Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging

Abstract

We demonstrate stimulated emission depletion (STED) microscopy implemented in a laser scanning confocal microscope using excitation light derived from supercontinuum generation in a microstructured optical fiber. Images with resolution improvement beyond the far-field diffraction limit in both the lateral and axial directions were acquired by scanning overlapped excitation and depletion beams in two dimensions using the flying spot scanner of a commercially available laser scanning confocal microscope. The spatial properties of the depletion beam were controlled holographically using a programmable spatial light modulator, which can rapidly change between different STED imaging modes and also compensate for aberrations in the optical path. STED fluorescence lifetime imaging microscopy is demonstrated through the use of time-correlated single photon counting.

3‐D stimulated emission depletion microscopy with programmable aberration correction

“…Here we demonstrate the simultaneous application of two depletion PSFs corresponding to different phase profiles implemented using a single SLM. The use of SLMs to manipulate the phase profiles for STED microscopy has been previously demonstrated [29,30] and provides the facility to program arbitrary phase profiles that can include pre-compensation for distortion of the STED beam caused by optical aberrations in the micro-scope or sample [30,31]. We note that spherical aberration can also be corrected manually with static optics in a STED microscope [32] but this needs to be adjusted for each imaging depth and so is impractical for x-z scanning.…”

“…This setup is based on a STED-FLIM microscope described previously [30] for which the depletion beam is provided by a mode-locked Ti : Sapphire laser and the excitation beam is obtained by coupling a fraction of the modelocked (~150 fs duration) pulse train into a~1 m length of microstructured optical fibre (MOF) in which a supercontinuum is generated. This supercontinuum typically spans from~500 nm to beyond 1 mm, from which appropriate excitation radiation is selected using a bandpass filter (628/40 or 609/54).…”

Fluorescence nanoscopy. Methods and applications

“…FCS is a technique that allows the diffusion coefficient of a molecule to be determined from the analysis of the fluctuations of its fluorescence emission as it traverses a known volume: The lower the number of fluorophores within the focal volume, the more accurate the measurements. Also, fluorescence lifetime imaging with subdiffraction resolution has been demonstrated on a STED microscope [21], paving the way for functional nanoscopy.…”

“…Fluorescence nanoscopy makes possible the investigation of the nanoscale dynamic organisation of proteins within subsynaptic domains: The fate of synaptic vesicles after exocytosis and fusion with the membrane has been studied by STED microscopy in fixed [199,200] and live neurons using video-rate (20)(21)(22)(23)(24)(25)(26)(27)(28) [27,201] and time-lapse STED microscopy [202,203]. AMPA receptors trafficking in neuronal cells have been investigated by single particle tracking PALM [204].…”

Supercontinuum ultra wide range confocal microscope for reflectance spectroscopy of living matter and material science surfaces

“…5 Among confocal microscopy methods, the reflectance approach has recently gained great attention especially in the field of dermatology diagnostic thanks to the possibility to image skin tissue structures at different depths using two or three different wavelengths. 6 The introduction of recently commercially available supercontinuum sources, usually in combination with AOTF (acoustic optical tunable filters), has led to advances in several of these fields, [7][8][9][10][11] and opens the possibility to work in the near infrared (IR) spectral range, which has been previously used mainly to have new sources for excitation in the two-photon visible fluorescence microscopy. Recent commercial confocal laser scanning microscopes also present a spectral mode, but, since acquisition is performed via PMT or APD detection, it is possible to obtain the whole spectrum only by sequentially scanning AOTFs, prisms or gratings.…”