20–25 However, until today there exists no systematic investigation on the influence of fluorescence on these particle size measurement techniques. There are several publications on the comparison of DLS and SAXS size measurements for different particle systems such as micelles, proteins, and polymers. 18,19 However, many researcher that work with fluorescent nanoparticles for bioanalytical applications still use common DLS systems to characterize their particle samples, due to the wide availability and broad application range of this established technique. To overcome the limitations of common DLS, modified and improved optical scattering techniques have been developed, such as 3D cross-correlation DLS, 12–15 DLS using a flat cell light scattering instrument, 16 diffusing wave spectroscopy, 17 or photon density wave spectroscopy. 11 Thus, DLS measurements of strongly absorbing samples and highly turbid colloidal systems can be challenging. Moreover, strong absorption can cause unwanted side effects such as local sample heating, beam expansion, and convection, that interfere with the sample characterization. Depending on the optical properties and the wavelength of the laser used for the DLS measurements, absorption can result in a partial loss of the coherent incident light and the subsequently emitted non-coherent fluorescence may also affect the measured signal. 10 Whereas SAXS depends on the scattering of X-rays based upon the electron density of the particles, which should be independent of any fluorescence signals, DLS relies on light scattering and may thus be affected by the absorption and emission of labelled dyes or self-luminescent nanomaterials.
Two commonly used techniques to determine the sizes of nanoparticles are dynamic light scattering (DLS), available in many different laboratories preparing or working with nanoparticles, and the more sophisticated small angle X-ray scattering (SAXS), that can be made traceable to the International System of Units (SI).
number of functional groups at the surface) and particle charge.
1–7 Particle size is a key parameter that determines the behavior and colloidal stability of particle reporters and their interaction with biological systems together with the surface chemistry ( i.e. Introduction Fluorescent nanoparticles such as dye-stained polymer particles, dye-labelled silica-particles and semiconductor quantum dots are increasingly used as reporters in various bioanalytical applications for in vivo and in vitro spectroscopy and imaging. only measurable for the dye-stained particles that absorb at the laser wavelength used for the DLS measurements. This effect was wavelength dependent, i.e. Nevertheless, a significant decrease of the detected correlation coefficients was observed with increasing dye concentration, due to the increased absorption of the incident light and thus, less coherent light scattering.
#DYNAMIC LIGHT SCATTERING INSTRUMENT USING MALVERN ZETASIZER SERIES#
DLS measurements carried out at three different laboratories using four different DLS instruments and two different laser wavelengths, i.e., 532 nm and 633 nm, revealed also no significant changes in size (intensity-weighted harmonic mean diameter, Z-Average) and size distribution (polydispersity index, PI) within and between the two dye-stained particle series and the blank sample. SAXS measurements of these particle series and a corresponding blank control (without dye) revealed similar sizes of all particles within an uncertainty of 1 nm. For this purpose, two series of 100 nm-sized polymer nanoparticles stained with different concentrations of the fluorescent dyes DY555 and DY680 were prepared, absorbing/emitting at around 560 nm/590 nm and 695 nm/715 nm, respectively. The influence of fluorescence on nanoparticle size measurements using dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) was investigated.