Fluorescence interferometry: principles and applications in biology

Abstract

The use of fluorescence radiation is of fundamental importance for tackling measurement problems in the life sciences, with recent demonstrations of probing biological systems at the nanoscale. Usually, fluorescent light-based tools and techniques use the intensity of light waves, which is easily measured by detectors. However, the phase of a fluorescence wave contains subtle, but no less important, information about the wave; yet, it has been largely unexplored. Here, we introduce the concept of fluorescence interferometry to allow the measurement of phase information of fluorescent light waves. In principle, fluorescence interferometry can be considered a unique form of optical low-coherence interferometry that uses fluorophores as a light source of low temporal coherence. Fluorescence interferometry opens up new avenues for developing new fluorescent light-based imaging, sensing, ranging, and profiling methods that to some extent resemble interferometric techniques based on white light sources. We propose two experimental realizations of fluorescence interferometry that detect the interference pattern cast by the fluorescence fields. This article discusses their measurement capabilities and limitations and compares them with those offered by optical low-coherence interferometric schemes. We also describe applications of fluorescence interferometry to imaging, ranging, and profiling tasks and present experimental evidences of wide-field cross-sectional imaging with high resolution and large range of depth, as well as quantitative profiling with nanometer-level precision. Finally, we point out future research directions in fluorescence interferometry, such as fluorescence tomography of whole organisms and the extension to molecular interferometry by means of quantum dots and bioluminescence.

FIGURE 1.

Dependence of various fluorescence imaging techniques on spatial resolution and depth of penetration. FRET, fluorescence resonance energy transfer; NSOM, near-field scanning optical microscopy; PALM, photoactivated localization microscopy; STORM, stochastic reconstruction microscopy; STED, stimulated emission depletion; I⁵M, image interference and incoherent interference illumination microscopy; OPT, optical projection tomography; SPIM, selective plane illumination microscopy; FMT, fluorescence molecular tomography; BLI/BLT, bioluminescence imaging/tomography.

FIGURE 2.

Operational principles of fluorescence interferometry. Fluorescence waves emitted from an excited fluorophore located between two matched, opposing lenses are directed using mirrors to a beam splitter, where they combine. An interference pattern is detected when the fluorophore is close to point 2, that is, near the zero-differential path length point (z₀) of the interferometer. For locations far from z₀ (points 1 and 3), only the constant fluorescence intensity is recorded.

FIGURE 3.

Time-domain fluorescence interferometry. (A) Experimental realization, (B) detected signal for wide-field excitation, and (C) detected signal for point excitation.

FIGURE 4.

Fourier domain fluorescence interferometry. (A) Experimental realization, (B) line-focus excitation, and (C) self-interference fluorescence from the sample is imaged along the transversal dimension and spectrally resolved in the spectral dimension of the two-dimensional detection array. The axial (depth) ranging profile of the fluorophore distribution along each transversal sample location is obtained by inverse discrete Fourier transforming (DFT⁻¹) each horizontal CCD line. (Reproduced from Ref. , figure 1, with permission of the Optical Society of America.)

FIGURE 5.

SD-FCT cross-sectional imaging of fluorescent samples. (A) Dual-layer fluorescent phantom, (B) fluorescence emission distribution along the axial (z) and transversal (y) coordinates of the phantom, and (C) SD-FCT tomogram of the two-layered fluorescent sample. (Reproduced from Ref. , figure 6, with permission of the Optical Society of America.)

FIGURE 6.

SD-FCT nanometer-level profiling of transparent surfaces. (A) Axial displacement sensitivity of SD-FCT and (B) SD-FCT profile of a nanoetched surface.