A full fiber-optic fluorescence correlation spectroscopy (FF-FCS) technique has been developed without the use of objectives and dichroic mirrors. To achieve this, an excitation laser has been focused onto a sample by a lensed optical fiber or a gradient index lens attached on the terminal surface of the optical fiber. The FF-FCS system does not exhibit a higher sensitivity than the conventional FCS system; however, it is much simpler and smaller. This work demonstrates the feasibility of FF-FCS by measuring fluorescent beads. In the future, we expect FF-FCS to be widely used as a laboratory tool and an embedded tool for quality-control systems, such as cytometers.
© 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
Fluorescence correlation spectroscopy (FCS) [1,2] is a powerful tool for studying intermolecular interactions and microenvironments even in living cells. The molecules of interest are fluorescently labeled, and the random fluctuation of the fluorescence intensity caused by the fluorescent molecule is analyzed inside a measured volume element. As a result, FCS determines the speed of translational diffusion and the number of fluorescently labeled molecules. The translational diffusion provides information about intermolecular interactions and the surrounding environment.
FCS is now widely used, especially in biological contexts, to measure the protein concentration, dissociation constant of homodimers, and interactions between proteins and DNA in living cells [3–5]. Fluorescence cross-correlation spectroscopy (FCCS) is more appropriate for measuring the dissociation constant between two specific molecules because of its two-color measurement [6–10]. Advanced techniques, such as polarization-dependent FCS (Pol-FCS) [11,12], fluorescence lifetime correlation spectroscopy (FLCS) [13,14], multipoint FCS [14–16], and STED-FCS , have recently been developed for more specific fields in life science. However, most commercial FCS systems are still too expensive for use in all laboratories. One of the reasons for this is the use of high-NA objectives and sophisticated dichroic mirror systems equipped in fluorescence microscopy to generate sub-femtoliter confocal volumes and high-efficiency fluorescence detection. A smaller confocal volume is generally better in FCS systems because the smaller number of molecules in the confocal volume leads to a larger fluorescence intensity fluctuation. In addition, high-NA objectives can effectively correct a weak fluorescence from single fluorescent molecules. Therefore, to this end, the commercial FCS setup is mounted on a high-end fluorescence confocal microscope using high-NA objectives. Accordingly, commercial FCS systems tend to be expensive and large in size.
Meanwhile, some efforts have been made toward achieving compact FCS systems mainly based on optical fiber systems [18–20]. In principal, their design concept is the same as that of the conventional FCS system using an objective, a dichroic mirror, and a pinhole. The pinhole is sometimes replaced by an entry of optical fiber with an appropriate core diameter.
We propose herein a simple and small FCS system based on fiber optics, called full fiber-optic FCS (FF-FCS). The FF-FCS system does not contain any objectives, dichroic mirrors, or pinhole apparatus; instead, it uses lensed optical fiber and an optical fiber coupler with a high coupling ratio. The lensed fiber cannot focus the excitation laser tightly like objectives; however, it is sufficient for dilute and bright particles that contain several fluorophores. The optical fiber coupler plays the role of an optical circulator for visible light, and the fluorescence emitted from samples can be effectively separated without a dichroic mirror.
We discuss the FF-FCS measurements for polystyrene fluorescence beads and demonstrate the feasibility of FF-FCS in this paper.
2. Full fiber-optic fluorescence correlation spectroscopy (FF-FCS)
2.1. Experimental setup
Figure 1 shows the schematic for the FF-FCS setup. A fiber output of the laser diode (LD) with a wavelength of 488 nm (LP488-SF20, Thorlabs, USA) was connected to port A of a 2 × 2 optical fiber coupler with a branching ratio of 99:1 (FC488-99B-FC, Thorlabs, USA). Subsequently, 99% of the excitation laser was outputted from port C, and the laser was absorbed by a fiber-optic light terminator (LT) (FTFC1, Thorlabs, USA). The remaining 1% of the excitation laser was guided to a lensed optical fiber (CL1-FC3, WT&T, Canada) connected to port D. The lensed end was directly dipped into the samples, and 1% of the excitation laser was focused on the sample. Only the fluorescence emitted from the focal region propagated back to the 2 × 2 fiber coupler. After which, 99% of the fluorescence was outputted from port B. In FCS, the excitation laser is usually used with ND filters to avoid photobleaching by an excitation laser that is too strong. In this system, the 2 × 2 fiber coupler plays the role of the dichroic mirror and the ND filter.
The fluorescence at port B was filtered by an emission filter (EF) (FF01-530/43-25, Semrock, USA) to eliminate the scattered/reflected light that could act as a possible background noise. The emission filter was mounted on an in-line multimode fiber optic filter mount (FOFMF/M, Thorlabs, USA), where the light outputted from port B was collimated and re-coupled to the multimode fiber (MMF) by two parabolic mirrors. Finally, the fluorescence was guided to a photomultiplier tube (PMT) (Photon counting head, H7421-40, Hamamatsu, Japan) by an MMF. The autocorrelation function (ACF) of the photon counting signal from the PMT was calculated by a laboratory-made software correlator using a laboratory-made hardware counter based on a field-programmable gate array (FPGA) (10M08SAE144C8G, Intel, USA).
In this system, all optical elements are connected by an FC/PC connector of optical fiber, and no parts require position adjustment of the optical axis. Furthermore, the fluorescence from the focal point is inevitably corrected by the lensed fiber without any adjustment, and the shape of confocal volume is hard to change because the optical fiber for illumination and correction of fluorescence is the same. In other words, the FF-FCS is very robust and easy to construct and operate, which is a very big benefit for users and embedded systems.
Unfortunately, the lensed fiber is not a well-designed lens like the objective lens, and the core size of the optical fiber for fluorescence correction cannot independently optimize, thereby degrading the system sensitivity. In the conventional FCS system, the pinhole for excitation and that for fluorescence detection are not usually the same. The general FCS system can collect fluorescence with high sensitivity, but it needs pinhole adjustment before the experiment, and the shape of the confocal volume can be easily deformed.
A control FCS measurement was performed herein with an LSM 510 ConforCor3 (Carl Zeiss)-equipped objective (NA 1.2, 40 × ) acting as the conventional FCS system.
2.2. Data analysis
FF-FCS essentially involves the same analytical procedure as that of a conventional FCS. Therefore, the ACF for a single diffusing species for FF-FCS is expressed as follows :4]:
The ACF obtained by FF-FCS was analyzed by fitting all obtained values to Eq. (1) using the nonlinear least-squares method. In the following experiments, the structure parameter was fixed at 1.0. Eleven measurements were independently repeated on the fluorescence latex beads (FluoSpheresTM, F13081, Molecular Probes, USA) to determine the structure parameter. The results with the structure parameter were then fitted as a free parameter. The result was 0.9981 ± 0.0011 (average ± standard error). In the conventional FCS system, the structure parameter is usually approximately 5 to 10 using well-designed objective lens. The structure parameter of the lensed fiber might be affected by spherical and chromatic aberration.
3. Results and discussion
3.1. Particle size dependence
FF-FCS measurements were performed for the dispersion of fluorescent latex beads with diameters of 20, 40, and 100 nm in water (FluoSpheresTM, F8787, F8795, and F13081, Molecular Probes, USA). The measurement of a monomeric green fluorescent protein by FF-FCS was not successful because of its weak fluorescence compared with that of fluorescent beads (data not shown). The excitation laser power at the focal plane of the lensed fiber was 7.29, 7.29, and 5.20 μW for these beads, respectively.
Figures 2(a) and 2(c) show the ACF and the normalized ACF, respectively. Figure 2(b) shows the residuals of fitting for each ACFs. A shift in the relaxation time of the normalized ACF with an increase in the particle diameter can be clearly observed in Fig. 2(c). The relatively high deviation of the ACF of 20 nm was caused by the low fluorescence brightness. Figure 2(d) shows the relationship between the diffusion time and the diameter of the particles. According to the Einstein–Stokes equation, the diffusion coefficient is inversely proportional to the particle radius; therefore, the diffusion time is linearly proportional to the particle radius. In other words, the linear relationship in Fig. 2(d) is in good agreement with theory, indicating that FF-FCS can quantitatively measure the diameter of fluorescent particles.
3.2. Concentration dependence
The concentration dependence on the ACF amplitude in FF-FCS was confirmed using a dilution series of fluorescent latex beads (FluoSpheresTM, F13081, Molecular Probes, USA), an excitation laser power of 5.20 μW, and a measurement duration of 100 s (10 s measurements repeated in 10 loops). Figures 3(a) and 3(b) show the ACFs obtained by FF-FCS and residuals of fitting analysis. The amplitude was observed to increase with the decreasing particle concentration because it was inversely proportional to the average number of particles inside the volume element. Figure 3(c) shows the relationship between the number of particles obtained by FF-FCS and the given concentration. Clear linearity and small standard errors were observed. Figure 3(d) shows that the diffusion time was independent of the concentration. The results indicated that FF-FCS can quantify the concentration of fluorescent particles independent of the diffusion time. Using Eq. (2) and the Einstein–Stokes equation, the lateral radius of the measurement volume was estimated as 1.41 μm for τD = 0.1 s, which is over three times larger than that of a water immersion objective lens with a numerical aperture of 1.2 used in conventional FCS systems (LSM 510 CofoCor3). The estimated measurement volume was 15.4 fL, which is approximately 100 times larger than that of the objective lens.
The lensed fiber diameter is smaller than 1 mm, implying that the FF-FCS system can be combined into endoscopes/laparoscopes, making FF-FCS measurements in living animals possible in the future. The lensed fiber is much cheaper than the objective lens, and is disposable; therefore, FF-FCS can be applied to extreme environments, such as those with very low temperatures or very low/high pressures, and medical diagnosis as a disposable medical equipment. FF-FCS used an emission filter (EF in Fig. 1) to eliminate scattered light and background noise at long wavelengths, which will be replaced by the wavelength division multiplexer or fiber Bragg grating in the near future to complete the fiber system.
In this work, we developed and demonstrated FF-FCS, which does not need an objective lens, a dichroic mirror, or a fluorescence microscope. FF-FCS uses a much simpler and smaller system than a conventional confocal FCS system. Furthermore, it can be realized by connecting all the fiber components, thereby making laborious pinhole adjustment unnecessary.
The fluorescence detection efficiency of FF-FCS is much lower than that of the conventional FCS. FF-FCS is not so sensitive that the large size of a structural object, such as cells, cell debris, or particle of exosome, would be the target in the deal for applications. GRIN lens-attached optical fiber can also be used instead of the lensed fiber. A suitably designed GRIN lens would improve the FF-FCS sensitivity.
The FF-FCS system is cheap, compact, and robust; hence, the technique can be widely used in many laboratories and measurement systems, such as in cytometers, production lines, and garage laboratories.
Canon Foundation; Uehara Memorial Foundation; JSPS KAKENHI (JP16K07312).
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