Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution

Abstract

Using ultralow light intensities that are well suited for investigating biological samples, we demonstrate whole-cell superresolution imaging by nonlinear structured-illumination microscopy. Structured-illumination microscopy can increase the spatial resolution of a wide-field light microscope by a factor of two, with greater resolution extension possible if the emission rate of the sample responds nonlinearly to the illumination intensity. Saturating the fluorophore excited state is one such nonlinear response, and a realization of this idea, saturated structured-illumination microscopy, has achieved approximately 50-nm resolution on dye-filled polystyrene beads. Unfortunately, because saturation requires extremely high light intensities that are likely to accelerate photobleaching and damage even fixed tissue, this implementation is of limited use for studying biological samples. Here, reversible photoswitching of a fluorescent protein provides the required nonlinearity at light intensities six orders of magnitude lower than those needed for saturation. We experimentally demonstrate approximately 40-nm resolution on purified microtubules labeled with the fluorescent photoswitchable protein Dronpa, and we visualize cellular structures by imaging the mammalian nuclear pore and actin cytoskeleton. As a result, nonlinear structured-illumination microscopy is now a biologically compatible superresolution imaging method.patterned excitation | moiré | subdiffraction

Highly photostable, reversibly photoswitchable fluorescent protein with high contrast ratio for live-cell superresolution microscopy

“…For transitions in either direction, the population of RSFP molecules remaining in the initial state is well approximated by a reciprocal function of both time and illumination intensity (13) that encompasses both fast and slow components of exponential decay. Thus, either state can be saturated, which results in the required nonlinear dependence of fluorescence emission rate on the illumination intensity (5,12). Moreover, there is no other competing process such as spontaneous transition between the two states, and the absorption crosssection for photoinduced switching from either state is very high.…”

“…Recently, reversibly photoswitchable fluorescent proteins (RSFPs) have demonstrated the ability to significantly reduce SR illumination intensities when applied to reversible saturable optical fluorescence transition (RESOLFT) microscopy (5) and nonlinear SIM (NL-SIM) (12). RSFPs can be repeatedly transited between fluorescent bright and dark states via photoinduced reversible cis-trans isomerization.…”

Saturated excitation of fluorescent proteins for subdiffraction-limited imaging of living cells in three dimensions

“…However, spatial resolution is limited by the wave nature of light, which makes it difficult to resolve details in cells smaller than about half the illumination wavelength. In the past 20 years, the diffraction barrier has been overcome by several advanced techniques in far-field fluorescence microscopy such as stimulated emission depletion microscopy [1,2], photoactivated localization microscopy [3,4], stochastic optical reconstruction microscopy [5][6][7], saturated structured illumination microscopy [8][9][10][11] and so on. Recently, superresolution fluorescence imaging of thick specimens was also achieved [12,13].…”

Fluorescence nanoscopy. Methods and applications

“…Upon detection, the pattern is demodulated and the fine details are recovered. Resolution is limited to twice the resolution of diffraction-limited microscopy Most fluorophores [100] Nonlinear SIM or sSIM Nonlinear molecular behaviour is exploited for improved SIM resolution Photoswitchable proteins [103] Resolution of STED microscopy scales as the inverse square root of the depletion beam intensity I max (Eq. 2; ≈1 GW cm −2 is required for 65 nm lateral resolution).…”

“…Reversible photoswitching of a fluorescent protein provides the required nonlinear response at light intensities six orders of magnitude lower and has very recently been shown to be a biologically compatible super-resolution imaging method, achieving a resolution of 50 nm in TIRF configuration [103]. SSIM acquisition only comprises 25 images at moderate intensities on the order of 1-10 W cm −2 and has, thus, great potential for live-cell imaging.…”