Correlational analysis of proteins and nonmetallic nanoparticles in a deep-nulling microscope.
We present a method for label-free microscopic analysis of nonmetallic nanoparticles such as biopolymers or technical polymers diffusing freely in an aqueous environment. We demonstrate the principal feasibility of the approach with first measurements of 20-200 nm sized polystyrene spheres and of the approximately 10 nm protein complex Photosystem I (PS I) of Thermosynechococcus elongatus. The approach is based on the combination of a microscope setup with a deep-nulling interferometer for measuring minute refractive index changes or absorptions in the focal area of the microscope. It is possible to obtain transient nulls in a microscope setup on the order of 10(-5), corresponding to optical pathway differences of less than 0.6 nm and to stabilize the nulls to about 5.10(-4). With this level of stabilization it is possible to perform a fast (1 s) correlational analysis of aqueous solutions containing the protein complex PS I or 20 nm spheres and to detect in real time single diffusional transits of larger nanospheres through the focal area of the microscope. A modulated heating of the samples in the microscope focus is not necessary for detection. The interferometer correlation data correspond well to conventional two-photon excited fluorescence correlation (FCS) data measured in the same setup, providing evidence that the detection volumes are of a similar size (approximately 200 nm). We also conducted first nulling experiments using coherent near-field sources of about 30 nm diameter. Theoretical considerations indicate that this combination with nanometric near-field sources will even allow label-free single-protein analysis.
Hilbert
Michael
M
Max-Planck-Institute for Biophysical Chemistry, Department 010, Spectroscopy and Photochemical Kinetics, Am Fassberg 11, D-37077 Göttingen, Germany.
Hippchen
Hendrik
H
Wehling
Axel
A
Walla
Peter J
PJ
D001426:Bacterial Proteins / Q000737:chemistry; D007368:Interferometry / Q000379:methods; D008853:Microscopy / Q000379:methods; D053758:Nanoparticles / Q000737:chemistry*; D045331:Photosystem I Protein Complex / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D046940:Synechococcus / Q000737:chemistry
D001426:Bacterial Proteins; D045331:Photosystem I Protein Complex; D011506:Proteins