TomoSTED – faster STED imaging at lower light doses

Tomographic STED (TomoSTED) uses a rotating 1D depletion pattern instead of the classical 2D donut shaped STED-beam. The reduction from 2D to 1D depletion results in a more robust depletion with a favourable light intensity distribution pattern, which allows to significantly reduce the overall light dose while simultaneously increasing the signal to noise ratio. Thus TomoSTED allows faster imaging while causing less bleaching while maintaining image quality.

Challenge

Super-resolution microscopy allows imaging of objects which are way smaller than the optical diffraction limit. This is performed by reducing the detection volume with a high intensity STED-Beam, which is wrapped around the focus in the shape of a 2D or 3D donut and switches off the fluorophores in the periphery. However, the tremendous advantage of an increased resolution comes with the downside, that it imposes high demands on the sample. The increased spatial sampling rate and the high light intensities result in an increased fluorophore bleaching. Furthermore, the reduced detection volume also results in a reduction of fluorescence signal in the focus. Thus, for many applications in the life sciences, the achievable resolution is not limited by the microscope itself but by the available fluorescence signal or the tolerable bleaching rate.

Our Solution

In tomographic STED microscopy, the light dose can be significantly reduced, while simultaneously the fluorescence signal from the focus is increased. This is realized by confining the fluorescence in only one lateral direction at the same time. A two-dimensional (2D) super-resolved image can be reconstructed from a series of sub-images with a rotated high-resolution axis.s.

Figure 1: Left: 1D STED focus, middle: classical STED focus. Right: Correlation between resolution and Power. The 1D focus used in tomoSTED microscopy features a more beneficial intensity distribution than the doughnut-shaped focus. Only half of the STED power is needed to achieve the same resolution with a 1D depletion pattern.

TomoSTED has two striking advantages over the classical STED-Approach using a 2D donut. First, the one-dimensional depletion pattern focuses the laser power to a smaller region than the well-known donut-shaped STED focus. Thus tomoSTED microscopy achieves identical super-resolution conditions at significantly lower laser powers (Fig. 1 graph), significantly reducing bleaching. Second, more signal can be gathered from the focus, since fluorescence is confined only in one dimension, which increases the signal to noise ratio and allows imaging of dimmer samples.

Advantages

Same resolution at reduced light dose (4x less for 2D and potentially 10x less for 3D imaging)
better signal to noise allows faster imaging and/or dimmer samples
significantly reduced photobleaching
The robust depletion pattern higher stability of the microscope system
A combination with reconstruction algorithms or ISM can further increase resolution and/or decrease the light dose
TomoSTED principle is applicable to other scanning super-resolution techniques

Development Status

The inventors have successfully detected and compensated different kinds of aberrations in technical and simple biological samples. Currently, they extend the method to relevant biomedical samples and validate its robust and simple applicability.