Nobel Lecture: Nanoscopy with freely propagating light^*

Figure 1

Length scales and spatial resolution limits of visual inspection (human eye), light (optical) microscopy, and electron microscopy. Far-field optical nanoscopy extends the resolution much beyond Abbe’s limit of half the wavelength of light ($>200\mathrm{nm}$), down to the single-digit nanometer range.

Figure 2

Light microscopy remains the most popular microscopy method in the life sciences, due to a number of distinct advantages such as live-cell imaging and biomolecular specificity. The latter is provided by labeling the biomolecules of interest with fluorescent markers, allowing their species-specific detection in the microscope.

Figure 3

Focusing of light by the microscope (objective) lens cannot occur more tightly than the diffraction (Abbe’s) limit. As a result, all molecules within this diffraction-limited region are illuminated together, emit virtually together, and cannot be told apart.

Figure 4

The diffraction resolution limit carved in stone (top: memorial in honor of Ernst Abbe in Jena, Germany), and resolution increase brought about by STED nanoscopy (top of recording) over confocal imaging (bottom) when imaging the cytoskeleton in a cell.

Figure 5

Realization in the early 1990s that the key to surpassing the diffraction resolution limit lies in fluorophore properties (quote from a manuscript submitted in 1993, top). The photograph (bottom) shows page 20 of the book “The Quantum Theory of Light” by Rodney Loudon (Oxford Science Publications), where I found a reminder of the phenomenon of stimulated emission, which I, of course, knew about from my physics studies, on Saturday morning, 6 November 1993. My copy of the book is now on display at the Nobel Museum, Stockholm.

Figure 6

Switching molecules within the diffraction-limited region transiently “off” (i.e. effectively keeping them in a nonsignaling state) enables the separate detection of neighboring molecules residing within the same diffraction region. (a) In fluorescence microscopy operating with conventional lenses (e.g. confocal microscopy), all molecules within the region covered by the main diffraction maximum of the excitation light are flooded with excitation light simultaneously and emit fluorescence together. This is because they are simultaneously allowed to assume the fluorescent (signaling) state. (b) Keeping most molecules–except the one(s) one aims to register–in a dark state solves the problem. The dark state is a state from which no signal is produced at the detector. Such a transition to the dark “off” state is most simply realized by inducing stimulated emission, which instantaneously forces molecules to their dark (“off”) ground state.

Figure 7

STED microscopy. (a) Setup schematic. (b) Region where the molecule can occupy the “on” state (green) and where it has to occupy the “off” state (red). (c) Molecular transitions. (d) For intensities of the STED light (red) equaling or in excess of the threshold intensity $I_s$, molecules are effectively switched “off.” This is because the STED light will always provide a photon that will stimulate the molecule to instantly assume the ground state, even in the presence of excitation light (green). Thus, the presence of STED light with intensity greater than $I_s$ switches the ability of the molecules to fluoresce off.

Figure 8

Nuclear pore complex architecture in an intact cell nucleus imaged by (a) confocal microscopy (diffraction-limited), and (b) STED nanoscopy.

Figure 9

STED nanoscopy of the HIV Envelope protein Env on single virions. Confocal microscopy is not able to reveal the nanoscale spatial distribution of the Env proteins; the images of the Env proteins on the virus particles look like 250–350 nm sized blurred spots (orange, left column). STED microscopy reveals that the Env proteins form spatial patterns (center column, orange), with mature particles having their Env strongly concentrated in space (panel in top row of center column, orange).

Figure 10

Video-rate STED imaging of synaptic vesicle motion in axon of living hippocampal neuron. (Lecture contains movie.)

Figure 11

STED nanoscopy in living mouse brain. The recording shows a part of a dendrite of a neuron expressing a yellow fluorescent protein (EYFP) in the cytosol, thus highlighting the neuron amidst surrounding (nonlabeled) brain tissue. The 3–4 fold improved resolution over confocal and multiphoton excitation fluorescence microscopy reveals the dendritic spines (encircled) with superior clarity, particularly the cuplike shape of some of their terminals containing the receiving side of the synapses.

Figure 12

Rendition of three-dimensional STED nanoscopy data showing the dendritic actin from a neuron of a living organotypical hippocampal brain slice (mouse).

Figure 13

Resolution scaling in the STED/RESOLFT concepts: an extension of Abbe’s equation. The resolution scales inversely with the square root of the ratio between the maximum intensity at the doughnut crest and the fluorophore-characteristic threshold intensity $I_s$.

Figure 14

Fluorophores affording virtually unlimited repetitions of the resolution-enabling on-off state transitions provide the present resolution records in far-field optical imaging using STED, in the single-digit nanometer regime. Color centers (charged nitrogen vacancy centers) in diamond hold great potential for various other applications, notably in magnetic sensing and quantum information, which may be eventually read out with diffraction-unlimited spatial resolution using conventional lenses, i.e. even when packed very densely at the nanometer scale.

Figure 15

States and state transitions utilized in (a) STED, (b) GSD, and (c) RESOLFT nanoscopy. (d) The modified expression for the resolution describes the spatial region in which molecules can still reside in the on state. (e) The intensity $I_s$ for guaranteeing the transition from the ON to the OFF state is inversely related to the state liftetime. The longer the lifetime of the involved states, the fewer photons per second are needed to establish the on-off state difference which is required to separate features residing within the diffraction barrier.

Figure 16

Parallelization of the STED/RESOLFT concept holds the key to faster imaging. The diffraction problem has to be addressed only for molecules residing within a diffraction-limited region. Thus, many intensity minima (“doughnuts”) are produced, at mutual distances greater than the diffraction limit, for highly efficient scanning of large sample areas. The use of highly parallelized schemes is greatly facilitated by harnessing transitions between long-lived molecular on-off states, such as cis/trans.

Figure 17

Massively parallelized RESOLFT nanoscopy. Here, an array of $~114000$ intensity minima (zeros) was used to image a living cell in 2 s.

Figure 18

Paradigm shift in the use of the physical phenomenon by which features are discerned in a far-field optical (fluorescence) microscope: from focusing of light, which is inherently diffraction limited, to using a molecular state transition, such as a transition between an “on” and an “off” state, which is not.

Figure 19

Both in coordinate-targeted and in coordinate-stochastic nanoscopy methods, many photons are required to define or establish, respectively, molecular coordinates at subdiffraction scales. In the coordinate-targeted mode (STED, RESOLFT, etc.), the coordinates of (e.g.) the “on” state are established by illuminating the sample with a pattern of light featuring an intensity zero; the location of the zero and the pattern intensity define the coordinates with subdiffraction precision. In the coordinate-stochastic mode (PALM, STORM, etc.), the coordinates of the randomly emerging “on”-state molecules are established by analyzing the light patterns emitted by the molecules (localization). Precision of the spatial coordinate increases in both cases with the number of photons in the patterns, i.e. by the intensity of the pattern. In both families of methods, neighboring molecules are discerned by transiently creating different molecular states in the sample.

Figure 20

To parallelize STED/RESOLFT scanning, a “widefield” arrangement with an array of intensity minima (e.g. an array of doughnuts) may be used. The numbers of molecules at these readout target coordinates do not matter, while PALM requires that there may be only a single “on”-state molecule within a diffraction zone, i.e. within the distance dictated by the diffraction barrier. (More precisely: the number of molecules per diffraction zone has to be so low that each molecule is recognized individually.) The position of each on-state molecule is however completely random in space. $I_s$ can be regarded as the number of photons that one needs to ensure that there is at least one photon interacting with the molecule, pushing it from one state to the other in order to create the required difference in molecular states. $I/I_s$ is, so to speak, the number of photons which really elicit the (on/off) state transition at the molecule, while most of the others just “pass by.” Similarly, in the PALM concept, the number of photons $n$ in $1/\sqrt{n}$ is the number of those photons that are really detected at the coordinate-giving pixilated detector (camera), i.e. that really contribute to revealing the position of the emitting molecule. In other words, in both concepts, to attain a high coordinate precision, one needs many photons that act.

Figure 21

From lenses to molecular switches. Whereas in the 20th century, the focusing quality of the lenses (i.e. excellent aberration correction) was decisive for gaining the best spatial resolution, now it is the molecules and their state transitions which become central to achieving the best resolution. Optimizing molecules toward providing robust and repeatedly executable (on/off) state transitions (STED/RESOLFT) and photon emissions (PALM) is primarily a chemistry problem.

Figure 22

The limiting role of diffraction is overcome by utilizing at least two molecular states to separate features residing closer than the diffraction barrier during the brief period in which the features are jointly illuminated for detection. While fluorescent molecules have been the first type of molecules which have provided such states, other molecules and states are conceivable that are not necessarily of the fluorescent type. This is why one could imagine breaking the diffraction barrier also in a nonfluorescence far-field optical microscope, provided suitable states and state transitions are identified.