The diffraction limit isn’t the barrier it used to be. Researchers around the world have developed schemes that allow optical imaging far below the traditional diffraction limit of half a wavelength of light. Many of these techniques, however, can be used only on thin samples. Now researchers at the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany, have come up with a new dye, 5-NHSS, and an associated technique that overcome this problem.The method promises far-field optical microscopy with a resolution of 10 nm. Because it can be done using multiphoton excitation, it can section a specimen optically and isn’t confined to thin samples.At the heart of many subdiffraction-limit imaging techniques is the use of single molecules of fluorescent dye. If the molecules are far enough apart, their images don’t overlap, and they act like point sources of light. Every photon captured provides diffraction-limited information concerning a particular molecule’s location. Once a number of photons are collected, the localization is the diffraction limit divided by the square root of the number of captured photons.Increasing the resolution tenfold requires collecting 100 times as many photons. As a consequence, a high photon flux per molecule is important, and a mostly uniform photon count for individual dye molecules is also helpful.Another constraint on the fluorescent molecules in these methods is that they must be sparse throughout the sample — but not too sparse. Too high a density makes the scheme fail because the images from the molecules overlap. Too low a density makes the picture painted of the sample incomplete. Each dye molecule is essentially being used to precisely pinpoint the location of a single point in the specimen.On the left is a wide-field image of a tubulin network in a PtK2 cell stained with the photochromic rhodamine dye 5-NHSS. In the middle is a single-photon image of the same network, reconstructed from turning on the dye selectively and mapping the location of each single molecule. On the right is a two-photon image done the same way. As can be seen, the single-molecule fluorescence readout improves resolution markedly because it avoids the diffraction limit. Images reprinted with permission of Angewandte Chemie.In its research, the team developed a photochromic rhodamine derivative, an easily controllable photoswitchable compound with a high fluorescence quantum yield and high photochemical stability under single-molecule conditions. A photochromic reaction of rhodamine amides had been first reported decades ago, the researchers noted. The reaction had not been pursued, in part because the molecules’ characteristics made them undesirable for applications such as optical memories and switches. Those same attributes, on the other hand, make them good candidates for single-molecule microscopy markers. The synthesis process that produces the dye 5-NHSS starts with a closed five-ring isomer. In this form the molecule is colorless and nonfluorescent, but illuminating the molecule with light in the 313- to 380-nm range with the right intensity transforms it. One ring of the isomer opens, and, in this form, the molecule appears red, absorbing in the green and emitting at around 580 nm. The same activation can be achieved using two-photon absorption in the 650- to 800-nm range. Whether produced by one- or two-photon absorption, the isomer is brightly fluorescent, with the researchers reporting up to 900 photons detected per molecule in polyvinylalcohol before photobleaching.The open isomer spontaneously reverts to the closed form, on a time-scale that depends upon the environment. In a non-polar solvent such as polyvinylalcohol, the process can take hours. In a polar solvent such as water, the reversion takes only 20 to 100 ms. For their demonstration imaging setup, the researchers used a home-built wide-field microscope with a two-photon beam scanning system. They photoswitched the dye via two methods. In the case of the single-photon absorption activation, they used a 375-nm pulse from a diode laser from Toptica Photonics of Gräfelfing, Germany, with wide-field illumination to cover a 12-μm-diameter spot. For two-photon absorption activation, they used a Ti:sapphire laser from Coherent of Santa Clara, Calif., tuned to 747 nm and supplying 5-ps pulses. They focused the beam to a diffraction-limited focal spot of 280 nm.They used a Coherent laser to provide the 532-nm excitation light in a wide-field illumination mode and captured the resulting point sources with an Andor electron-multiplying CCD camera.With this setup, they tested the dye in a number of situations. Using polyvinylalcohol and the dye alone, they found that they could collect the maximum value of about 900 photons per molecule in 20 ms per frame. The molecule localization accuracy would then be about 10.5 nm. In contrast, the accuracy at 2-ms exposure time was 14 nm. However, a 20-ms exposure increased the background signal and the overall acquisition time — the frame exposure time multiplied by the number of frames. A typical image would require 10,000 to 20,000 frames. Shortening the exposure time to 10 ms led to 11-nm localization, little background noise and a total acquisition time of one to three minutes. This acquisition time was short enough that no special stabilization or drift compensation was needed.In a test, the researchers labeled a tubular network of mammalian cells with 5-NHSS and imaged the cells using single-photon absorption, and two-photon absorption for photoactivation. They found the two sets of images virtually identical.Two-photon activation offers a number of advantages, such as avoiding damaging ultraviolet light and allowing optical sectioning, which enables a greater density of markers and deeper penetration into tissue.Combining two-photon imaging with single-molecule fluorescence-based location readout, researchers mapped the surface of 5-μm silica beads surface-stained with the photochromic rhodamine dye 5-NHSS. A reconstruction based on 17 slices in the Z direction is shown here.The investigators verified the viability of two-photon absorption for sectioning by imaging 5-μm silica beads surface-stained with the dye. They also performed axial sectioning by imaging lamin proteins stained with the dye in the nucleus of human glioma. Both cases showed good lateral resolution with optical sections of less than 1 μm and, in the case of the cells, did so with a sample greater than 6 μm thick. The work is detailed in the Aug. 20 issue of Angewandte Chemie.The researchers expect multicolor applications, a possibility because the emission spectrum of the dye can be shifted by changing the rhodamine in the compound. In polar solvents, the markers can be localized several times by introducing a lag time between frames, which will allow the thermal relaxation to the non-fluorescent state to take place between images. That could be useful during live-cell experiments, the researchers noted.