Single-shot, shadowless total internal reflection fluorescence microscopy via annular fiber bundle

Abstract

We demonstrate a method of generating instantaneous and uniform total internal reflection fluorescence (TIRF) excitation by using an annular fiber bundle and spatially incoherent light sources. We show the flexibility of our method in that it can generate TIRF excitation with either a laser light source or an LED of different wavelengths, and in that it can facilitate switching between TIRF and epi illumination. In this report we detail the design of the fiber bundle, then demonstrate the performance via single-molecule imaging in the presence of high background and high-throughput, uniform TIRF imaging of cells over a large field-of-view. Our versatile method will enable quantitative shadowless TIRF imaging.

Total internal reflection fluorescence (TIRF) microscopy [1] is a popular and useful tool for studying surface features of biological specimens [2] and imaging single-molecules [3, 4] with a high signal-to-noise ratio. When an excitation beam impinges on the glass-sample interface with an incidence angle that is greater than the critical angle, the illumination beam generates an evanescent field that selectively excites fluorescently labeled biomolecules near the surface with a penetration depth of 50–200 nm [5].

There are a variety of methods to generate TIRF excitation [6, 7], with the most common in cellular imaging being objective TIRF. Typically, objective TIRF is achieved by tightly focusing a single laser excitation beam to the periphery of the back focal plane (BFP) of an objective with a numerical aperture (NA) of 1.4 or greater (Fig. 1a) [8]. However, this method can cause uneven illumination due to interference fringes from the coherent laser beam and shadowing caused by obscuring objects in cells [9]. These issues have been mitigated via spinning TIRF where the excitation spot is rapidly rotated to various positions around the TIRF annulus of the objective within a single camera exposure (Fig. 1b) using refractive optics [10], piezo/galvo mirrors [11–13], acousto-optic deflectors [14] or a digital micromirror device [15]. The result is an incoherent superimposition of the excitation intensity from each azimuthal angle.

Fig. 1.

Illustrations of (a) single-spot, (b) variable-angle, and (c) annular TIRF described at the back focal plane (BFP) and imaging plane (IP). (d) Illustrations of fiber bundle input and output facets, showing the outer (D_o), inner (D_i), and multimode fiber (D_MMF) diameters.

Alternatively, an annular mask can be placed at a plane conjugated to the BFP, so that only the TIRF annulus is illuminated and a uniform TIRF excitation field is instantly generated (Fig. 1c) [8, 16, 17]. This implementation is appealing due to the simplified setup with no moving parts and its compatibility with incoherent light sources; however, the use of a mask incurs significant power losses which can be greater than 99% in some cases [17]. Whereas uniform TIRF illumination with a higher efficiency has been proposed by generating annular beams with axicon optics [18, 19], these approaches have a limited field-of-view (FOV), suffer from a strong zero-order spot that prevents quantitative analysis or are difficult to implement in a commercial imaging system.

In this report, we demonstrate a novel method of generating instantaneous, uniform and efficient TIRF by coupling an excitation source into a tailored fiber bundle. The basic concepts of our approach are outlined as follows: (i) The individual fibers in the bundle are arranged into a ring at the output end which is focused and re-imaged within the TIRF annulus of the BFP of the objective, and (ii) the beam exiting from each fiber is spatially incoherent such that they are incoherently summed at the image plane. Figure 1 shows its working principle in comparison with single-spot and spinning TIRF and illustrations of the input and output ends of the fiber bundle.

We designed the annular fiber bundle to be compatible with a 60×/NA1.45 objective (PLAPON60XOTIRFM, Olympus). Our fiber bundle consisted of 137 individual multimode fibers that were close-packed at the input end and arranged in a single ring around a spacer at the output end (Fig. 1d). Considering the size of the BFP of our objective, we designed the bundle such that when the output was magnified 3× it would be relayed to the outer annular region of the BFP that generates TIRF illumination. A detailed description of the design process is given in Supplement 1.

The fiber bundle was fed into a custom made TIRF microscope built around an Olympus IX73 body (Fig. 2). The bundle output was collimated by a lens L₁ (f₁ = 100 mm) and focused to the BFP of the objective by a lens L₂ (f₂ = 300 mm) which was mounted on a manual xy and motorized z translation stage. By translating L₂ one inch along the optic axis, we can change the size of the beam at the BFP to achieve TIRF or weakly focused epi illumination. The beam was reflected by a filter cube (TRF89901v2, Chroma) and the fluorescence signal was detected by a scientific complementary metal oxide semiconductor (sCMOS, Zyla 4.2 PLUS; Andor) camera or an electron multiplying charged-coupled device (EMCCD, iXon Ultra 897; Andor). The sCMOS was used to demonstrate large FOV (222 × 222 μm²) TIRF, but the EMCCD was used in all other experiments. An additional 1.66× magnification system was installed prior to the EMCCD for a total image magnification of 100× to satisfy the Nyquist criterion for single-molecule imaging. An image of the fiber bundle output taken at the BFP is shown in an inset of Fig. 2.

Fig. 2.

Experimental setup. FB_i/o: Fiber bundle input/output, SMF: Single mode fiber, MMF: multimode fiber, L: Lens, TL: Tube lens, M: Mirror, FM: Flip mirror, FC: Filter cube. Lower-left inset: Image of fiber bundle beam at the BFP. Scale bar, 2 mm.

488 nm and 638 nm lasers (06-MLD, Cobolt) as well as a 470 nm light emitting diode (LED, M470F3, Thorlabs), which was directly coupled via SMA connectors to the fiber bundle, were the light sources used with the fiber bundle to demonstrate multicolor imaging and TIRF with coherent or incoherent sources. The diode lasers were first coupled into a 400 μm core multi-mode fiber (MMF, M28L01, Thorlabs) that was attached to a shaker motor (JRF370–18260, ASLONG) to degrade the coherence of the beam [20] before coupling into the fiber bundle input. For comparison with single-spot TIRF, a 491 nm or 640 nm laser (04–01 Calypso, 05–01 Bolero, Cobolt) was coupled to a single-mode fiber (P5–488PM-FC-1, Thorlabs) and collimated by a lens (f = 300 mm) and directed to the microscope by a flip mirror. We observed a total power efficiency of ~30% when using the 638 nm laser, with 76% of the total -loss occurring at the coupling of the MMF into the fiber bundle. This was expected, as ~50% of the fiber bundle input is void and the MMF was roughly butt-coupled to the fiber bundle input with an intentional gap between the MMF and fiber bundle to compensate for the difference in their core sizes. A further optimized design will greatly mitigate the power losses.

We first demonstrated the shallow excitation depth of our TIRF field using the 638 nm diode laser coupled into the annular fiber bundle. Single-molecule images were recorded of surface-immobilized IgG antibodies labeled with Alexa Fluor 647 (AF647) at a degree of labeling of ~1.1 [21] in the presence of 10 nM STAR635 diluted in an imaging buffer as fluorescent background. Our result shows that the single-molecules are able to be resolved with an average signal to background ratio of 2.3 (n = 20) when imaged in TIRF (Fig. 3a), whereas the background overwhelms the single-molecule signal when imaged under epi illumination (Fig. 3b). Note that at this concentration of fluorescent background, even a slight degree of far field excitation due to partial TIRF could substantially increase the background level [20], indicating that our annular fiber bundle produces clean TIRF excitation. The effective penetration depth was roughly 235 nm, which was estimated by measuring the diameter of 1 μm fluorescent beads (F8816, ThermoFisher) [22]. In addition, we obtained single-molecule images without fluorescent background (Fig. 3c) and analyzed the intensity of each spot, comparing annular and single-spot TIRF illumination. The annular TIRF intensity distribution has narrower peaks and two observable populations, while the single-spot TIRF distribution is broader and less defined (Fig. 3d). This demonstrates that our annular TIRF excitation field enabled the distinction between antibodies labeled with one or two fluorophores, which is only possible with uniform excitation [20].

Fig. 3.

Single-molecule imaging. Images of the same field-of-view taken in the presence of 10 nM fluorescent background using (a) TIRF and (b) epi illumination. (c) TIRF image without fluorescent background in a different field-of-view than in (a) and (b). (d) Intensity distributions generated from 20 images comparing annular TIRF and single-spot TIRF illumination. Inset: illustration of excitation penetration depth while imaging in high background. Scale bars, 5 μm.

We further examined the uniformity of our illumination by measuring beam profiles taken by exciting a ~5 μm thick dye layer (Atto488 or STAR635) sandwiched between a microscope slide and coverslip and imaged with the sCMOS detector for a 222 × 222 μm² FOV (Fig. 4). The uniformity was characterized by the root mean square (RMS) of the line profile intensity [20]. Detector noise from the sCMOS degraded the RMS values of the diode laser (Fig. 4a) and LED (Fig. 4c) to 0.79 each, but after smoothing via adjacent-averaging the RMS values were 0.84 and 0.86, respectively. In the central 82 × 82 μm² region representing the EMCCD FOV the respective smoothed RMS values were 0.91 and 0.92. Without shaking, the diode laser had an RMS of 0.70 before and 0.77 after smoothing. Raw and smoothed line profiles are plotted in Fig. 4d for the LED and diode laser with shaking.

Fig. 4.

Beam characterization. Dye layer images recorded in TIRF using a (a) diode laser with shaker motor, (b) diode laser without shaker motor, and (c) LED. (d) Line profiles taken along diagonal as indicated by solid lines in (a, c). Scale bars, 50 μm.

Artifacts from single-spot TIRF illumination are often more severe when imaging subcellular structures in cells. To demonstrate the homogeneity of the TIRF excitation generated by our fiber bundle we imaged U2OS cells that were stained with Alexa Fluor 488 phalloidin (A12379, ThermoFisher) to label filamentous actin. Images taken with the 470 nm LED or 488 nm diode laser coupled with our fiber bundle are compared with single-spot TIRF. Figure 5 shows that our annular fiber bundle generates uniform TIRF illumination for artifact-free imaging regardless of the light source, whereas single-spot TIRF results in strong interference fringes from the excitation source, scattering and/or shadowing artifacts from the unidirectional excitation.

Fig. 5.

TIRF imaging of actin in U2OS cells using (a) LED annular TIRF, (b) laser diode annular TIRF, and (c) single-spot objective TIRF. Arrow indicates direction of single-spot TIRF excitation. Scale bars, 10 μm

Finally, we demonstrated high-throughput stitched imaging with our annular fiber bundle using a 15% image overlap on the phalloidin stained U2OS cells to record a 550 × 550 μm² area. To demonstrate the ease of switching between illumination modes, Fig. 6 shows a comparison of the same FOV taken in TIRF and epi illumination using the LED light source, where the inset shows a magnified view of the boxed region. Surface features such as focal adhesions are clearly resolved under TIRF excitation, whereas these features are less visible under epi illumination due to elevated background fluorescence from non-surface actin structures.

Fig. 6.

High-throughput stitched imaging of actin stained U2OS cells imaged under (a) TIRF and (b) epi illumination using the LED source and fiber bundle. Upper-right inset: Zoomed view of boxed region. Scale bars, 50 μm.

In this report we demonstrated a method of instantly achieving shadowless TIRF excitation using an annular fiber bundle. We showed that this method is suitable for multicolor imaging and generates a uniform and shallow excitation field. It is possible to use other popular TIRF objectives such as a 100×/NA1.49 objective if one designs a new fiber bundle and modifies the imaging system slightly. Our annular fiber bundle was designed to be suitable with both a laser or LED; however, LED excitation has a very limited power throughput [23] and thus is not suitable for imaging weakly fluorescent samples such as single-molecules. If one only uses a laser as an excitation source, a more optimized design, for example, utilizing a shorter focal length lens L₁ and fewer MMF fibers is likely to increase the power throughput. Versatile control of the incidence angle is possible via calibration of the motorized translation stage on L₂, which will be useful for depth-matched multi-color TIRF illumination [12] and 3D reconstruction by multi-angle TIRF [24]. Polarization-based TIRF experiments [25] may be feasible by generating radially or azimuthally polarized light using a segmented half wave plate [26]. With no moving parts, our method is compatible with video-rate live-cell TIRF imaging. We expect our method will make quantitative TIRF imaging systems more accessible.