Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins

Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins
approved October 11, 2005 (received for review July 15, 2005)
Published online before print November 28, 2005
Michael Hofmann , Christian Eggeling , Stefan Jakobs, and Stefan W. Hell
Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, D-37077 G?ttingen, Germany
Edited by Erich P. Ippen, Massachusetts Institute of Technology, Cambridge, MA
Fluorescence microscopy is indispensable in many areas of science, but until recently, diffraction has limited the resolution of its lens-based variant. The diffraction barrier has been broken by a saturated depletion of the marker's fluorescent state by stimulated emission, but this approach requires picosecond laser pulses of GW/cm2 intensity. Here, we demonstrate the surpassing of the diffraction barrier in fluorescence microscopy with illumination intensities that are eight orders of magnitude smaller. The subdiffraction resolution results from reversible photoswitching of a marker protein between a fluorescence-activated and a nonactivated state, whereby one of the transitions is accomplished by means of a spatial intensity distribution featuring a zero. After characterizing the switching kinetics of the used marker protein asFP595, we demonstrate the current capability of this RESOLFT (reversible saturable optical fluorescence transitions) type of concept to resolve 50?100 nm in the focal plane. The observed resolution is limited only by the photokinetics of the protein and the perfection of the zero. Our results underscore the potential to finally achieve molecular resolution in fluorescence microscopy by technical optimization.
photoswitching | nanoscopy | resolution | saturation | photochromic
Owing to its specificity and sensitivity, fluorescence microscopy would be extremely powerful for biological imaging (1, 2) if diffraction (3) did not pose a limit on the minimal distance x at which similarly marked objects can be discerned. In the focal plane, x is well approximated by Abbe's equation, x /(2nsin), where is the wavelength of light and nsin is the numerical aperture of the lens (3). With typical values of nsin < 1.5, it follows that x will never be smaller than /3. However, in an emerging family of microscopes using reversible saturable optical fluorescence transitions (RESOLFT) between two marker states (A and B), the resolution is governed by
with denoting the saturation factor of the saturated transition. In a RESOLFT microscope, yields x 0, meaning that the resolution is no longer limited by diffraction (4, 5).
The simplest variant of RESOLFT microscopy is readily explained as follows. If we illuminate with a (diffraction-limited) intensity I(x) that features a point x0 with I(x0) = 0 and I(x0 ? ) > 0, to induce A B, this transition will occur everywhere except at x0. Saturating A B by increasing max[I(x)] creates narrow regions of state A delimited by x0 ? x/2, even though I(x) is limited by diffraction. For example, if the state A is a fluorescent state, the fluorescence will be possible only in this narrow region around x0 whose extent x can be squeezed down to the molecular scale. Images can now be obtained by moving the intensity zero across the specimen and subsequently reading out the fluorescence for each coordinate. This concept is not restricted to a single zero but can be extended to include many zero points or lines, in which case, one can use a camera for sequential read-out and image buildup (4?6).
With denoting the cross section of A B, the rate kAB is given by I(x). In a RESOLFT microscope, the resolution and the effective spot size x depend on the rate of possible competing processes that may counteract the saturation of A B. If the competing process is, for example, a (spontaneous) transition B A occurring at rate kBA, this rate must be outperformed (kAB >> kBA) by applying I(x) >> kBA/ Isat. The saturation intensity Isat thus classifies the intensity magnitude required to prepare small x. For a given form of I(x), x merely depends on max[I(x)] Isat. Calculation shows that for >> 1, the state A is confined to x as given by Eq. 1 (4, 5).
Stimulated emission depletion (STED) microscopy is a RESOLFT type of microscopy, where the fluorescent molecular state (A) is deexcited to the ground state (B) by stimulated emission (6, 7). Because the saturation of stimulated emission (typical 10?17 cm2) is opposed by the nanosecond fluorescent decay kfl (1 ns)?1, STED necessitates Isat = kfl/ 100 MW/cm2. Although the potential of this method to resolve /50 (16 nm) has been confirmed, as has Eq. 1 (8), a disadvantage of STED is the requirement for intense (picosecond) pulses tending to boost multiphoton-induced bleaching of the dye (9, 10). Here, we break the diffraction barrier by using ultralow levels of light by employing a saturable transition between two conformational states of a fluorescent protein. The weak spontaneous interstate conversion results in weak competing rates and hence low Isat.
Photochromic fluorescent proteins have recently become a target of research because of their ability to visualize protein tracking (11?16). In this study, we use asFP595 (11) from Anemonia sulcata, featuring a fluorescence-activated metastable "on" state (state A) and a fluorescence-inhibited metastable "off" state (state B), between which the protein can be "switched" by using blue (on off) and yellow (off on) light. After characterizing the transition rates of asFP595, we prove the breaking of the diffraction barrier with a few nanowatts of light.
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