Subdiffraction-Limit Two-Photon Fluorescence Microscopy for GFP-Tagged Cell Imaging

Abstract

We report applications of two-photon excitation fluorescence (2PEF) microscopy with subdiffraction-limit resolution for green-fluorescent-protein-tagged cell imaging. The microscope integrates 2PEF microscopy and stimulated emission depletion microscopy in one microscope that has the benefits of both techniques: intrinsic three-dimensional resolution, confined photobleaching, and subdiffraction-limit resolution. The subdiffraction-limit resolution was demonstrated by resolving green-fluorescent-protein-tagged caveolar vesicles located within a distance shorter than the diffraction limit of a regular 2PEF microscope, which is approximately 250 nm even with the best optics. The full width at half-maximum of the effective point-spread function for the 2PEF microscope was estimated to be approximately 54 nm.

Imaging Living Synapses at the Nanoscale by STED Microscopy

“…STED has been shown to work in thicker tissue sections such as brain slices (Ding et al, 2009). A combination of 2-photon excitation and pulsed STED de-excitation, as previously shown (Li et al, 2009), should extend depth penetration even further without sacrificing spatial resolution. A unique advantage of STED over other nanoscopy techniques is its potential for targeted and tunable photoactivation or uncaging at nanoscale dimensions, possibly allowing the instant creation of high concentrations of physiologically relevant molecules at predefined locations in biological tissues.…”

Review of recent developments in stimulated emission depletion microscopy: applications on cell imaging

“…63 The 2P-p-STED microscopy was first demonstrated by resolving GFP-tagged caveolar vesicles in a chemically fixed CHO cell. 64 A simplified version of 2P-p-STED that uses a single laser line for both 2P excitation and 1P depletion was also demonstrated recently for 4-(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran (DCM) dyes. 65 The possibility of using this setup in cellular imaging was demonstrated by imaging the antibody-ATTO 647N-labeled microtubules in fixed PtK2 cells.…”

STED microscopy for nanoscale imaging in living brain slices

“…Subsequently, two-photon STED (using pulsed two-photon excitation and pulsed single-photon quenching) was developed for imaging GFP [59], and two-photon STED time-lapse imaging of neural structures was demonstrated in acute brain slices [50,52]. Notably, the latter two studies achieved a resolution of 50 nm some 50 lm below tissue surface using long-working distance water objectives, facilitating the combination of STED microscopy with neurophysiological approaches.…”