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Biomedical Optics & Medical Imaging

Demonstration of an all-fiber stimulated emission depletion illumination system

Stable spatially and spectrally differentiated laser beams can be delivered to remote locations and may thus enable endoscopic in vivo imaging, with resolution beyond the diffraction limit.
3 November 2016, SPIE Newsroom. DOI: 10.1117/2.1201610.006672

Imaging at the nanoscale holds great promise for many life science studies. Among several nanoscale imaging techniques that have been developed in the past decade, stimulated emission depletion (STED) microscopy (invented by Stefan Hell)1 has received a significant amount of attention. In the STED technique, two laser beams—a Gaussian-shaped beam and a red-shifted donut-shaped beam—are used to excite a fluorophore, and deplete it (through stimulated emission), respectively. A series of breakthroughs in the past decade have dramatically advanced this technique, and sub-50nm resolution can now be routinely obtained with carefully engineered STED microscopes. All current (including commercially available) implementations of STED microscopy, however, involve the use of free-space beam-shaping devices2–7 to achieve the Gaussian and donut-shaped beams at desired colors. This can be problematic because it usually requires precise co-alignment of two or more spatially and spectrally separated lasers. Moreover, obtaining a donut-shaped depletion pattern, with a well-defined zero central intensity at the focal plane of a high-numerical-aperture objective, requires the generation of a very specific free-space beam, i.e., which carries orbital angular momentum (OAM) with helicity that is aligned with the orientation of its (circular) polarization. Additional free-space polarization optics in the beam path are therefore usually needed to ensure the generation and alignment of such beams (which increases the complexity, and compromises the stability, of the microscope).

Purchase SPIE Field Guide to MicroscopyIt is thought that an all-fiber STED microscope could address the engineering challenges associated with this technique. Such a device would also enable field-deployable instruments, with which nanoscale microscopy could be performed. Perhaps even more excitingly, a fiber-compatible STED microscope may facilitate endoscopic implementation of in vivo STED imaging. This would allow access to sub-cellular structures at larger penetration depths, a prospect that has thus far not been realized because optical fibers do not typically carry (in a stable manner) the specific kinds of OAM beams that are required for STED. In the absence of the inherent stability of OAM modes in regular fibers, attempts to achieve fiber-based STED have generally involved interferometrically combining multiple modes in a fiber to obtain the desired beam patterns at the output.8–10 These approaches, however, involve phase-sensitive beam shaping, and the fibers therefore cannot be bent, perturbed, or otherwise treated like a fiber. These methods thus do not provide any significant advantages over current free-space STED microscopes.

Borrowing from principles developed for OAM-based fiber telecommunications,11, 12 we have designed and fabricated a specialty fiber—see Figure 1(a)—that features a high-refractive-index ring structure.13 Our structure supports OAM beams in the visible spectral range (i.e., where most dyes that are used in STED microscopy operate). We use a long-period fiber grating (LPG) as a resonant mode converter to realize an OAM mode at the depletion wavelength. We can then build an all-fiber STED illumination system—see Figure 1(b)—in which both the excitation and the STED beams are naturally co-aligned. We can achieve this co-alignment because both the Gaussian-shaped excitation beam and the donut-shaped depletion beam are stably guided eigenmodes of the same circular fiber structure. No alignment of the two beams is thus necessary once they have been generated in the fiber (i.e., with the aforementioned grating). In our system, the excitation (632nm) and depletion (776nm) laser beams are coupled through a fiber-pigtailed wavelength multiplexer (WDM). This WDM is spliced to our fiber, in which we use the LPG to convert the fundamental mode into the desired donut-shaped OAM mode at the depletion wavelength (while leaving the excitation beam unaltered).


Figure 1. (a) Microscope image of the facet of the orbital angular momentum (OAM)-carrying fiber. Inset shows the high-index ring structure, where the OAM mode is primarily located. (b) Schematic diagram of the all-fiber stimulated emission depletion (STED) illumination system, which includes a wavelength division multiplexer (WDM), tilted long-period fiber grating (LPG), vortex fiber, beamsplitter (BS) or dichroic mirror (DC), detector, and an objective (obj.) lens.

We have tested our device with the use of a home-built confocal microscope. In particular, we characterized the point spread functions (PSFs) for both the excitation and the depletion beams, under different fiber-bending conditions. Two of the typical fiber loops we used for these tests are shown in Figure 2(a), along with the corresponding near-field mode images of the depletion beam at the output facet of the fiber. These images indicate that the OAM mode barely deteriorates, in terms of mode purity, in the presence of tight bends. In addition, we used spatial interferometry14 to quantitatively measure the mode purity of the output OAM beams. We found that the mode purity remains greater than 98% under all the perturbation conditions. Our PSF measurements, for the tightest fiber bends we used (6mm, i.e., considerably smaller than the 1mm bend radii that are specified for endoscopy applications15), are shown in Figure 2(b). As expected, based on our mode purity measurements, we readily obtained well co-aligned PSFs for the Gaussian-shaped excitation and the donut-shaped depletion beams.


Figure 2. (a) Photographs (top) of two of the typical fiber loops (with radius, r, of 6.5 and 3cm) used to characterize the point spread function (PSF) of the excitation and depletion beams of the all-fiber STED illumination system. Corresponding near-field OAM-mode images, at the fiber output facet, are also shown (bottom). (b) Experimentally measured PSFs in the lateral (top left) and axial (bottom left) planes of a sharply bent fiber (6mm radius), shown top right. The line-cut profiles (bottom right) show the co-aligned Gaussian-shaped excitation beam at 623nm (green) and the donut-shaped depletion beam at 776nm (red). a.u.: Arbitrary units.

In summary, we have demonstrated an all-fiber compact STED illumination system with which we can achieve fiber modes that are tolerant to perturbations. Since the outputs of our system are stable modes of the fiber itself, we obtain naturally self-aligned PSFs for the excitation and depletion beams. The fiber we use can easily be cleaved and spliced, and it is compatible with various termination options. With such fibers and fiber gratings, we thus provide an all-fiber platform that can be used to build simplified, mechanically robust STED systems and that can be used to realize other imaging systems (i.e., where the exploitation of spatio-spectral beam shaping is required). For our future work, we anticipate further integration of micro-optics (such as gradient index objectives and fiber raster scanners) into our device, which should potentially enable endoscopic implementations of STED imaging.

The authors acknowledge financial support from the National Science Foundation (ECCS-1310493) and Office of Naval Research (N00014-13-1-0627).


Lu Yan, Siddharth Ramachandran
Department of Electrical and Computer Engineering & Photonics Center
Boston University
Boston, MA

Lu Yan is a PhD student in the Nanostructured Fibers and Nonlinear Optics Laboratory. His research focus is on optical-fiber higher-order modes and their applications in biomedical imaging.

Siddharth Ramachandran is a professor of electrical and computer engineering, and the director of the Nanostructured Fibers and Nonlinear Optics Laboratory.


References:
1. S. W. Hell, J. Wichmann, Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy, Opt. Lett. 19, p. 780-782, 1994.
2. K. I. Willig, R. R. Kellner, R. Medda, B. Hein, S. Jakobs, S. W. Hell, Nanoscale resolution in GFP-based microscopy, Nat. Methods 3, p. 721-723, 2006.
3. E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, S. W. Hell, STED microscopy reveals crystal colour centres with nanometric resolution, Nat. Photon. 3, p. 144-147, 2009.
4. M. Reuss, J. Engelhardt, S. W. Hell, Birefringent device converts a standard scanning microscope into a STED microscope that also maps molecular orientation, Opt. Express 18, p. 1049-1058, 2010.
5. http://www.leica-microsystems.com/products/confocal-microscopes/details/product/leica-tcs-sp8-sted-3x/ Product details for the Leica TCS SP8 STED 3X super-resolution microscope. Accessed 9 October 2016.
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15. C. M. Lee, C. J. Engelbrecht, T. D. Soper, F. Helmchen, E. J. Seibel, Scanning fiber endoscopy with highly flexible, 1mm catheterscopes for wide-field, full-color imaging, J. Biophoton. 3, p. 385-407, 2010.