Breaking the diffraction limit without fluorescence labels

Combining three laser beams, including a doughnut-shaped beam, permits ‘super-resolution’ microscopy of nanostructures with the contrast of optical absorption.
07 April 2014
Pu Wang and Ji-xin Cheng

Conventional optical microscopes can resolve objects no smaller than about 300nm, a restriction known as the ‘diffraction limit,’ defined as approximately half the wavelength of light being used to view the specimen. However, researchers want to view subcellular structures, as well as synthetic nanostructures such as carbon nanotubes, which are a few nanometers in diameter.

Many approaches for breaking the diffraction limit have been proposed.1–5 However, they generally require fluorescence labeling, which itself perturbs the system under investigation. We recently developed a new approach, named saturated transient absorption microscopy (STAM), to view the nanoscopic world without labeling.6 With this method, we can see the signal directly from the target nanostructures or molecules rather than from the labels.

The principle of STAM is to create an effective focal area that is much smaller than the diffraction limit by playing with the electronic states of the target nanostructure or molecule. To achieve this, three laser beams, including a doughnut-shaped laser beam (the saturation beam) that selectively illuminates the peripheral area but not the very central area, are collinearly aligned (see Figure 1). Electrons illuminated by the doughnut-shaped laser beam are kicked temporarily into a higher energy level and are said to be excited, while the others (at the very center) remain in their ground state. When the doughnut-shaped beam is strong enough, the ‘excited state’ is fully filled (saturated) and no more molecules can be kicked up to it. The signals are generated using the other two lasers, called pump and probe. The ‘pump-probe’ signal can only come from the molecules in the ground state, not the ones in the saturated state at the very center of the doughnut-shaped beam. In this way, the effective focal size of this system can be significantly reduced, thus reaching subdiffraction-limit resolution.

Figure 1. Principle of saturated transient absorption microscopy (STAM). The effective focal area is decreased because the pump-probe signal can only be generated at the center region where the excited state is not saturated.

Using this principle, we have constructed a STAM system (see Figure 2).6 Briefly, a 1064nm beam was split into the pump and saturation beams, with an 830nm beam collinearly aligned with them. We employed a phase-only spatial light modulator to generate a helical phase pattern with a 0–2π continuous phase gradient around 360° (see Figure 2). With a Gaussian incident beam, the wave front of the reflected output from the phase pattern was engineered with spatial phase delay, resulting in the doughnut-shaped beam.

Figure 2. Diagram of the saturated transient absorption microscope. Upper-right inset: The helix phase pattern sent to a spatial light modulator (SLM) to generate the doughnut shape focus of the saturation beam. Lower-left inset: The measured point spread function of the saturation beam. Ti: Titanium. OPO: Optical parametric oscillator. AOM: Acousto-optic modulator. BS: Beam splitter. PD: Photodiode. DM: Dichroic mirror.

Applying this STAM system, we have imaged graphite nanoplatelets to demonstrate its ability to break the diffraction limit. Nanoplatelets that cannot be resolved by a conventional pump-probe are resolved by STAM (see Figure 3). Moreover, we have imaged a small single nanostructure to measure the spatial resolution of our microscope system. With a de-convolution analysis, we confirmed that a spatial resolution of λ/4 (∼200nm) was achieved, where λ is the wavelength of the incident light.

Figure 3. Subdiffraction-limit STAM imaging of graphite nanoplatelets. Nanofeatures that cannot be resolved by conventional pump-probe microscopy are resolved by STAM. Scale bar: 1μm.

In summary, we have demonstrated an approach for imaging non-fluorescent nanostructures. Subdiffraction-limited imaging of graphite nanoplatelets achieved a resolution of λ/4. The STAM technique enables super-resolution imaging of nanomaterials and nonfluorescent chromophores that may remain out of reach for fluorescence-based super-resolution methods. We are now working to optimize the STAM system, for instance, by using a shorter wavelength to further improve the resolution. We also intend to apply STAM to image other nanomaterials with saturable absorption, such as single-wall carbon nanotubes,7–9 as well as iron oxide and zinc oxide nanoparticles,10, 11 which are biocompatible imaging agents.12,13

Pu Wang, Ji-xin Cheng
Purdue University
West Lafayette, IN

Pu Wang received a BS in physics from Fudan University, China, and a master's in medical biophysics from the Indiana University School of Medicine. He is currently working toward a PhD in biomedical engineering. His research interests include nonlinear optical microscopy and photoacoustic imaging and spectroscopy.

Ji-xin Cheng received a BS and PhD from the Department of Chemical Physics at the University of Science and Technology, China. He is currently a professor at the Weldon School of Biomedical Engineering at Purdue. His lab develops label-free imaging tools and nanotechnologies for challenging biomedical applications.

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. S. Bretschneider, C. Eggeling, S. W. Hell, Breaking the diffraction barrier in fluorescence microscopy by optical shelving, Phys. Rev. Lett. 98, p. 218103, 2007.
3. M. G. L. Gustafsson, Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution, Proc. Nat'l Acad. Sci. USA 102, p. 13081-13086, 2005.
4. M. J. Rust, M. Bates, X. Zhuang, Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (Storm), Nat. Methods 3, p. 793-796, 2006.
5. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, H. F. Hess, Imaging intracellular fluorescent proteins at nanometer resolution, Science 313, p. 1642-1645, 2006.
6. P. Wang, M. N. Slipchenko, J. Mitchell, C. Yang, E. O. Potma, X. Xu, J.-X. Cheng, Far-field imaging of non-fluorescent species with subdiffraction resolution, Nat. Photon. 7, p. 449-453, 2013.
7. A. G. Rozhin, Y. Sakakibara, H. Kataura, S. Matsuzaki, K. Ishida, Y. Achiba, M. Tokumoto, Anisotropic saturable absorption of single-wall carbon nanotubes aligned in polyvinyl alcohol, Chem. Phys. Lett. 405, p. 288-293, 2005.
8. P. Avouris, M. Freitag, V. Perebeinos, Carbon-nanotube photonics and optoelectronics, Nat. Photon. 2, p. 341-350, 2008.
9. I. H. Baek, S. Y. Choi, H. W. Lee, W. B. Cho, V. Petrov, A. Agnesi, V. Pasiskevicius, D.-I. Yeom, K. Kim, F. Rotermund, Single-walled carbon nanotube saturable absorber assisted high-power mode-locking of a Ti:sapphire laser, Opt. Express 19, p. 7833-7838, 2011.
10. C. P. Singh, K. S. Bindra, G. M. Bhalerao, S. M. Oak, Investigation of optical limiting in iron oxide nanoparticles, Opt. Express 16, p. 8440-8450, 2008.
11. L. Irimpan, V. P. N. Nampoori, P. Radhakrishnan, Spectral and nonlinear optical characteristics of ZnO nanocomposites, Sci. Adv. Mater. 2, p. 117-137, 2010.
12. T. K. Jain, M. K. Reddy, M. A. Morales, D. L. Leslie-Pelecky, V. Labhasetwar, Biodistribution, clearance, and biocompatibility of iron oxide magnetic nanoparticles in rats, Mol. Pharm. 5, p. 316-327, 2008.
13. J. Zhou, N. S. Xu, Z. L. Wang, Dissolving behavior and stability of ZnO wires in biofluids: a study on biodegradability and biocompatibility of ZnO nanostructures, Adv. Mater. 18, p. 2432-2435, 2006.
Sign in to read the full article
Create a free SPIE account to get access to
premium articles and original research
Forgot your username?