Microspheres Support—Getting Started

The polystyrene microspheres are swelled in a solvent containing a hydrophobic dye specific to the FluoSpheres™ product of choice. The dye is then able to diffuse into the polystyrene matrix. The beads are then removed from the solvent and dialyzed into an aqueous environment; this reverses the swelling and traps the dye in the polystyrene. Once trapped, the dye is fairly protected from the external environment. The exact protocol and identity of the hydrophobic dyes are proprietary.

We do not determine the exact number of dye molecules per microsphere. The number of dye molecules is a function of the diameter of the particle and the properties of the dye. 

The concentration and surface charge of the microspheres is typically found in the specific lot Certificate of Analysis. Most certificates of analysis are available on the product web page.
Search for the CofA for your product.

We recommend using dialysis with a MWCO of ~500,000 to remove the sodium azide from the microsphere-containing buffer.

The stability in alcohol or water/alcohol mixtures is dependent on the alcohol chain length. The beads are fairly stable in 40% methanol, but they will dissolve much more readily in long chain alcohols. Prolonged storage of beads in an alcohol containing solution will tend to leach out the dye.

We recommend using a ProLong™ product or any other aqueous mounting media. You should avoid organic-based mountants such as xylene-based mounting media.

Point spread functions will require a subresolution bead, and we have two different subresolution bead products that could be used for this purpose. TetraSpeck™ Microspheres, 0.1 µm (Cat. No. T7279), are single subresolution microspheres that are simultaneously stained with blue, green, orange, and dark red dyes. The PS-Speck™ Microscope Point Source Kit (Cat. No. P7220) consists of individually-stained blue, green, orange, and dark red fluorescent microspheres that are ~175 nm in diameter.

We offer a variety of FluoSphere™ microspheres and kits specifically designed for blood flow determination; if the available colors and/or sizes do not suit your needs, we recommend using beads modified with carboxylate groups for blood flow determination applications. We give some basic usage guidelines in the FluoSpheres™ Fluorescent Micospheres for Tracer Studies product manual. Additionally the University of Washington Seattle offers the Fluorescent Microsphere Resource Center (FMRC) website with detailed applications manuals and references for using fluorescent microspheres to evaluate regional blood flow.

TransFluoSpheres™ products utilize two or more fluorophores that are carefully chosen to allow excited-state energy transfer, thus producing fluorescent microspheres with large Stokes shifts. 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). Because these TransFluoSpheres™ beads fluoresce at a wavelength that is considerably longer 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.

Time-resolved luminescence reagents like our europium and platinum luminescent FluoSpheres™ allow for detection of low levels of protein or DNA targets in the midst of nonspecific signals and autofluorescence. The FluoSpheres™ europium beads contain Eu³⁺ coordination complexes with luminescence decay times of >600 µsec, much longer than the <50 nsec 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 µsec. Thus, time-gated fluorescence detection using these microspheres avoids signal from autofluorescence. In addition, the europium luminescent microspheres feature long-wavelength emission (610–650 nm) that is well separated from their excitation peak (340–390 nm). The platinum luminescent microspheres are maximally excited near 390 nm with narrow emission that is maximal near 650 nm. 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.

Microspheres >1 µm in diameter can be centrifuged at 1,300 rpm. Centrifugation is not an effective way to collect smaller microspheres; for beads <1 µm in diameter, we recommend washing by either cross-flow filtration or dialysis with a 500 kDa MWCO.

We recommend using surface-modified microspheres in the following pH ranges:

Note: The polystyrene microspheres can tolerate up to pH 13, but handling the microspheres at this pH is difficult due to excessive aggregation.