Microspheres and Qdot Nanocrystals for Tracing—Section 14.6

Page Contents

• Fluorescent Microspheres for Regional Blood Flow Studies
• Particle and Cell Tracking with Fluorescent Microspheres
• Qdot Nanocrystal Tracers
• Ordering Information

FluoSpheres and TransFluoSpheres polystyrene microspheres satisfy several prerequisites of ideal long-term biological tracers. Because the dyes in our microspheres are incorporated throughout the microsphere rather than just on its surface, the fluorescence output per microsphere is significantly greater than that obtained from protein or dextran conjugates (Fluorescein equivalents in Molecular Probes yellow-green-fluorescent FluoSpheres beads—Table 6.6) and is relatively immune to photobleaching and other environment-dependent effects. FluoSpheres and TransFluoSpheres microspheres are also biologically inert and physically durable, and they are available with a large number of uniform sizes and surface properties. Furthermore, their spectral properties can be freely manipulated during manufacture without altering their surface properties. See Microspheres—Section 6.5 for an extensive discussion of the properties of our FluoSpheres and TransFluoSpheres polystyrene beads.

Measuring the effect of various interventions on regional blood flow is an important quantitative application of fluorescent microspheres. Relatively large radiolabeled microspheres (10–15 µm in diameter) have long been used for regional blood flow studies in tissues and organs. Fluorescent microspheres, however, have been shown to be superior to radioactive microspheres in chronic blood flow measurements. In most cases, the microspheres are injected at desired locations in the circulatory system and eventually lodge in the capillaries, where they can later be counted in dissected tissue sections. To eliminate the hazards, expense and disposal problems of the radiolabeled microspheres, researchers have turned to fluorescent and colored microspheres for measuring myocardial and cortical blood flow. Blood flow measurements using fluorescent microspheres in other organs, including the kidney, lung, spleen, adrenal glands, bone and teeth, are equally feasible. Our 0.2 µm FluoSpheres microspheres have been used to trace new blood vessel development, which is important for the study of tumor angiogenesis and microvascular continuity.

FluoSpheres Microspheres for Blood Flow Determination

We have used Molecular Probes fluorescent dye technology to produce a range of intensely fluorescent FluoSpheres microspheres specifically designed for regional blood flow determination (FluoSpheres microspheres for blood flow determination—Table 14.5). Regional blood flow studies using our FluoSpheres polystyrene microspheres for blood flow determination (Figure 14.6.1) have been validated in several side-by-side comparisons with radioactively labeled microspheres. The two methods exhibit equivalent detection sensitivity, and excellent correlation between the flow measurements has been reported. In addition, techniques have been developed to extract the microspheres and the fluorescent dyes they contain from tissue samples, allowing blood flow quantitation to be performed using readily available instrumentation such as spectrofluorometers and fluorescence microplate readers. Up to seven simultaneously circulating tracers can be discriminated based on uncompensated fluorescent color differences. With spectral spillover corrections, the number of simultaneously detectable tracers can be increased to thirteen. Microspheres that have been perfused or injected into tissues and organs retain their fluorescence following histological serial sectioning. Our FluoSpheres microspheres for blood flow determination, which are available in eleven distinguishable fluorescent colors (Figure 14.6.1), are also compatible with blood flow analyzer systems that perform automated extraction and analysis.

Figure 14.6.1 Normalized fluorescence emission spectra of the dyes contained in FluoSpheres polystyrene microspheres for blood flow determination, after extraction into 2-ethoxyethyl acetate (Cellosolve acetate). The eleven colors of fluorescent microspheres represented are: 1) blue, 2) blue-green, 3) green, 4) yellow-green, 5) yellow, 6) orange, 7) red-orange, 8) red, 9) carmine, 10) crimson and 11) scarlet (FluoSpheres microspheres for blood flow determination—Table 14.5).

FluoSpheres Color Kits for Regional Blood Flow Studies

We offer four different FluoSpheres Blood Flow Determination Fluorescent Color Kits (FluoSpheres blood flow and color kits—Table 14.6):

• Kit #1 (F8890) contains 10 mL vials of 10 µm microspheres in seven fluorescent colors (blue, blue-green, yellow-green, orange, red, crimson and scarlet).
• Kit #2 (F8891) contains 10 mL vials of 15 µm microspheres in seven fluorescent colors (blue, blue-green, yellow-green, orange, red, crimson and scarlet).
• Kit #3 (F8892) contains 10 mL vials of 15 µm microspheres in five fluorescent colors (blue-green, yellow-green, orange, red and crimson).
• Kit #4 (F21015) contains 10 mL vials of 15 µm microspheres in four additional fluorescent colors (green, yellow, carmine and red-orange), which are spectrally distinguishable from the dyes in Kits #2 and #3 (Figure 14.6.1).

The aqueous suspensions of 10 µm and 15 µm beads contain 3.6 million and 1 million microspheres per mL, respectively. All kits include a detailed protocol. Each of the colors of FluoSpheres microspheres for blood flow studies can also be purchased separately (FluoSpheres microspheres for blood flow determination—Table 14.5).

Fluorescent Microsphere Resource Center

Additional technical support, including a detailed applications manual on the use of fluorescent microspheres for blood flow determination, is available from the Fluorescent Microsphere Resource Center (FMRC) at the University of Washington, Seattle (fmrc.pulmcc.washington.edu).

Availability of intensely fluorescent, highly uniform microspheres in different colors and sizes permits diverse applications in tracking particles and cells, tracing fluid dynamics and amplifying signals. Using a mixture of beads of different sizes, each labeled with a different fluorescent color, researchers can discriminate the size dependence of uptake or transport of microspheres in vivo in cells, capillaries, lung or other tissues. In addition to our microspheres specially designed for blood flow studies, we offer a wide range of fluorescent FluoSpheres carboxylate-modified microspheres in different fluorescent colors, bead diameters and surface functional groups (see Summary of FluoSpheres fluorescent microspheres—Table 6.7 in Microspheres—Section 6.5 for a complete list). Of particular interest are our FluoSpheres beads with 0.04 µm diameters and FluoSpheres beads for tracer studies with 1 µm diameters, which have been specially formulated to perform well in cell tracking and particle tracking studies, respectively. Our smallest microspheres can be microinjected into cells (see below) or are actively taken up by phagocytosis (Probes for Following Receptor Binding and Phagocytosis—Section 16.1).

FluoSpheres Beads with 0.04 µm Diameters for Microinjection

Unlike our other fluorescent microspheres, most of which come in aqueous suspensions containing 2% solids and 2 mM sodium azide as a preservative, FluoSpheres beads with 0.04 µm diameters are prepared as aqueous suspensions containing 5% solids without preservatives. At more than double the concentration of our standard FluoSpheres microspheres, these carboxylate-modified 0.04 µm beads (F8795, F8792, F8794, F8793, F8789, F8794) are well suited to applications requiring microinjectable tracers. Yellow-green–, orange-, red-orange–, red- and dark red–fluorescent colors are available in the FluoSpheres Fluorescent Color Kit (F10720). In many biological systems, the concentrated fluorescence and spherical shape of the FluoSpheres beads permit them to be detected against a relatively high but diffuse background fluorescence. However, our TransFluoSpheres microspheres (FluoSpheres and TransFluoSpheres microspheres for tracing—Table 14.7)—microspheres with an extremely large Stokes shifts—are preferred for some studies because their fluorescence may be better resolved from the tissue's autofluorescence (Figure 14.6.2).

Figure 14.6.2 Schematic diagram of the advantages of the large Stokes shift exhibited by our TransFluoSpheres beads. A1 and E1 represent the absorption and emission bands of a typical TransFluoSpheres bead. The large separation of the absorption and emission maxima (Stokes shift) is characteristic of our TransFluoSpheres beads. Unlike most low molecular weight fluorescent dyes, which show considerable overlap of their absorption and emission spectra, the TransFluoSpheres beads can be excited (EX) across the entire absorption band A1 and the resulting fluorescence can be detected across the full emission band E1, thereby allowing the researcher to maximize the signal (S1). Moreover, because of the large Stokes shifts of the TransFluoSpheres beads, researchers can often avoid problems associated with autofluorescence. The absorption and emission bands of a typical autofluorescent component are represented in this figure by A2 and E2. Although the endogenous fluorescent species will be excited simultaneously with the TransFluoSpheres beads, the resulting emission (E2) does not coincide with E1 and is therefore readily rejected by suitably chosen optical filters.

FluoSpheres Beads with 1.0 µm Diameters for Tracer Studies

Our FluoSpheres fluorescent microspheres for tracer studies are 1.0 µm polystyrene beads for analysis by tissue extraction (FluoSpheres and TransFluoSpheres microspheres for tracing—Table 14.7). Because the dye content of these microspheres is much higher than that of other fluorescent microspheres, stronger signals can be generated using fewer microspheres per tracing experiment. These heavily dye-loaded beads are available in four well-resolved fluorescent colors: blue-green (F13080), yellow-green (F13081), orange (F13082) and red (F13083), with excitation/emission maxima at approximately 430/465, 505/515, 540/560 and 580/605 nm, respectively (the emission spectra are shown as peaks 2, 4, 6 and 8 of Figure 14.6.1). Aerosols containing these fluorescent microspheres have been used to acquire high-resolution maps of regional pulmonary ventilation. Transport of these fluorescent microspheres through tissues can be determined using methods that have been developed for regional blood flow determination or by confocal laser-scanning microscopy. As with the FluoSpheres beads for blood flow determination, the microspheres and the fluorescent dyes they contain are first extracted from the tissue sample, and then the fluorescence is quantitated on a spectrofluorometer or fluorescence microplate reader.

SAIVI 715 Injectable Contrast Agents with 0.1 µm and 2 µm Diameters

SAIVI 715 injectable contrast agents (S31201, S31203) are specially formulated for small-animal in vivo imaging of regions of inflammation, blood pooling and wound healing. These contrast agents are polymeric microspheres that have been labeled with a fluorescent dye; each microsphere particle contains many dye molecules protected within the polymer sphere. Their use for in vivo imaging offers many advantages over existing contrast agents, including 1) no known intrinsic toxicity, 2) a high degree of localization within diseased vasculature and 3) longer in vivo residence times than organic dye–labeled proteins Formulated to resist liver accumulation, SAIVI injectable contrast agents have been observed to circulate throughout the blood and to accumulate in tissues that exhibit damaged, excessive or otherwise abnormal blood vessel development as part of the disease process. These agents are optimized for long-wavelength emission and tested by in vivo imaging after injection in disease models established in mice. We also offer a set of complementary contrast agents (SAIVI Alexa Fluor 680 and SAIVI Alexa Fluor 750 conjugates of transferrin and albumin, Protein Conjugates—Section 14.7) for rapid imaging of early-onset events.

Europium and Platinum Luminescent Microspheres for Time-Resolved Fluorometry

Detecting a low level of protein or DNA targets in a tissue sample or on a membrane using classic fluorochromes is sometimes difficult and prone to errors because specific fluorescence signals tend to be low and are usually mixed with nonspecific signals and autofluorescence. One approach to improve detectability is the use of time-resolved luminescence reagents, such as our FluoSpheres europium luminescent microspheres and FluoSpheres platinum luminescent microspheres (Molecular Probes europium and platinum luminescent FluoSpheres microspheres—Table 14.8). The FluoSpheres europium beads contain Eu³⁺ coordination complexes with luminescence decay times of >600 microseconds—much longer than the <50 nanosecond decay time of conventional fluorophores and autofluorescence. The luminescence of the Pt²⁺ chelate in the FluoSpheres platinum luminescent microspheres has a decay time of >40 microseconds. Thus, time-gated fluorescence detection using these microspheres results in complete rejection of autofluorescence signals. In addition, the europium luminescent microspheres feature long-wavelength emission (610–650 nm) that is well separated from their excitation peak (340–390 nm) (Figure 14.6.3). The platinum luminescent microspheres are maximally excited near 390 nm with narrow emission that is maximal near 650 nm (Figure 14.6.4). Because of these unusually large Stokes shifts, filter combinations can be chosen that effectively isolate the desired luminescence signal. The narrow emissions and different lifetimes permit simultaneous use of the europium and platinum luminescent microspheres as tracers. These microspheres are available with nominal diameters of 0.04 µm or 0.2 µm (FluoSpheres and TransFluoSpheres microspheres for tracing—Table 14.7).

Figure 14.6.3 Fluorescence excitation and emission maxima of the FluoSpheres europium luminescent microspheres (F20880, F20881, F20883, F20884).

Figure 14.6.4 Luminescence excitation and emission spectra of the FluoSpheres platinum luminescent microspheres (F20886).

Microspheres for Monitoring Airflow

A cubic foot of air may contain hundreds of thousands of airborne particles such as viruses, bacteria, pollen, mold spores and gaseous chemicals (Figure 14.6.5). To combat harmful airborne particles, many types of air filters and instruments for detecting airborne particles have been developed for medical and defense purposes. Because most airborne particles are small (typically 10–150 nm, below microscopic resolution) and are difficult to detect, verification of the function of these filters and instruments can be a challenge. Our FluoSpheres microspheres are specially stained fluorescent polystyrene beads that can mimic airborne particles and be used as unique markers to verify equipment reliability. A single "virus-sized" 20 nm diameter FluoSpheres microsphere carries 100–200 fluorophore molecules and so emits sufficiently bright fluorescence for easy detection.

Figure 14.6.5 FluoSpheres microsphere sizes relative to common airborne particles.

Transplantation and Migration Studies with Fluorescent Microspheres

Labeling cells with fluorescent microspheres prior to transplantation enables researchers to distinguish cell types and analyze graft migration in the host over extended time periods. Unlike other tracers, most of which rapidly diffuse or leach from their site of application in tissues, fluorescent microspheres tend to remain in place for periods of at least months. Their stability and biological inertness give them considerable potential for transplantation studies. Moreover, the intense fluorescence, high uniformity and low debris content of our FluoSpheres polystyrene microspheres will circumvent many of the problems encountered in use of fluorescent microspheres from other sources. Cells are generally labeled with microspheres by microinjection or other invasive methods (Techniques for loading molecules into the cytoplasm—Table 14.1). Potential problems of sterility, unequal uptake and translocation of the beads by the cells need to be considered before using fluorescent microspheres in transplantation and other cell tracing studies. It has been reported that polystyrene microspheres for transplantation studies can be sterilized by heating below their softening temperature.

Neuronal Tracing with Fluorescent Microspheres

Katz, the first scientist to use fluorescent microspheres as neuronal tracers, demonstrated that rhodamine-labeled microspheres could undergo retrograde transport. Although the mechanism of microsphere transport is not completely understood, the process can apparently be facilitated by using a high concentration of particles with small diameters (<0.05 µm) and high negative surface-charge densities. Fluorescent microspheres are also suitable for retrograde tracing because they are not cytotoxic and persist for extraordinarily long periods in nerve cells. The fluorescence intensity of microsphere-labeled neuronal perikarya in rats was found to be undiminished one year after injection. Adsorption of neuroactive proteins, including neurotrophins, on small-diameter fluorescent microspheres provides a means of locating their in vivo and in vitro microinjection sites and of following retrograde transport in the central nervous system. Our fluorescent microspheres are also becoming important tracers in ophthalmological studies where they undergo retrograde transport.

Our red-fluorescent, carboxylate-modified FluoSpheres beads (F8793) are retrogradely transported in rat lumbosacral ventral root axons that have been subjected to peripheral crush injury. In this study, the fluorescent beads were used to photoconvert diaminobenzidine (DAB) into an insoluble, electron-dense reaction product in order to facilitate ultrastructural analysis by electron microscopy (Fluorescent Probes for Photoconversion of Diaminobenzidine Reagents—Note 14.2).

Following Phagocytosis with Fluorescent Microspheres

Many cell types actively ingest opsonized or nonopsonized fluorescent microspheres (Probes for Following Receptor Binding and Phagocytosis—Section 16.1). The preferred microspheres are about 0.5–2 µm in diameter. Confocal laser-scanning microscopy can distinguish between ingested beads and those simply bound to the surface. Flow cytometry, fluorescence microscopy and fluorescence spectrometry have been used to quantitate phagocytosis by macrophage cells and protozoan cells. Macrophage cells in primary cultures of rat cerebral cortex have been identified by their ability to selectively phagocytose fluorescent microspheres; macrophage cells have also been sorted based on the absolute number of microspheres phagocytosed. Fluorescent microsphere uptake has been used as a model for alveolar macrophage cell translocation and clearance of inhaled aerosols containing environmental particulates. As with our fluorescent microspheres for blood flow determination (see above), solvent extraction of the fluorescent dye from the beads can be used to quantitate microsphere uptake by macrophage cells.

For the study of collagen phagocytosis, we manufacture yellow-green–fluorescent FluoSpheres collagen I–labeled microspheres in either 1.0 µm or 2.0 µm diameters (F20892, F20893; Probes for Following Receptor Binding and Phagocytosis—Section 16.1). Fibroblasts phagocytose and then intracellularly digest collagen. These activities play an important role in the remodeling of the extracellular matrix during normal physiological turnover of connective tissues, in development, in wound repair and possibly in aging and various disorders. A well-established procedure for observing collagen phagocytosis by either flow cytometry or fluorescence microscopy involves the use of collagen-coated fluorescent microspheres, which attach to the cell surface and become engulfed by fibroblasts.

Tracking the Movement of Proteins and Other Biomolecules with Fluorescent Microspheres

FluoSpheres microspheres can serve as bright, inert and extremely photostable labels for tracking particle movement and other dynamic processes over extended time periods. The intense fluorescence of our FluoSpheres beads permits the detection and tracking of very small single particles in three dimensions. Examples of the diverse applications of fluorescent microspheres include studies designed to track or quantitate:

• Binding of kinetochores to microtubules in vitro
• Brownian motion in protein and dextran solutions
• Exchangeable GTP-binding sites on paclitaxel-stabilized fluorescent microtubules
• Direction and rate of axonal transport in the squid giant axon
• Fluid dynamics and flow in mucus and peripheral lymph
• Injection sites in tissues
• Lateral diffusion of lipids and receptors in membranes
• Movement of microinjected microspheres poleward during mitosis of sea urchin eggs and sand dollar eggs
• Particle flow through a model biofilm containing Pseudomonas aeruginosa, P. fluorescens and Klebsiella pneumoniae using confocal laser-scanning microscopy (Figure 14.6.6)
• Sentinel lymph node detection
• Three-dimensional motion of microspheres in order to assess water permeability in individual Chinese hamster ovary (CHO) cells expressing CHIP28 water channels

Fluorescent microspheres have been used with optical tweezers to control the movement of single myosin filaments or kinesin molecules and for imaging at suboptical resolution by scanning luminescence X-ray microscopy. In addition, our 0.1 µm red-fluorescent carboxylate-modified microspheres (F8801, Microspheres—Section 6.5) have been used to model particle distribution and penetration of adenovirus-mediated gene transfer in human bronchial submucosal glands using xenografts.

Figure 14.6.6 Superimposed time sequence image (2.3-second intervals), showing a single bead moving through a biofilm channel. Autofluorescence of the cell clusters can be seen as lighter areas relative to the channels. The direction of bulk fluid flow is indicated by the arrow. Photo contributed by Paul Stoodley, Exeter University, and Dirk DeBeer, Max Planck Institute of Marine Biology.

Qtracker Cell Labeling Kits

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. We offer the following eight kits:

• Qtracker 525 Cell Labeling Kit (Q25041MP)
• Qtracker 565 Cell Labeling Kit (Q25031MP)
• Qtracker 585 Cell Labeling Kit (Q25011MP)
• Qtracker 605 Cell Labeling Kit (Q25001MP)
• Qtracker 625 Cell Labeling Kit (A10198)
• Qtracker 655 Cell Labeling Kit (Q25021MP)
• Qtracker 705 Cell Labeling Kit (Q25061MP)
• Qtracker 800 Cell Labeling Kit (Q25071MP)

The mechanism whereby such cell penetrating peptides induce the cellular uptake of cargoes including oligonucleotides and proteins as well as Qdot nanocrystals remains a topic of active investigation and considerable debate. It is, however, reasonably well established that the uptake is passive and does not require specific receptors or active transport processes. Therefore, labeling of live cells is a simple matter of adding the Qtracker cell labeling reagents supplied in the kits to adherent or suspension cells in complete growth medium, incubating at 37°C for 45–60 minutes, followed by a wash step to remove the labeling reagents. The Qdot nanocrystals are stably incorporated in cytoplasmic vesicles (Figure 14.6.7) and are passed to daughter cells upon cell division but are not transferred to adjacent cells in mixed cultures or host tissues. Fluorescence is not impacted by complex and varying cellular environments including changes in intracellular pH, temperature and metabolic activity. A wide range of applications for Qtracker Cell Labeling Kits have been reported, including fiducial marking of cell populations for identification during or after downstream analysis, labeling of mouse cerebral vascular tissue for visualization of explantation, imaging the assembly of cultured pulmonary artery adventitial fibroblasts and endothelial cells into three-dimensional structures and tracking embryonic and mesenchymal stem cells.

Figure 14.6.7 Distribution of Qdot nanocrystals in cytoplasmic vesicles after labeling cells with the Qtracker 655 Cell Labeling Kit (Q25021MP). HeLa cells were labeled with the Qtracker 655 Cell Labeling Kit and then observed using a Leica TCS SP2 confocal microscope (excitation at 488 nm). This representative image shows the Qdot nanocrystals distributed in vesicles throughout the cytoplasm.

Qtracker Non-Targeted Quantum Dots

Qtracker non-targeted quantum dots are designed for small animal in vivo imaging, and especially for studying vascular structure after microinjection (Figure 14.6.8).  Our selection includes:

• Qtracker 565 non-targeted quantum dots (Q21031MP)
• Qtracker 655 non-targeted quantum dots (Q21021MP)
• Qtracker 705 non-targeted quantum dots (Q21061MP)
• Qtracker 800 non-targeted quantum dots (Q21071MP)

These nanocrystals exhibit intense red or near-infrared fluorescence emission for maximum transmission through tissues and avoidance of background autofluorescence. Qtracker non-targeted quantum dots have polyethylene glycol (PEG) surface coatings to minimize nonspecific binding interactions and associated inflammatory responses. Because the PEG surface coating does not contain reactive functional groups, the Qtracker non-targeted quantum dots are retained in circulation longer and can be imaged for up to 3 months without additional injections Qtracker non-targeted quantum dots are supplied as 2 µM solutions in 50 mM borate buffer, pH 8.3 in units of 200 µL.

Figure 14.6.8 Chick embryo injected through the major vitelline vein with Qtracker non-targeted quantum dots. Following a few minutes of circulation of the Qtracker 705 non-targeted quantum dots (Q21061MP), fluorescence images of the embryo were captured at increasing magnification using 460 nm excitation and a digital imaging system equipped with appropriate emission filters. These Qtracker reagents revealed highly detailed vascular structure at all levels of magnification. Images contributed by Greg Fisher, Carnegie Mellon University.