Microspheres—Section 6.5

Page Contents

• Properties of Molecular Probes Fluorescent and Nonfluorescent Microspheres
• Applications for Fluorescent Microspheres
• FluoSpheres Fluorescent Microspheres
• Fluorescent Microsphere Starter Kits
• Fluorescent Microspheres for Educational Purposes
• TransFluoSpheres Fluorescent Microspheres: Tools for Multicolor Detection
• BlockAid Blocking Solution
• Ordering Information

Although low molecular weight reactive dyes are versatile and easy to use, they are not without limitations. For example, the fluorescence output of the dye–biomolecule conjugate is often limited by the number of dyes that can be attached to the biomolecule without disrupting its function. Our highly fluorescent microspheres—both the FluoSpheres () and TransFluoSpheres () beads—provide a means of overcoming this limitation. Moreover, our TransFluoSpheres beads are designed to facilitate multicolor detection, particularly in applications that use lasers with their inherent limited number of excitation wavelengths. TransFluoSpheres beads contain a series of two or more proprietary dyes that have been carefully chosen to ensure excited-state energy transfer between the dyes. This strategy enables fine-tuning of both the excitation and emission wavelengths of the microspheres so that they match a particular instrument's excitation source and detection sensitivity, and complement the spectra of other fluorophores in a multicolor experiment.

Fluorescent FluoSpheres and TransFluoSpheres Microspheres

Molecular Probes FluoSpheres and TransFluoSpheres beads are manufactured using high-quality, ultraclean polystyrene microspheres. These microspheres are internally labeled with Molecular Probes dyes, making them among the brightest fluorescent microspheres available (Molecular Probes yellow-green-fluorescent FluoSpheres beads compared with other commercially available yellow-green-fluorescent microspheres—Table 6.5). We employ methods to ensure that each bead is heavily loaded with dye. The protective environment within the bead matrix shields the dyes from many of the environmental effects that cause photobleaching of exposed fluorophores. Not only are our yellow-green–fluorescent beads more photostable, but their emission spectra are not affected by changes in pH, as are conventional fluorescein-labeled microspheres. Fluorescent microspheres can be fixed in formalin and embedded in paraffin if care is used to avoid extraction of the noncovalently associated dyes from the microspheres. The stability, uniformity and reproducibility of fluorescent beads, as well as the extensive selection of colors available, make our microspheres the preferred tools for research and diagnostic assays that use fluorescence.

In addition to the microspheres described in this section, we have developed several important microsphere-based products for calibrating and aligning fluorescence microscopes (Fluorescence Microscopy Accessories and Reference Standards—Section 23.1) and flow cytometers (Flow Cytometry Reference Standards—Section 23.2). Custom preparation of microspheres with other colors, sizes and surface coatings is available; please contact Invitrogen Custom Services for more information.

Colored and Unstained Microspheres

We also offer a wide selection of colored and unstained microspheres for research applications as well as for water- and air-flow testing and other bead-based applications. Through the acquisition of Interfacial Dynamics Corporation (IDC), we provide milliliter to 500-liter quantities of ultraclean microspheres with diameters from 20 nm to 10.0 µm and with more than 20 different surface functionalities; view this is page for a complete listing of the colored and unstained microspheres available. IDC pioneered the commercial development of surfactant-free polymer particles used in bead-based assay systems and has been a key supplier of beads for Molecular Probes fluorescent microspheres. Our outstanding capabilities in microsphere manufacturing allow a high level of control of the colloid engineering employed in the particle synthesis, providing excellent batch-to-batch consistency. Other microsphere manufacturers often use surfactants to prevent aggregation. However, standards of surfactant purity are generally not very high, leading to a poorly defined particle surface and variable protein attachment. In the manufacturing of our ultraclean microspheres, no surfactants are required to prevent aggregation, taking much of the guesswork out of stability and adsorption experiments. We can tailor-make colored and unstained microspheres of many sizes, surface chemistries, densities and volumes to meet the diverse needs of customers, including academic, industrial and government laboratories, as well as major global diagnostic companies; please contact Invitrogen Custom Services for more information.

Fluorescent microspheres have been used as immunofluorescent reagents, retrograde neuronal tracers, microinjectable cell tracers (Microspheres and Qdot Nanocrystals for Tracing—Section 14.6) and standardization reagents for microscopy (Fluorescence Microscopy Accessories and Reference Standards—Section 23.1) and flow cytometry (Flow Cytometry Reference Standards—Section 23.2). Suspension arrays of fluorescent microspheres that differ in intensity, size or excited-state lifetime are extensively used for simultaneous assays to determine multiple analytes in a single sample. In addition, fluorescent microspheres are potentially more sensitive than colorimetric methods in most, if not all, of the major microsphere-based test systems presently in use, including latex-agglutination tests, filter-separation tests, particle-capture ELISA methods and two-particle sandwich techniques. Applications of our fluorescent microspheres for assessing tissue blood perfusion are described in Microspheres and Qdot Nanocrystals for Tracing—Section 14.6.

A Wide Array of Fluorescent Colors

Molecular Probes FluoSpheres fluorescent microspheres contain dyes with excitation and emission wavelengths that cover the entire spectrum from the near UV to the near-infrared. Figure 6.5.1 shows the normalized emission spectra for 9 fluorescent colors of FluoSpheres beads. Because long-wavelength (>680 nm) light can penetrate tissues, our infrared-fluorescent microspheres may allow researchers to conduct experiments that were not previously possible with beads that emit at shorter wavelengths. We would like to highlight the following FluoSpheres products:

• Blue-fluorescent FluoSpheres beads with excitation/emission maxima of 350/440 nm contain an improved blue-fluorescent dye that has exceptional brightness and a long shelf life. We also offer blue-fluorescent FluoSpheres beads with slightly shorter-wavelength fluorescence spectra (excitation/emission maxima ~365/415 nm).
• Yellow-green–fluorescent FluoSpheres beads have excitation/emission maxima of 505/515 nm and thus are excited very efficiently using the 488 nm spectral line of the argon-ion laser, resulting in exceptionally intense fluorescence (Molecular Probes yellow-green-fluorescent FluoSpheres beads compared with other commercially available yellow-green-fluorescent microspheres—Table 6.5).
• Orange-, red-orange– and red-fluorescent FluoSpheres beads have excitation maxima of 540 nm, 565 nm and 580 nm, respectively.
• Nile red–fluorescent FluoSpheres beads have broad excitation/emission bandwidths at 535/575 nm, making them compatible with filter sets appropriate for fluorescein, rhodamine and Texas Red dyes.
• Crimson- and dark red–fluorescent FluoSpheres beads with excitation/emission maxima of 625/645 nm and 660/680 nm, respectively, are efficiently excited by the 633 nm spectral line of the He-Ne laser. Although the dark red–fluorescent beads are significantly less fluorescent than the crimson-fluorescent particles, they fluoresce at wavelengths that are longer than, and clearly distinguishable from, those of the crimson-fluorescent particles.
• Infrared-fluorescent FluoSpheres beads with excitation/emission maxima of 715/755 nm are the longest-wavelength fluorescent microspheres currently available from any source. These beads absorb and emit at wavelengths at which most tissues are almost optically transparent.
• Europium luminescent and platinum luminescent FluoSpheres beads have excitation/emission maxima of 340–370/610 nm (Figure 6.5.2) and ~390/650 nm (Figure 6.5.3), respectively, and decay times of >40 microseconds for the platinum microspheres and >100 microseconds for the europium microspheres, far longer than those of conventional fluorescent probes and autofluorescent samples. These beads should be useful as standards for time-resolved microscopy and for tracing applications in highly autofluorescent samples. 

Our FluoSpheres beads are many times brighter than fluorescent microspheres from other companies (Molecular Probes yellow-green-fluorescent FluoSpheres beads compared with other commercially available yellow-green-fluorescent microspheres—Table 6.5). Fluorescein equivalents in our yellow-green-fluorescent FluoSpheres beads—Table 6.6 shows the approximate number of unquenched fluorescein equivalents in our yellow-green–fluorescent FluoSpheres beads. Quantitative imaging measurements on intact beads indicate that the fluorescence intensities of single 0.04 µm and 0.1 µm yellow-green FluoSpheres beads are equivalent to 61 and 3650 EGFP molecules, respectively. These numbers are lower than the corresponding fluorescein equivalents largely because of quenching effects in the intact beads that are avoided by extraction of the dye in the measurements reported in Fluorescein equivalents in our yellow-green-fluorescent FluoSpheres beads—Table 6.6. The intensity of the beads is sufficient to allow visualization of single particles, even for our smallest microspheres, which appear as point sources; see the description of the PS-Speck Microscope Point Source Kit (P7220) in Fluorescence Microscopy Accessories and Reference Standards—Section 23.1. Our FluoSpheres beads show little or no photobleaching, even when excited with the intense illumination required for fluorescence microscopy.

Although some of our FluoSpheres beads are available in limited sizes, colors and surface functions, we will prepare custom orders upon request (Invitrogen Custom Services). We also have considerable experience developing standards, including microsphere-based standards for companies selling fluorescence instrumentation. FluoSpheres beads can also be prepared with intensities that are lower than those of our regular products—a desirable feature in some multicolor applications. FluoSpheres beads with calibrated intensities are already available in our InSpeck Microscope Intensity Calibration Kits (Fluorescence Microscopy Accessories and Reference Standards—Section 23.1) and LinearFlow Flow Cytometry Intensity Calibration Kits (Flow Cytometry Reference Standards—Section 23.2).

Figure 6.5.1 Normalized fluorescence emission spectra of our FluoSpheres beads, named according to their excitation/emission maxima (nm): 1) blue (365/415), 2) blue (350/440), 3) yellow-green (505/515), 4) orange (540/560), 5) red-orange (565/580), 6) red (580/605), 7) crimson (625/645), 8) dark red (660/680) and 9) infrared (715/755) FluoSpheres beads.

A Wide Range of Sizes

To meet the diverse needs of academic and industry laboratories, we offer FluoSpheres beads in a variety of sizes (Summary of FluoSpheres fluorescent microspheres—Table 6.7). The smallest microspheres are currently about 0.02 µm in diameter, with a coefficient of variation (CV) of about 20% as determined by electron microscopy. The size uniformity improves with increasing size, with the CV decreasing from ~5% for 0.1 µm FluoSpheres beads to ~1% for those with 10–15 µm diameters. The sizes specified in the product names are nominal bead diameters; because of batch-to-batch variation in the undyed microspheres, the actual mean diameters shown on the product labels may differ from the nominal diameters, especially for the smaller microspheres. Because of their small size, 0.02–0.04 µm microspheres are effectively transparent to light in aqueous suspensions and behave very much like true solutions.

Four Different Surface Functional Groups

We prepare FluoSpheres beads with four different surface functional groups, making them compatible with a variety of conjugation strategies. Our fluorescent dyes have negligible effect on the surface properties of the polystyrene beads or on their protein adsorption. We caution, however, that the surface properties have an important role in the functional utility of the microspheres; we cannot guarantee the suitability of a particular bead type for all applications.

• Carboxylate-modified FluoSpheres beads have pendent carboxylic acids, making them suitable for covalent coupling of proteins and other amine-containing biomolecules using water-soluble carbodiimide reagents such as EDAC (E2247, Derivatization Reagents for Carboxylic Acids and Carboxamides—Section 3.4). In order to both decrease nonspecific binding and provide additional functional groups for conjugation, these FluoSpheres beads have a high density of carboxylic acids on their surfaces.
• Sulfate FluoSpheres beads are relatively hydrophobic particles that will passively and nearly irreversibly adsorb almost any protein, including albumin, IgG, avidin and streptavidin.
• Aldehyde–sulfate FluoSpheres beads, which are sulfate microspheres that have been modified to add surface aldehyde groups, are designed to react with proteins and other amines under very mild conditions.
• Amine-modified FluoSpheres beads can be coupled to a wide variety of amine-reactive molecules, including the succinimidyl esters and isothiocyanates of haptens and drugs or the carboxylic acids of proteins, using a water-soluble carbodiimide. The amine surface groups can also be reacted with SPDP (S1531, Chemical Crosslinking Reagents—Section 5.2) to yield (after reduction) microspheres with pendent sulfhydryl groups.

Fluorescent Microspheres Conjugated to Biotin, Avidin and Streptavidin

We offer yellow-green–fluorescent FluoSpheres microspheres conjugated to biotin or streptavidin, and yellow-green–fluorescent, red-fluorescent, europium luminescent and nonfluorescent microspheres conjugated to NeutrAvidin biotin-binding protein (Summary of biotin-, streptavidin- and NeutrAvidin biotin-binding protein–labeled FluoSpheres microspheres—Table 6.8). NeutrAvidin biotin-binding protein has been specially processed to remove carbohydrates and lower the isoelectric point, resulting in a near-neutral protein that has significantly lower nonspecific binding than conventional avidin. These microsphere conjugates provide valuable tools for improving the sensitivity of flow cytometry applications and immunochemical assays. They are also useful as tracers that can be detected with standard enzyme-mediated avidin/streptavidin methods. Additional sizes and colors of these microspheres can be custom-ordered through Invitrogen Custom Services.

Fluorescent Microspheres Coated with Collagen

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 aging and in 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. We offer yellow-green–fluorescent FluoSpheres collagen I–labeled microspheres in either 1.0 µm or 2.0 µm diameter (F20892, F20893) for use in these applications. These microspheres have collagen I from calf skin attached covalently to their surface.

Europium and Platinum Luminescent Microspheres for Time-Resolved Fluorometry

Detecting low levels 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. The FluoSpheres europium luminescent beads contain Eu³⁺ coordination complexes with luminescence decay times of >100 microseconds —much longer than the nanosecond decay times 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, both the europium luminescent and platinum luminescent microspheres feature long-wavelength emissions (610–650 nm) that are well separated from their excitation peaks (340–390 nm) (Figure 6.5.2, Figure 6.5.3). Because of this exceptionally large Stokes shift, filter combinations can be chosen that effectively isolate the desired luminescence signal.

These microspheres are available uncoated (F20880, F20881, F20886) or conjugated to NeutrAvidin biotin-binding protein (F20883, F20884), with nominal diameters of 0.04 µm or 0.2 µm. Beads labeled with NeutrAvidin biotin-binding protein can be used for the indirect detection of antigens and DNA targets in many biotin/avidin-based assays.

Figure 6.5.2 Luminescence excitation and emission spectra of the FluoSpheres europium luminescent microspheres (F20880, F20881).

Figure 6.5.3 Luminescence excitation and emission spectra of the FluoSpheres platinum luminescent microspheres (F20886).

For applications requiring several different microsphere colors or sizes, we offer three types of fluorescent microsphere starter kits:

• The FluoSpheres Fluorescent Color Kit (F10720) consists of 1 mL samples of yellow-green–, orange-, red- and dark red–fluorescent, carboxylate-modified 0.04 µm FluoSpheres beads packaged as high-density, azide-free suspensions for microinjection.
• The FluoSpheres Size Kits contain 1 mL samples of carboxylate-modified FluoSpheres beads in 0.02, 0.1, 0.2, 0.5, 1.0 and 2.0 µm sizes. These beads are available in yellow-green– (F8888) or red- (F8887) fluorescent colors.
• The FluoSpheres Blood Flow Determination Fluorescent Color Kits provide several different fluorescent colors of 10 µm (F8890) or 15 µm (F8891, F8892, F21015) FluoSpheres microspheres; see Microspheres and Qdot Nanocrystals for Tracing—Section 14.6 for a description of these kits.

Constellation microspheres for imaging (C14837) can be used to demonstrate hands-on techniques with a fluorescence microscope. Constellation microspheres consist of a selected mixture of beads in assorted sizes and colors () that can be used to practice adjusting the focus and switching filters on a fluorescence microscope. These microspheres are stable at room temperature, so they can be conveniently stored.

Advantages of TransFluoSpheres Fluorescent Microspheres

TransFluoSpheres fluorescent microspheres (Summary of TransFluoSpheres fluorescent microspheres—Table 6.9; Figure 6.5.4, ) are specially designed to overcome the limitations imposed by modern fluorescence instrumentation. Many flow cytometers, confocal laser-scanning microscopes and laser scanners incorporate the argon-ion laser as the excitation source, thereby limiting the available excitation wavelengths to the laser's 488 nm and 514 nm spectral lines and severely restricting simultaneous multicolor detection. Ideally, it would be useful to have a series of fluorescent dyes with absorption maxima close to the argon-ion laser's spectral lines, but with emission maxima at a variety of longer wavelengths. This approach would require that some of the dyes exhibit large Stokes shifts—defined as the separation of the absorption and emission maxima. Unfortunately, very few low molecular weight dyes have a combination of a large Stokes shift and a high molar absorptivity. For example, the Texas Red fluorophore—often used in combination with fluorescein—has particularly weak absorption at 488 nm and 514 nm. In applications that employ the argon-ion laser as an excitation source, Texas Red conjugates have a low fluorescence output that is easily obscured by the more intense fluorescein fluorescence, even when detected past 600 nm.

Our TransFluoSpheres beads, which incorporate two or more fluorescent dyes that undergo excited-state energy transfer, exhibit Stokes shifts that can be extremely large. Each microsphere contains a dye with an excitation peak that maximally overlaps the spectral output of a commonly used excitation source (for example, the 488 nm spectral line of the argon-ion laser; Figure 6.5.5). In addition, each microsphere contains one or more longer-wavelength dyes that are carefully chosen to create a relay series that can efficiently transfer the energy from the initially excited dye to the longest-wavelength acceptor dye. The proprietary dyes used in the TransFluoSpheres beads are optimally loaded to ensure that the excitation energy is efficiently transferred from dye to dye so that essentially only the longest-wavelength dye in the series exhibits significant fluorescence. Because these TransFluoSpheres beads fluoresce at a considerably longer wavelength than the excitation wavelength, they provide a signal that can be detected in samples with significant Rayleigh or Raman scattering or with endogenous fluorescent compounds such as bilins, flavins and certain drugs. Also, the large Stokes shifts exhibited by the TransFluoSpheres beads allow the use of broadband filters, both to excite the sample and to detect the emission, resulting in a greater fluorescent signal (Figure 6.5.4).

Figure 6.5.4 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.

Figure 6.5.5 Fluorescence emission spectra of our five 488 nm light–excitable TransFluoSpheres beads, named according to their excitation/emission maxima (nm): A) 488/560, B) 488/605, C) 488/645, D) 488/685 and E) 488/720. The arrow in each spectrum represents the 488 nm spectral line of the argon-ion laser. The beads shown in panels B, D and E are no longer routinely available; please inquire through Invitrogen Custom Services.

TransFluoSpheres Beads to Match Different Excitation Sources

We offer TransFluoSpheres beads compatible with two different laser excitation sources. The argon-ion laser–excitable TransFluoSpheres beads have an excitation maximum near 488 nm but emit at 560 nm (T8864, T8872, T8880) or 645 nm (T8883). The red He-Ne laser–excitable TransFluoSpheres beads have excitation/emission maxima of 633/720 nm (T8870). TransFluoSpheres beads can also be combined with our more traditional FluoSpheres beads or with low molecular weight dyes for multicolor detection.

Using carbodiimide reagents such as EDAC (E2247, Derivatization Reagents for Carboxylic Acids and Carboxamides—Section 3.4), researchers can couple protein or other amine-containing molecules to our carboxylate-modified TransFluoSpheres beads, making these microspheres suitable for a wide range of applications. TransFluoSpheres beads can be used in the major microsphere-based test systems and in experiments that currently employ standard fluorescent microspheres to measure regional blood flow (Microspheres and Qdot Nanocrystals for Tracing—Section 14.6), to study phagocytosis (Probes for Following Receptor Binding and Phagocytosis—Section 16.1), to detect cell-surface antigens and to trace neurons.

In addition, we offer TransFluoSpheres microspheres with excitation/emission maxima of 488/605 nm (T8860, T8861) conjugated to NeutrAvidin biotin- binding protein, as well as TransFluoSpheres beads with excitation/emission maxima of 488/645 nm (T10711) conjugated to streptavidin. Flow cytometry studies demonstrate that the sensitivity of our 40 nm TransFluoSpheres beads conjugated to streptavidin is superior to that of fluorescein streptavidin and comparable to that of R-phycoerythrin–streptavidin for detecting biotinylated epidermal growth factor (EGF) bound to EGF receptors. In multicolor experiments, the long-wavelength fluorescence emission of these TransFluoSpheres beads permits their use simultaneously with fluorescein- and R-phycoerythrin–labeled probes. For all applications requiring protein-coated microspheres, we strongly recommend using our BlockAid blocking solution (B10710, see below) to reduce nonspecific binding.

If wavelength or bead-size requirements are not met by our current selection of products, we invite inquiries about custom synthesis by contacting Invitrogen Custom Services. We can fine-tune the excitation and emission to match a particular excitation source or detection wavelength. In addition, we can covalently conjugate our TransFluoSpheres beads to other target-specific proteins to provide detection reagents that have potentially greater sensitivity in flow cytometry applications and immunochemical assays.

The intensely fluorescent and highly photostable FluoSpheres and TransFluoSpheres microspheres have significant potential for applications requiring probes that can deliver a strong signal. Unfortunately, microspheres conjugated to proteins have hydrophobic regions that may cause them to bind to nontarget surfaces in some experimental systems. This nonspecific binding can often be relieved by the use of a blocking solution. However, we have found that microspheres require a stronger blocking solution than those in common use, and therefore we have developed the BlockAid blocking solution (B10710).

The BlockAid reagent is a protein-based blocking solution designed for use with Fluospheres and TransFluoSpheres microspheres conjugated to biotin, streptavidin, NeutrAvidin biotin-binding protein or other proteins. In our tests, BlockAid blocking solution has been found to be effective in flow cytometry applications with the NIH 3T3, A431, RAW and Jurkat cell lines; however, with the HMC-1 cell line, it did not appear to offer any advantages over standard blocking solutions. The BlockAid blocking solution has proven useful for reducing the nonspecific binding of protein-coated or other macromolecule-coated microspheres in a wide variety of flow cytometry, microscopy and microarray applications.