Article pubs.acs.org/Langmuir
Size of Submicrometer Particles Measured by FCS: Correction of the Confocal Volume Tobiasz Deptuła,†,‡ Johan Buitenhuis,§ Maciej Jarzębski,†,‡ Adam Patkowski,†,‡ and Jacek Gapinski*,†,‡ †
NanoBioMedical Center and ‡Faculty of Physics, A. Mickiewicz University, Umultowska 85, 61-614 Poznań, Poland § Institute of Complex Systems, ICS-3, Forschungszentrum Jülich, D-52425 Jülich, Germany ABSTRACT: When fluorescence correlation spectroscopy (FCS) in combination with a confocal microscope is used to determine the hydrodynamic radius a of particles comparable to or larger than the linear size σ of the confocal volume of the microscope, a correction must be used that depends on the a2/ σ2 ratio and the distribution of the dye within the particle. Here we present the experimental validation of the theoretically predicted approximate correction necessary for appropriate measurements of the size of uniformly fluorescently labeled spheres of radius comparable to the size of the confocal volume. We also test the approximate correction formula for different ranges of the a/σ ratio and propose a simple procedure to obtain the correct nanoparticle size from such a measurement. of radius a comparable to the lateral radius σ of the confocal volume is studied, a larger effective confocal volume has to be used for the calculation of the diffusion coefficient. This results from the fact that a large NP can be seen by the detector of the confocal microscope, even when its center is located substantially outside the confocal volume. The size of the effective confocal volume for a uniformly labeled spherical NP has been previously calculated,2,3 and the approximate simple correction formula was proposed. We first discuss the theoretical estimation of the effective confocal volume for spherical particles that are uniformly labeled and verify the simple correction formula by numerical simulations. Then we show an experimental test of the theoretical prediction using two kinds of uniformly labeled monodisperse fluorescent spheres of radius of about 200 and 600 nm, three objectives, and different pinhole sizes in the confocal microscope that result in different sizes of the confocal volume. We show that the simple correction formula holds in a broad range of the ratio of a/σ.
1. INTRODUCTION Nanoparticles (NPs) of sizes in the range of 1 nm to 1 μm became very important in medicine (drug delivery systems and diagnostics), food industry, and cosmetics. Therefore, many different kinds of nanoparticles are synthesized. Usually the crucial issue for their successful application is their size. Many different experimental techniques are used for a fast and accurate NP size determination. Several of them: scanning ion occlusion sensing (SIOS), differential centrifugal sedimentation (DCS), nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), scanning mobility particle sizing (SMPS), and dynamic light scattering (DLS), have been compared in a recent work.1 Another modern and important technique of NP size determination operating on a single molecule level is fluorescence correlation spectroscopy (FCS). Its great advantage is that it operates on small (∼20 μL) amounts of sample at nanomolar (1−10 nM) concentrations, and thus only a very small amount of sample is needed. Another advantage of FCS is that the effective volume used in this method is of the linear size of
