Correlational Analysis of Proteins and Nonmetallic Nanoparticles in a Deep-Nulling Microscope

Abstract

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 ∼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 (∼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.

Photothermal Detection of Individual Gold Nanoparticles: Perspectives for High‐Throughput Screening

“…Recently, several techniques have emerged which allow the analysis of individual nanoparticles by means of photothermally detected absorption [13][14][15][16][17][18][19][20] or interferometry. [21][22][23][24] Here, we show that a specific implementation of the photothermal heterodyne detection technique [17,18] can address a dramatically extended detection volume while still not requiring too long integration times to discriminate quickly and reliably between a target species and the background signal from the empty solution.…”

Generation of Nanopores Down to 10 nm for Use in Deep‐Nulling Interferometry

“…[12] The nulling depth D is defined as D = I exit /I max , where I max is the total light intensity passing the detection area and I exit is the actual residual intensity detected at the exit of the interferometer. Using a 3D piezo stage (P611, E664, PI Systems, Karlsruhe, Germany) the nanopores were adjusted into the focus of coherent light emerging from a He-Ne laser (632.8 nm, 1 mW, Melles Griot, Bensheim, Germany).…”

“…[12] These results constitute important steps towards the ultimate goal of detecting label-free single biomolecules or other nanoparticles diffusing freely in a physiological, aqueous environment. …”

“…[12] These improvements were accomplished simply by significantly enhancing the vibration isolation of the setup.…”

“…[11] Herein, we present the combination of coherent near-field light sources with a deep-nulling interferometer. Deep-nulling microscopy [12] enables the label-free detection of biomolecules, or other nanoparticles that diffuse quickly through the focal volume of a microscope, with a sensitivity up to the single-particle level.…”