Single-molecule measurements calibrate green fluorescent protein surface densities on transparent beads for use with ‘knock-in’ animals and other expression systems
Introduction
Several problems in synaptic transmission call for knowledge about the absolute surface density of receptors, channels, and transporters (Anglister et al., 1994, Lester et al., 1996, Nusser et al., 1998). In one potential route to such measurements, the gene for the membrane protein is replaced by a construct containing the protein fused to green fluorescent protein (GFP). If the GFP is maintained in a monomeric state and otherwise prevented from interacting with other chromophores, the fluorescence properties are independent of ionic strength, polarity of the solution, and other conditions that might be encountered in living cells (Tsien, 1998, Lippincott-Schwartz et al., 1999, Piston et al., 1999). Furthermore, the resolution of the fluorescence microscope (better than ∼0.5 μm) corresponds well (1) to the size of individual synapses and (2) to the distance neurotransmitter molecules are expected to diffuse during the time of chemical synaptic transmission (∼3 μm during ∼10 ms).
Such ‘knock-in’ animals will yield useful data if one has methods for absolute quantification of the fluorescence protein density. For this purpose, we have chosen to couple GFP to the surface of transparent beads large enough (∼90 μm average diameter) to present a functionally flat surface on the distance scale of μm. Such calibrated beads can be introduced into microscopic preparations as internal standards. Commercially available fluorescent beads are available from several sources with precise diameters, and the FocalCheck microspheres from Molecular Probes have dye on the surface only; but dye densities are neither controlled or specified.
This study addresses the challenge of calibrating GFP-coated beads. It would be inappropriate to rely solely on the macroscopic method of coupling known masses of GFP — and therefore known numbers of GFP molecules — to the beads. Such measurements could be distorted if an appreciable fraction of the coupled molecules do not fluoresce, for instance because of changes during purification or because of interactions with the bead surface. The most rigorous method for calibration employs recently developed procedures to measure the fluorescence of single GFP molecules at low surface densities under microscopes in common laboratory use (Unger et al., 1999). Although it is unlikely that densities this low would be generally interesting, the linearity of CCD detectors and the use of proper neutral-density filters allow one to extrapolate to much higher surface densities.
We have compared this single-molecule fluorescence method to the macroscopic method, which we optimize by amino-acid analyses to measure the number of GFP molecules most accurately. We report both excellent linearity and excellent agreement between these two methods, providing known densities of GFP molecules over a range of nearly four orders of magnitude that span expected membrane densities of channels, receptors, and transporters. As a result, the beads provide a simple and versatile tool for absolute quantification of GFP densities.
Section snippets
His6-GFP/Ni-NTA beads preparation
We employed GFP37, a GFP mutant containing the S65T, V163A, I167T, and S175G mutations (Siemering et al., 1996, Grabner et al., 1998). The S65T mutation increases the brightness and shifts the absorbance peak from 397 to 488 nm; the emission peak (at 509 nm) remains close to that of the wild type (504 nm). The additional three mutations allow for more efficient GFP expression at 37°C.
The procedures for preparing histidine-tagged GFP (His6-GFP), for cleaning slides, and for minimizing background
Single-molecule GFP Images
Fig. 1 presents images of single His6-GFP molecules. The GFP fluorescent spots were not observed for blank beads, increased in density roughly in proportion to the increased expected density and were observed only when the GFP specific filters were used. We observed all-or-none bleaching and occasionally blinking (Unger et al., 1999). These observations support our interpretation that we observed fluorescence from single GFP molecules in the beads.
Two imaging systems were used for both the
Overview of GFP calibrations
The major result of this study is a set of procedures for calibrating transparent beads with surface densities of His6-GFP. These procedures yield results that are internally consistent in two ways. First, fluorescence intensities are linearly proportional to the amount of GFP coupled to the beads, over nearly 4 orders of magnitude. Second, the absolute calibration (GFP/μm2) based on single-molecule fluorescence agrees, with an rms deviation of 11–21%, with the absolute calibration based on the
Acknowledgements
We thank K. Beam for the GFP37 construct, N. Dinh and M. Young (City of Hope National Medical Center) for amino acid analysis, M. Simon for providing the Nikon microscope, and N. Davidson, R. Farley, K. Philipson, and B. Khakh for discussion. This work was supported by grants from the NIH (NS-11756, DA-09121).
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