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

Abstract

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. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Measurement of nanoscale three-dimensional diffusion in the interior of living cells by STED-FCS

“…This is a notable difference between gated STED-FCS and SPLIT-FLCS. In gated STED-FCS the observation volume is tuned by changing a temporal detection window that, alone, is not sufficient to remove the uncorrelated background from the signal 21 . In the SPLIT-FLCS method, we are able to isolate the uncorrelated background from the total signal by considering it as an additional fluorescence intensity “decay”.…”

“…This feature makes SPLIT particularly attractive for STED-FCS in 3D. In CW-STED-FCS the two most common sources of temporally uncorrelated background are (i) the detector afterpulse and (ii) the anti-Stokes fluorescence emission induced directly by the STED beam 21 . Detector afterpulse can be removed by cross-correlating the signal from two detectors 22 , whereas STED-induced background is more difficult to deal with.…”

“…Detector afterpulse can be removed by cross-correlating the signal from two detectors 22 , whereas STED-induced background is more difficult to deal with. For instance, in gated STED-FCS the temporal gate is not sufficient to eliminate the uncorrelated background from the signal 21 .…”

Resonant-scanning dual-color STED microscopy with ultrafast photon counting: A concise guide

“…This problem can be partially resolved by implementing time-gated detection [7–9]. Recently, a lock-in detection technique [10] and a filtering method [11], both based on temporally differentiating background caused the CW depletion light from the pulsed fluorescence signal, have been invented to improve the signal-to-noise ratio in time-gated CW STED microscopy.…”

Low photobleaching and high emission depletion efficiency: the potential of AIE luminogen as fluorescent probe for STED microscopy

“…It can achieve very high lateral spatial resolution (<50 nm) and be used to probe fine biological structure [1][2][3][4]. In STED microscopy, two laser beams are adopted.…”