinjection, whole-cell patch clamping, scrape loading, microprojectile bombardment, electroporation or osmotic shock (Techniques for loading molecules into the cytoplasm—Table 14.1). Availability of dextrans in a range of molecular weights makes them useful as size-exclusion probes for determining pore sizes in membranes.
- Fluorescent microspheres (Microspheres and Qdot Nanocrystals for Tracing—Section 14.6). Molecular Probes FluoSpheres and TransFluoSpheres fluorescent microspheres—which contain ~102 to ~1010 fluorescent dyes per bead are intensely fluorescent tracers (). Although other multiply labeled particles such as our BioParticles fluorescent bacteria (Probes for Following Receptor Binding and Phagocytosis—Section 16.1) may be used as tracers, they are often not biologically inert nor are they as physically durable as fluorescent microspheres. These properties make fluorescent beads particularly useful as long-term markers for transplantation studies. Submicron microspheres can be injected into cells or taken up by phagocytosis. Much larger (10–15 µm) beads provide an alternative to radioactive microspheres for determination of organ blood flow, and intermediate-sized (1–5 µm) microspheres are useful for studies that trace inhaled particles.
- Qdot nanocrystal tracers (Microspheres and Qdot Nanocrystals for Tracing—Section 14.6). Qtracker Cell Labeling Kits provide spectrally distinct Qdot nanocrystals that have been functionalized on their surface with polyarginine peptides to facilitate spontaneous uptake by live cells. Qtracker non-targeted quantum dots are designed for small animal in vivo imaging, and especially for studying vascular structure after microinjection. These nanocrystals exhibit intense red or near-infrared fluorescence emission for maximum transmission through tissues and avoidance of background autofluorescence
- Proteins and protein conjugates (Protein Conjugates—Section 14.7). Our fluorescent protein tracers have molecular weights between ~12,000 (cholera toxin subunit B conjugates) and ~240,000 daltons (B- and R-phycoerythrin). Their applications are sometimes similar to those of the fluorescent dextrans; however, unlike the polydisperse dextrans, fluorescent protein tracers have molecular weights that are reasonably well defined.