Evanescent-wave fluorescence excitation in aqueous solutions using symmetric planar waveguides

We describe a method of delivering light to biological samples using a planar waveguide structure with a symmetric cladding environment. The symmetry of the waveguiding structure allows the penetration depth of the evanescent field to be tuned over a wide range by varying the thickness and/or refractive index of the waveguide. Furthermore, the symmetrical structure facilitates efficient excitation of waveguide modes using, e.g., optical fibers. We have performed fluorescence excitation experiments using of dielectric as well as metallic (surface plasmon) waveguides. The method is well suited for evanescent wave microscopy of biological samples, surface sensing and other applications that require illumination spread over macroscopic areas but confined to penetration depths ranging from 100 nm up to several μm. Introduction Fluorescence microscopy (FM) can be regarded as one of the most important characterization techniques within cell biology, molecular biology and related fields [1]. Although most FM studies are carried out using conventional epi-fluorescence microscopes, there exists a wide range of more specialized techniques, including confocal laser scanning microscopy, two-photon absorption, resonance energy transfer and total internal reflection fluorescence microscopy (TIR-FM). In TIR-FM, the excitation light is usually incident on the sample from a substrate with higher refractive index (e.g. a thin glass slide) under an angle large enough for it to undergo total internal reflection at the substrate-sample interface. Fluorescence in the sample is therefore excited only within the penetration depth of the exponentially decaying evanescent field associated with the total internal reflection, typically extending approximately 100 nm into the low-index medium. A review of TIR-FM can be found in Ref. [2]. Fluorescence excitation by an evanescent field can also be accomplished using a planar waveguiding structure, where the sample (typically an aqueous solution) forms one of the waveguide cladding layers. The penetration depth of the evanescent field into the sample in this case is defined by the relationship between the refractive index of the sample, substrate and waveguide materials, as well as the thickness of the waveguide layer. In this paper, we will present a new type of planar waveguide structure developed specifically for fluorescence microscopy applications [3], where the cladding material is chosen to match the refractive index of the sample. We discuss the special features of this waveguiding geometry and present experimental results obtained on samples containing fluorescent beads. In particular, we will focus on surfaceplasmon excited fluorescence which currently attracts considerable interest in biological research [4]. Waveguide-excitation fluorescence microscopy The use of planar waveguide structures for fluorescence excitation and microscopy has been previously reported in the literature [5]. The waveguide structure consisted of a glass substrate coated with a highindex thin film supporting a guided mode with 100– 200 nm penetration depth of the evanescent field. A similar waveguide configuration was used to realize two-photon fluorescence excitation over a macroscopic surface area [6]. Conversely, Horváth et al. [7] developed a so-called “reverse-symmetry” waveguide sensor using nanoporous silica (n=1.2) as bottom cladding in order to greatly increase the penetration depth of the guided mode into the sample. This type of sensor geometry has been used to monitor cell attachment and spreading [8] but, to our knowledge, it has not been used for FM applications. In the abovementioned experiments, light was coupled into the waveguides using surface gratings illuminated by light incident under a specific resonance angle that depends on the grating period, effective waveguide index and the excitation wavelength. Due to the asymmetry in the refractive index of the top and bottom cladding layers, the waveguides have a cut-off thickness below which no guided mode is supported by the structure, limiting the range of possible penetration depths. The symmetric waveguide structures demonstrated in the present paper provide several advantages over previous designs. The penetration depth of the symmetric mode can be tuned over a wide range without cut-off. The guided mode can be efficiently excited directly from an optical fiber, eliminating the need for patterning of gratings and resonance-angle excitation. This also implies that the sample can be excited with multiple wavelengths through the same fiber. Aside from the fiber-coupled light source, the necessary components are no more than a few millimeters in height, meaning that a standard light microscope (normal or inverted) can be configured to deliver TIR-FM performance. ThG01