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Supercritical Angle Fluorescence Characterization Using Spatially Resolved Fourier Plane Spectroscopy Finub James Shirley, Pieter Neutens, Rita Vos, Md MahmudUl-Hasan, Liesbet Lagae, Niels Verellen, and Pol Van Dorpe Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ acs.analchem.7b04822 • Publication Date (Web): 12 Mar 2018 Downloaded from http://pubs.acs.org on March 12, 2018
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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Analytical Chemistry
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Analytical Chemistry Page 2 of 11 Bulk fluorophores Bound fluorophores
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Analytical Chemistry
Supercritical Angle Fluorescence Characterization Using Spatially Resolved Fourier Plane Spectroscopy Finub James Shirley,†,‡ Pieter Neutens,‡ Rita Vos,‡ Md. Mahmud-Ul-Hasan,†,‡ Liesbet Lagae,‡,† Niels Verellen,‡,† and Pol Van Dorpe∗,‡,† †KU Leuven, Laboratory of Solid-State Physics and Magnetism, Celestijnenlaan 200D, B-3001 Leuven, Belgium. ‡imec, Kapeldreef 75, 3001 Heverlee, Belgium. E-mail:
[email protected] Abstract
Introduction
Most fluorescent immunoassays require a wash step prior to read-out due to the otherwise overwhelming signal of the large number of unbound (bulk) fluorescent molecules, that dominate over the signal from the molecules of interest, usually bound to a substrate. Supercritical angle fluorescence (SAF) sensing is one of the most promising alternatives to total internal reflection fluorescence for fluorescence imaging and sensing. However, detailed experimental investigation of the influence of collection angle on the SAF surface sensitivity, i.e. signal to background ratio (SBR), is still lacking. In this letter, we present a novel technique that allows to discriminate the emission patterns of free and bound fluorophores simultaneously, by collecting both angular and spectral information. The spectrum was probed at multiple positions in the back focal plane using a multimode fiber connected to a spectrometer and the difference in intensity between two fluorophores was used to calculate the SBR. Our study clearly reveals that increasing the angle of SAF collection enhances the surface sensitivity, albeit at the cost of decreased signal intensity. Furthermore, our findings are fully supported by full-field 3D simulations.
Fluorescence is one of the most widely used methods for the study of biological specimen. 1,2 The fluorescence microscope has undergone a number of improvements over the years to improve its sensitivity towards specific applications. 3,4 One of the most widely used instruments for measuring fluorophore labeled surfaces is the Total Internal Reflection Fluorescence (TIRF) microscope. 5–7 Here, total internal reflection of light creates a non-propagating evanescent field extending out of a, typically glass, surface. This field is then used to excite fluorophores with a very high surface selectivity due to the exponentially decaying nature of the light field intensity away from the interface surface. Biosensors based on TIRF have been created using waveguides. 8 In contrast to TIRF, which only excites fluorophores close to the surface, supercritical angle fluorescence (SAF) only collects fluorescence generated close to the surface. 9,10 SAF is observed when the evanescent field surrounding an emitter becomes propagating in close proximity to i.e. distance d < emission wavelength λ, a high refractive index contrast interface. The emitter has to be within the low refractive index medium and the SAF is emitted above the critical angle (θc ) into the higher refractive index
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Analytical Chemistry
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Figure 1: a) Schematic showing the angular distribution of undercritical angle fluorescence (UAF) and supercritical angle fluorescence (SAF) emission of a fluorophore close to an interface. b) Expected evolution of SAF intensity with increasing d, the distance between the fluorophore and the interface.
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Figure 2: Schematic showing the emission of fluorophores at different distances (d) from the interface where: a) d > λ and b) d < λ, where λ is the wavelength of light. Two different fluorophores are used to represent the two positions. The combined contribution of the two fluorophores is represented in panel c. The corresponding images to the right show the angular distribution of fluorophore emission in the back focal plane. The center of the circles is at θ = 0◦ . A line drawn starting from the center towards the outside increases in θ with increasing distance from the center. In this paper, we propose a method to measure the surface sensitivity of SAF sensing (Figure 2). Two different fluorophores were used for the experiment and their spectrum was measured at different positions in the back focal plane. At the back focal plane or Fourier plane, light traveling at like angles from multiple point sources combines to form points where each point corresponds to a unique angle. The two fluorophores were selected such that they had emission peaks that could be distinguished from
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Analytical Chemistry
each other, while still being excitable with a single excitation source. One fluorophore was bound to the surface and the other was suspended in the bulk solution. The bulk fluorophore represents the excess detection antibodies i.e. antibodies tagged with fluorophores, that are present in a fluorescent immunoassay while the bound fluorophores represent the detection antibodies that are bound to the antigen and captured on the surface. This technique gives a more biologically relevant measure of the background exclusion as a function of SAF collection angle (θ) because the two fluorophores make it possible to simultaneously measure the fluorescence of the unbound fluorophores in the bulk against that of the fluorophores bound to the surface. Numerical simulations were done to validate the results of the experiment.
rophore that is away from the sample surface is out of focus. The effective numerical aperture (NA) of fluorescence collection decreases when the fluorophore is further away from the focal plane. A correction for this NA change is implemented in the bulk fluorescence calculation. The surface bound fluorophores were simulated with a dipole placed at a distance of 5nm from the interface. A distance of 5nm was chosen because it is approximately the length of the SAM layer to which the fluorophores are bound. The number of fluorophores in the bulk and on the surface was known and the simulated values were multiplied by the number of fluorophores and the excitation efficiency for our excitation wavelength. From the simulations, we were able to calculate the angular emission profiles of all the bound and bulk fluorophores. The equations used for these calculations can be found in the supporting information. The goal of the experiment was to collect the angular emission profile of the two different fluorophores in the sample and compare it with the simulation results. Therefore, a custom built free-space back focal plane (BFP) setup was used (Figure 3). A Thorlabs LP637-SF70 637 nm diode laser is used at a power of 45mW. A Thorlabs PAF-X-15-B is used to collimate the laser output. A set of mirrors is used to direct the collimated laser beam on the sample in transmission mode to excite the fluorophores. The objective is a Nikon CFI Apo TIRF 60X Oil objective with a NA of 1.49. The objective is therefore able to collect angles up to θ = 79.37◦ . A Thorlabs FELH0650 emission filter is used to block the excitation photons while allowing the fluorescence to transmit through. A 100 mm focal length (f) lens L1 is placed at a distance of 2f from the BFP of the objective. Lens L1 forms the image plane (IP) at f and projects the BFP at 2f. A circular mask is placed at the center of the BFP to block the excitation photons that got through the filter. A second 150 mm lens L2 can be placed at two positions P1 and P2 to image the IP and BFP, respectively. Behind L2, there is a removable mirror M1 that can be used to switch between imaging and spectrometry. Without M1, the beam travels to a 100 mm tube lens L3 that fo-
Materials and methods Fluorophores can be approximated as point dipole emitters in electromagnetic simulations. 17–19 Simulations were done using a full field 3D finite difference time domain solver from Lumerical. 20 The dipole source was positioned above a glass substrate 21 and inside a water medium (n = 1.33). The critical angle for this interface is θc = 61.3◦ . The dipole was placed in all 3 orientations and at different distances away from the glass-water interface to simulate bound (Atto 633) and bulk (Atto 680) fluorophores. The far-field emission profile was collected for each simulation. The emission of the dipole at positions between 10 nm and 1400 nm from the interface represents the emission of the bulk fluorophores close to the surface. The emission of the bulk fluorophores that are far away from the interface was simulated using a dipole 4 µm away from the interface. This dipole was sufficiently far away from the influence of the interface and it’s emission profile was similar to a free dipole suspended in a water medium. In the actual experiment, the bulk extends to a height of 600 µm from the sample surface. The sample surface was the focal plane of the optical setup used for the experiment. Therefore, an unbound fluo-
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face and Atto 680 suspended in water was mounted on the sample holder. The sample surface (water-glass interface) was brought into focus by using the setup in the imaging configuration. The lens was switched to image the BFP and a mirror was introduced to project the BFP onto the optical fiber of the spectrometer. Movement of the stage mounted fiber was synchronized with the acquisition of the spectrometer. The spectrum was collected with increments of 50 µm across 10 mm of the BFP and an integration time of 1 second at each position. This spectrum was then compared to the measured individual spectra of the two fluorophores.
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Figure 3: Schematic of the back focal plane (BFP) setup. The inset at the top illustrates the sample with the bound and bulk fluorophores represented by the red and green dots, respectively. The inset at the bottom shows a representation of the distinct angular emission profile of the two fluorophores. The fiber of the spectrometer (blue line) is scanned across the BFP as indicated by the arrow. Switching between the observation of the image plane (IP) and BFP is done by moving lens L2. cuses the IP or BFP onto a Hamamatsu c910013 EMCCD camera. Introducing M1 into the beam path, directs the beam to a 100mm lens L4 after reflecting off M2. L4 focuses the BFP at the position where a 100 µm multimode optical fiber is mounted onto a single axis stage that can be scanned across the BFP. The fiber guides the light into an ocean optics QE pro spectrometer. The top inset in Figure 3 shows an illustration of the bulk and bound fluorophores on the sample. The bottom inset shows a depiction of the contribution of the two fluorophores at different angles in the BFP. The blue line represents the fiber connected to a spectrometer, that is scanned across the BFP, in the direction of the arrow, while collecting the spectrum at every position. The sample with Atto 633 bound to the surACS Paragon Plus Environment
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Analytical Chemistry
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the experiment can be compared with the spectrum of the individual Atto 633 (red) and Atto 680 (green) fluorophores. We can see that the spectrum is dominated by the far-field emission of the Atto 680 which was in the bulk, with a little shoulder that shows the far-field emission of the Atto 633 bound to the surface. The blue curve in Figure 4b shows the spectrum that was collected at θ = 68◦ in the SAF region and it looks similar to the spectrum from the Atto 633 (red) which was bound to the surface. The contribution from the Atto 633 in this region comes from the near-field emission propagating into the supercritical angle of the glass-water interface. The contribution of the bulk fluorophore Atto 680 (red) completely disappears from the spectrum in this region because it’s contribution in the near-field is negligible compared to the contribution from the surface bound Atto 633. A supercritical angle of 68◦ was chosen because that was where we observed the greatest SAF signal. Figure 5a and b show the results of the simulation for both bulk and surface bound fluorophores respectively. The signal from the bulk fluorophores starts to drop quickly around θ = 60◦ and almost completely disappears just above the critical angle at θ = 62◦ . The surface fluorophores show a very different behavior with the signal being quite uniform below the undercritical angle and increasing rapidly above the critical angle. This can clearly be seen in Figure 5 c which shows the ratio of the surface signal to the bulk signal which is also called the signal to background ratio (SBR). The surface signal is negligible compared to the bulk signal in the undercritical angle region, with a total SBR of 0.3 in this region. Above the critical angle, the SBR increases rapidly and the surface fluorophores begin to dominate over the bulk fluorophores. Therefore, this region has a higher total SBR value of 58.7. It must be noted that the simulated emission profiles do not look smooth because of artifacts from the far-field calculation. The far-field calculation assumes that all radiation propagating in the far-field will pass through the monitor. However, since the simulation area and hence the size of the monitor cannot be infinite, we cannot capture
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