Single-molecule measurements calibrate green fluorescent protein surface densities on transparent beads for use with ‘knock-in’ animals and other expression systems

doi:10.1016/S0165-0270(00)00354-X

Journal of Neuroscience Methods

Volume 105, Issue 1, 30 January 2001, Pages 55-63

https://doi.org/10.1016/S0165-0270(00)00354-X Get rights and content

Abstract

Quantitative aspects of synaptic transmission can be studied by inserting green fluorescent protein (GFP) moieties into the genes encoding membrane proteins. To provide calibrations for measurements on synapses expressing such proteins, we developed methods to quantify histidine-tagged GFP molecules (His₆-GFP) bound to Ni-NTA moieties on transparent beads (80–120 μm diameter) over a density range comprising nearly four orders of magnitude (to 30 000 GFP/μm²). The procedures employ commonly available Hg lamps, fluorescent microscopes, and CCD cameras. Two independent routes are employed: (1) single-molecule fluorescence measurements are made at the lowest GFP densities, providing an absolute calibration for macroscopic signals at higher GFP densities; (2) known numbers of His₆-GFP molecules are coupled quantitatively to the beads. Each of the two independent routes provides linear data over the measured density range, and the two independent methods agree with root mean square (rms) deviation of 11–21% over this range. These satisfactory results are obtained on two separate microscope systems. The data can be corrected for bleaching rates, which are linear with light intensity and become appreciable at intensities >∼1 W/cm². If a suitable GFP-tagged protein can be chosen and incorporated into a ‘knock-in’ animal, the density of the protein can be measured with an absolute accuracy on the order of 20%.

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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Single-molecule measurements calibrate green fluorescent protein surface densities on transparent beads for use with ‘knock-in’ animals and other expression systems

Abstract

Introduction

Section snippets

His6-GFP/Ni-NTA beads preparation

Single-molecule GFP Images

Overview of GFP calibrations

Acknowledgements

Neuron

J. Neurosci. Methods

Curr. Biol.

Biophys. J.

Neuron

Neuron

Biophys. J.

Curr. Opin. Struct. Biol.

Neuron

Curr. Biol.

Photophysics and optical switching in green fluorescent protein mutants

Proc. Natl. Acad. Sci. USA

His₆-GFP/Ni-NTA beads preparation