Live-Cell Superresolution Imaging by Pulsed STED Two-Photon Excitation Microscopy

Abstract

Two-photon laser scanning microscopy (2PLSM) allows fluorescence imaging in thick biological samples where absorption and scattering typically degrade resolution and signal collection of one-photon imaging approaches. The spatial resolution of conventional 2PLSM is limited by diffraction, and the near-infrared wavelengths used for excitation in 2PLSM preclude the accurate imaging of many small subcellular compartments of neurons. Stimulated emission depletion (STED) microscopy is a superresolution imaging modality that overcomes the resolution limit imposed by diffraction and allows fluorescence imaging of nanoscale features. Here, we describe the design and operation of a superresolution two-photon microscope using pulsed excitation and STED lasers. We examine the depth dependence of STED imaging in acute tissue slices and find enhancement of 2P resolution ranging from approximately fivefold at 20 μm to approximately twofold at 90-μm deep. The depth dependence of resolution is found to be consistent with the depth dependence of depletion efficiency, suggesting resolution is limited by STED laser propagation through turbid tissue. Finally, we achieve live imaging of dendritic spines with 60-nm resolution and demonstrate that our technique allows accurate quantification of neuronal morphology up to 30-μm deep in living brain tissue.

A new filtering technique for removing anti‐Stokes emission background in gated CW‐STED microscopy

“…Even if we shifted the STED beam wavelength to 577 nm, the resolution enhancement expected from 2PE-gCW-STED becomes relevant only when we applied the proposed filter (see Figure 4). The method represents an important step towards the implementation of an efficient but also cheap and low-complex implementation of 2PE-STED microscopy [13,14].…”

Recent applications of superresolution microscopy in neurobiology

“…Exchanging the fluorescent protein for Dronpa-M159T, a fast-switching variant of Dronpa, enabled the recording of similar images of actin distribution in slices at much lower light levels using RESOLFT microscopy [32]. STED microscopy of acute brain slices and imaging of dendritic spines has been used by two groups, who implemented a two-photon excitation in combination with pulsed stimulated emission depletion [33,34], one using cytosolic fluorescent protein labels and establishing also a second color to image protrusions of microglia [35 ], the other injecting Alexa Fluor 594 into individual neurons [34].…”

Accumulating advantages, reducing limitations: Multimodal nonlinear imaging in biomedical sciences – The synergy of multiple contrast mechanisms

“…Recently sub-diffraction limited imaging methods were adapted to TPEF microscopy, increasing the resolution beyond the diffraction limit. The combination of STED [77,[80][81][82][83] with nonlinear excitation requires a chromophore with large Stokes shift. In case of STED a significant increase in resolution has been realized, e.g., of 60 nm [82,83].…”

“…While two-photon-STED increases the resolution, the resolution enhancement decreases with depth. So far twofold increase in resolution at 100 mm depth has been reported [83]. Here structured illumination in combination with temporal focusing is an alternative method to increase the resolution in two-photon fluorescence microscopy deeper in tissue.…”