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Applied Optics

Prism-based excitation wavelength selection for multicolor fluorescence coincidence measurements

Roel Kassies, Aufried T. M. Lenferink, Ine M. J. Segers‐Nolten, Cees Otto

Published Feb 20, 2005• 11 citations
Publisher

Abstract

We have designed and constructed a prism setup for multiple-color confocal fluorescence experiments. The prism setup permits easy selection of any color or any combination of colors from a multicolor light source, such as a mixed-gas argon-krypton-ion laser. The selected colors emerging from the prism setup are, by design, optimally overlapped in the focus of a high-numerical-aperture objective, such as that commonly used in single-molecule fluorescence experiments. The various excitation powers can be easily adjusted in this setup. We will exemplify the potential of this setup in single-molecule fluorescence emission spectroscopy.

DOI
10.1364/ao.44.000893

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We have designed and constructed a prism setup for multiple-color confocal fluorescence experiments. The prism setup permits easy selection of any color or any combination of colors from a multicolor light source, such as a mixed-gas argon-krypton-ion laser. The selected colors emerging from the prism setup are, by design, optimally overlapped in the focus of a high-numerical-aperture objective, such as that commonly used in single-molecule fluorescence experiments. The various excitation powers can be easily adjusted in this setup. We will exemplify the potential of this setup in single-molecule fluorescence emission spectroscopy.
In this study, we demonstrate that the fluorescence of fluorescein dye molecules can be significantly modified in both the weak and strong coupling regimes by placing the molecules inside a perturbative or non-perturbative Fabry-Pérot cavity. By controlling the cavity thickness and dye concentration, we selectively altered the frequency and number of cavity transmission peaks, thereby establishing conditions for weak or strong coupling for fluorescein molecules. Using steady-state fluorescence spectroscopy, we confirmed that both the excitation and emission spectra were substantially tuned in these coupling regimes, enabling tunable and multicolor fluorescence from a single type of dye molecule, fluorescein.
We describe a dual-color three-dimensional stimulated emission depletion (3D-STED) microscopy employing a single laser source with a repetition rate of 80 MHz. Multiple excitation pulses synchronized with a STED pulse were generated by a photonic crystal fiber, and the desired wavelengths were selected by an acousto-optic tunable filter with high spectral purity. Selective excitation at different wavelengths permits simultaneous imaging of two fluorescent markers at a nanoscale resolution in three dimensions.
High-peak-power ultrafast lasers are widely used in nonlinear spectroscopy, but often limit its spectral resolution because of the broad frequency bandwidth of ultrashort laser pulses. Improving the resolution by achieving spectrally narrow excitation of, or emission from, the resonant medium by means of multiphoton interferences has been the focus of many recent developments in ultrafast spectroscopy. We demonstrate an alternative approach in which high resolution is exercised by detecting narrow spectral correlations between broadband excitation and emission optical fields. All-optical correlation analysis, easily incorporated into a traditional spectroscopic setup, enables direct, robust, and simultaneous detection of multiple narrow resonances in a single measurement.
Acquisition of quantitative information from microscopic biological samples is highly desirable in the context of the emerging field of systems biology. We derive a statistical approach to estimate the number of fluorescent molecules in the observation volume based on a confocal microscope for single-molecule detection. The method employs ps-pulsed laser sources for excitation and time-correlated single-photon counting with 4 avalanche photon diodes (APDs) for detection of individual photons. The feasibility for estimating the number of molecules is shown based on simultaneous emission and detection of multiple photons (photon-antibunching) under realistic experimental conditions. In theory, it should be possible to estimate the number of molecules with errors of less than 1% by using novel photo-stabilizing agents. The proposed method puts into perspective its application for high-resolution microscopy without the need for photo-switching or photo-activation of fluorescence dyes.
A single-photon source is an essential tool for the emerging field of quantum technologies. Ideally, it should be spectrally compatible with other photonic devices while providing a high flux of narrow-band photons. The single organic dye molecule dibenzanthanthrene under cryogenic conditions possesses the given characteristics and therefore constitutes a prominent single-photon source. Nevertheless, the implementation of such a single-photon source requires a complex experimental setup involving a cryostat with a confocal microscope for the effective collection of the molecular emission. In the approach presented here we use a single emitter coupled directly to the end facet of an optical fiber. This has the potential to transfer a single-photon source based on a quantum emitter from a proof-of-principle type of setup to a scalable ``plug-and-play'' device. We present successful coupling of a single organic molecule to an optical fiber and record the excitation spectrum, measure the saturation curve, and analyze the contributions of Raman background fluorescence. The single-photon nature is proven by an antibunched autocorrelation recording, which reveals coherent Rabi oscillations.
The use of highly monochromatic light permits the selective excitation of atoms in vapors if excitation and detection of the fluorescence is carried out collinearly. The atoms capable of absorbing light then form an atomic beam of well-defined velocity along the direction of the laser beam, but no velocity selection occurs perpendicular to it. The potential of the technique for Doppler-free atomic spectroscopy and for the study of excited atom collisions is demonstrated by using the Na D1 line as an example.