Single-molecule spectroscopy and imaging over the decades

Abstract

As of 2015, it has been 26 years since the first optical detection and spectroscopy of single molecules in condensed matter. This area of science has expanded far beyond the early low temperature studies in crystals to include single molecules in cells, polymers, and in solution. The early steps relied upon high-resolution spectroscopy of inhomogeneously broadened optical absorption profiles of molecular impurities in solids at low temperatures. Spectral fine structure arising directly from the position-dependent fluctuations of the number of molecules in resonance led to the attainment of the single-molecule limit in 1989 using frequency-modulation laser spectroscopy. In the early 1990s, a variety of fascinating physical effects were observed for individual molecules, including imaging of the light from single molecules as well as observations of spectral diffusion, optical switching and the ability to select different single molecules in the same focal volume simply by tuning the pumping laser frequency. In the room temperature regime, researchers showed that bursts of light from single molecules could be detected in solution, leading to imaging and microscopy by a variety of methods. Studies of single copies of the green fluorescent protein also uncovered surprises, especially the blinking and photoinduced recovery of emitters, which stimulated further development of photoswitchable fluorescent protein labels. All of these early steps provided important fundamentals underpinning the development of super-resolution microscopy based on single-molecule localization and active control of emitting concentration. Current thrust areas include extensions to three-dimensional imaging with high precision, orientational analysis of single molecules, and direct measurements of photodynamics and transport properties for single molecules trapped in solution by suppression of Brownian motion. Without question, a huge variety of studies of single molecules performed by many talented scientists all over the world have extended our knowledge of the nanoscale and many microscopic mechanisms previously hidden by ensemble averaging.

Figure 1

(a) Schematic of a focused optical beam pumping a single resonant molecule in a cell or other condensed phase sample. The molecule may emit fluorescence or its presence may be detected by carefully measuring the transmitted beam. (b) Typical energy level scheme for single-molecule spectroscopy showing the interaction with the pumping light. S₀, ground singlet state; S₁, first excited singlet; T₁, lowest triplet state or other intermediate state. For each electronic state, several levels in the vibrational progression are shown. Typical low-temperature studies use wavelength λ_LT to pump the dipole-allowed (0-0) transition, while at room temperature shorter wavelengths λ_RT which pump vibronic sidebands are more common. Fluorescence emission shown as dotted lines originates from S₁ and terminates on various vibrationally excited levels of S₀ or S₀ itself. Molecules are typically chosen to minimize entry into dark states such as the triplet state (illustrated), although this or other dark processes can lead to blinking useful in super-resolution microscopy. The intersystem crossing or intermediate production rate is k_ISC, and the triplet decay rate is k_T.

Figure 2

Observation of Statistical Fine Structure (SFS) (right) for pentacene in p-terphenyl (schematic structure) using laser FM spectroscopy. Reprinted with permission from Ref. (T. P. Carter, M. Manavi and W. E. Moerner, J. Chem. Phys., 1988, 89, 1768). Copyright 1988, AIP Publishing LLC.

Figure 3

(a,b) Spectral diffusion for pentacene in p-terphenyl and (c) light-induced spectral shifts for perylene in poly(ethylene). From Refs. ^,, respectively, by permission. Panel b reprinted from Ref. , copyright (1991) by The American Physical Society.

Figure 4

A selection from the huge variety of single-molecule methods and applications over the decades – with apologies for possible omission.

Figure 5

(a) Structure of the green fluorescent protein superimposed on a series of images of a single GFP trapped in a gel, 100 ms per image. (b) Schematic of energy-level structure consistent with the blinking and photoswitching effects . Reprinted with permission from Ref. . (c) Images (600nmx600nm) of 488-nm pumped emission from the long-lived dark state (odd panels) with the photoreactivated state (even panels) produced by 405 nm irradiation for the same single molecule of the T203F yellow mutant of GFP. Similar results occurred for the T203Y mutant . Reprinted with permission from Ref. . (d) Reactivation of EYFPMreB fusions in live C. crescentus cells. Fluorescence images show single EYFP-MreB molecules (white spots) overlaid on a reversed-contrast white-light image of the cell being examined. Only a few molecules are reactivated by 407nm light in each image. Bar, 1 μm. For details, see . Reprinted with permission from Ref. .

Figure 6

Illustration of the key ideas underlying super-resolution optical microscopy based on single-molecule localization combined with active control of the emitting concentration and sequential imaging.

Figure 7

Selected super-resolution images in cells. (a) The cell surface of Caulobacter crescentus bacteria. Scale bar = 1 μm. From Ref. by permission. (b) Two-color 3D imaging of the cell surface and a protein fiber-like structure in Caulobacter. Wikimedia file Bacteria-3D-Double-Helix.jpg Creative Commons Attribution-Share-Alike 4.0 International license. (c) Super-resolution and diffraction-limited (DL) images of the locations of voltage-gated sodium (NaV) channels in a differentiated PC12 cell. Reprinted from Ref. (A. E. Ondrus, H. D. Lee, S. Iwanaga, W. H. Parsons, B. M. Andresen, W. E. Moerner and J. Du Bois, Chem. Biol., 2012, 19, 902–912), with permission from Elsevier.

Figure 8

Various PSFs for 3D localization microscopy shown as a function of z-position of the emitter (experimentally measured). (a) Astigmatic. Scale bar=~0.5μm. From Ref. (B. Huang, W. Wang, M. Bates and X. Zhuang, Science, 2008, 319, 810–813). Reprinted with permission from AAAS. (b) Phase-ramp. Reprinted with kind permission from Springer Science and Business Media: Ref. (D. Baddeley, M. B. Cannell and C. Soeller, Nano Research, 2011, 4, 589–598). (c) Double-Helix. Scale bar=2μm. Reprinted with permission from Ref. . (d) Accelerating beam. Scale bar=1μm. Reprinted by permission from Macmillan Publishers Ltd: Nat. Photonics (S. Jia, J. C. Vaughan and X. Zhuang, Nat. Photonics, 2014, 8, 302–306), copyright (2014) (e) Saddlepoint . Scale bar=1μm. Reprinted from Ref. , copyright (2014) by The American Physical Society. (f)+(g) Tetrapods, scale bars=2μm and 5μm, respectively. The arrows (right) represent the z-ranges over which the PSFs on the left were imaged, which correspond to their applicable depth ranges. Top right: Experimental setup for pupil plane modulation-based PSF engineering . Adapted with permission from Ref. (Y. Shechtman, L. E. Weiss, A. S. Backer, S. J. Sahl and W. E. Moerner, Nano Lett., 2015, 15, 4194–4199). Copyright (2015) American Chemical Society.

Figure 9

The working principles of the ABEL trap: single-molecule motion in a microfluidic environment is tracked with high speed and Brownian motion is compensated by applying appropriate electrokinetic forces (black arrow) in 2D in a feedback loop. The motion of the molecule along the third dimension (z) is confined within the ~600 nm depth of the microfluidic chip (inset).

Figure 10

Examples of single-molecule dynamics probed in the ABEL trap. (a) Photodynamics of allophycocyanin. Different intensity and lifetime states represent partially photobleached and quenched intermediates. Adapted with permission from Ref. . (b) Redox cycling of nitrite reductase. Example molecules of nitrite reductase fluctuate between two digital intensity levels, corresponding to the oxidized (red) and reduced (blue) Cu redox states caused by single-electron transfer events. Adapted with permission from Ref. (c) Counting the number of ADP molecules on a multi-subunit chaperonin enzyme (TRiC). Digital steps represent photobleaching of the individual Cy3-ADP molecules bound to the protein. Adapted with permission from Ref. . (d) Correlated intensity-lifetime-spectral photodynamics of a single Atto647N fluorophore in solution. Shaded areas indicate the three typical emissive states of this molecule. Adapted with permission from Ref. (Q. Wang and W. E. Moerner, J. Phys. Chem. B, 2012, 117, 4641–4648). Copyright (2012) American Chemical Society. (E) Visualization of single DNA binding and unbinding dynamics by ABEL-trap measurements of the diffusion coefficient (D) and electrokinetic mobility (μ). Reproduced with permission from Ref. .

Figure 11

Broad impact of single-molecule spectroscopy and imaging.