W. E. Moerner: Seeing One Molecule at a Time

Almost everything chemists measure is an average. A UV-visible spectrum of a solution, for example, reports on some 10¹⁸ molecules at once, and the result is the ensemble average. Differences between individual molecules, such as one molecule being in a slightly different environment or conformation, are hidden. In 1989 William E. Moerner, working at the IBM Almaden Research Center in California, did what many thought impossible: he detected the optical absorption of a single molecule in a solid. A few years later he showed that single molecules of green fluorescent protein blink and can be switched on again by light. These two findings opened the door to super-resolution microscopy and earned Moerner a share of the 2014 Nobel Prize in Chemistry with Eric Betzig and Stefan Hell.

The Scientific Problem Before the Discovery

In a solid or liquid sample, each molecule sits in a slightly different local environment. This causes inhomogeneous broadening: the absorption line of an ensemble is broad, even if each molecule absorbs at a narrow frequency. In the 1970s and 1980s, chemists used a technique called spectral hole burning to look inside the broad line: a narrow laser burns a tiny "hole" in the absorption band by photochemically changing the molecules that absorb at that frequency. Moerner himself was an expert on this method, and he explored its use for high-density optical data storage.

Hole burning led to a question: if one narrows down the number of molecules probed, can one reach the limit of a single molecule? It was thought extremely difficult. A single molecule absorbs light weakly, with a cross-section of about 10⁻¹⁶ cm² at best, so the signal is tiny compared with background noise. Many scientists believed that single molecules could be detected only by mass spectrometry, scanning probes or electrons, not by optical methods.

The Discovery: Step by Step

1. Choosing the sample. Moerner and Lothar Kador used pentacene molecules doped at very low concentration in a crystal of p-terphenyl, cooled to about 1.8 K with liquid helium. At such a low temperature, the absorption of each pentacene molecule becomes extremely sharp (a zero-phonon line), a few tens of MHz wide.
2. Reducing the number of molecules. The concentration was so low that only a few molecules in the laser focus had a frequency within the range scanned. Because inhomogeneous broadening spreads molecules over a wide spectrum, tuning a narrow laser meant seeing just one molecule at a time.
3. Sensitive detection (1989). They used frequency-modulation spectroscopy, in which the laser frequency is rapidly modulated and the signal is detected at the modulation frequency, to reach the sensitivity needed to see the absorption of a single molecule. The result was a narrow peak: the spectrum of one molecule, published in Physical Review Letters in 1989.
4. Fluorescence detection (1990). Michel Orrit and Jacky Bernard in France reported fluorescence excitation of single molecules in 1990, a method with better signal-to-noise and the basis of most later work.
5. Single molecules at room temperature (1990s). Techniques with confocal microscopes and sensitive cameras brought single-molecule detection to room temperature and to living samples.
6. GFP blinking (1997). Moerner's group at UC San Diego, with Roger Tsien's lab, studied single molecules of a mutant of green fluorescent protein (GFP). They found that each molecule blinked on and off, and that violet light could switch non-fluorescent molecules back to a fluorescent state. This was a first demonstration that fluorescent proteins can be optically switched, which gave Betzig the idea of using such switches for PALM.

The Chemistry Behind It

The key concept is the narrow zero-phonon line of a chromophore at very low temperature. A molecule's absorption is governed by the Beer-Lambert law for an ensemble, A = εcl. For one molecule, the relevant quantity is the absorption cross-section σ. The fraction of laser light absorbed is roughly σ divided by the area A of the beam focus:

ΔI/I ≈ σ / A

For a broad room-temperature band σ is far too small for this to work. But at liquid-helium temperature, all the absorption strength of the molecule is concentrated in a very narrow zero-phonon line, so σ at resonance becomes much larger. Together with tight focusing of the laser to a spot a few micrometres wide, and with sensitive modulation detection, the signal rises to a measurable fraction of the beam, of the order of 10⁻⁴ or smaller.

In fluorescence mode, a molecule is excited from the ground state S₀ to the excited state S₁, and then can emit a photon (fluorescence) or cross to the triplet state T₁ (intersystem crossing). While in T₁, the molecule is dark until it returns to S₀; this gives blinking. Single-molecule traces show:

S₀ + hν → S₁ → S₀ + hν'      (fluorescence "on")
S₁ → T₁ (dark "off", lifetime µs–ms) → S₀
Photobleaching: irreversible chemical change (permanent "off")

An important result of single-molecule work is that the distribution of properties (rates, spectra, conformations) can be measured. In an ensemble one only sees the mean; for single molecules we see the full distribution and can observe rare states and intermediates in time, without synchronising the molecules.

Applications Today

• Super-resolution microscopy. The ability to turn molecules on and off is the basis of PALM, STORM and related methods.
• Biophysics. Single-molecule fluorescence resonance energy transfer (smFRET) shows how proteins and nucleic acids change shape during function.
• Molecular motors. Researchers track kinesin, myosin and polymerases, step by step.
• Anti-Brownian electrokinetic (ABEL) trap. Moerner's lab developed a device that holds a single molecule in solution by feedback-controlled electric fields, allowing long observations.
• DNA sequencing. Single-molecule real-time sequencing approaches monitor single enzymes as they incorporate fluorescent nucleotides.

Exam Corner

• Nobel Prize in Chemistry 2014: Betzig, Hell and Moerner, for super-resolved fluorescence microscopy.
• Moerner (1989) detected optical absorption of a single pentacene molecule in a p-terphenyl crystal at about 1.8 K.
• Inhomogeneous broadening: ensemble line is a sum of many narrow homogeneous lines.
• Spectral hole burning is a method for beating inhomogeneous broadening.
• Blinking of single molecules arises from intersystem crossing to the triplet state and other dark states.

Common Misconceptions

• "A single molecule cannot be seen with light." It can, if the background is reduced and the sample is arranged so that only one molecule is in resonance.
• "Ensemble averages are always enough." Averages hide heterogeneity and rare events that single-molecule methods reveal.
• "Moerner invented the super-resolution microscope." His discoveries provided the foundation; PALM and STED were developed by Betzig and Hell.

FAQ

Why liquid helium temperature?

At about 2 K, vibrations are frozen and molecular absorption lines become very narrow, which allows one molecule to be picked out spectrally.

What is spectral hole burning?

A narrow laser changes the molecules at a particular frequency, leaving a dip in the inhomogeneously broadened band.

Why is single-molecule data valuable?

It reveals distributions, intermediates and dynamics that ensemble measurements average out.

What is GFP?

Green fluorescent protein, from the jellyfish Aequorea victoria, is a protein that glows green. It was the subject of the 2008 Chemistry Nobel Prize (Shimomura, Chalfie and Tsien).

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