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

作者：Hernandez Ivan Coto; Peres Chiara; Zanacchi Francesca Cella; d'Amora Marta; Christodoulou Sotirios; Bianchini Paolo; Diaspro Alberto^*; Vicidomini Giuseppe

来源：Journal of Biophotonics, 2014, 7(6): 376-380.

DOI：10.1002/jbio.201300208

• fluorescence microscopy
• stimulated emission depletion (STED) microscopy
• super-resolution microscopy
• background subtraction

Stimulated emission depletion (STED) microscopy is a prominent approach of super-resolution optical microscopy, which allows cellular imaging with so far unprecedented unlimited spatial resolution. The introduction of time-gated detection in STED microscopy significantly reduces the (instantaneous) intensity required to obtain sub-diffraction spatial resolution. If the time-gating is combined with a STED beam operating in continuous wave (CW), a cheap and low labour demand implementation is obtained, the so called gated CW-STED microscope. However, time-gating also reduces the fluorescence signal which forms the image. Thereby, background sources such as fluorescence emission excited by the STED laser (anti-Stokes fluorescence) can reduce the effective resolution of the system. We propose a straightforward method for subtraction of anti-Stokes background. The method hinges on the uncorrelated nature of the anti-Stokes emission background with respect to the wanted fluorescence signal. The specific importance of the method towards the combination of two-photon-excitation with gated CW-STED microscopy is demonstrated.
[GRAPHICS]
Principle and time course of the measurement for the proposed anti-Stokes emission background subtraction method (right). Application of the method for a fluorescent beads calibration sample (left).

• 出版日期2014-6