Pump-Probe Fluorescence (Stimulated Emission) Microscopy with Integrated Electronic Delay Control and Lock-In Detection
Ankur Gogoi1*, Subir Das1, Ho Bo Wei1, Fu-Jen Kao1
1Institute of Biophotonics, National Yang Ming University, Taipei, Taiwan
* presenting author:ANKUR GOGOI, email:ankurgogoi@gmail.com
Pump-probe fluorescence microscopy, especially stimulated emission (SE) microscopy, is one of the emerging imaging modalities due to the enhanced sensitivity and specificity, better spatiotemporal resolution as well as its three dimensional sectioning capability. The intrinsic spatial coherence of the stimulated emission with the probe beam also allows for the long working distance fluorescence detection. One additional unique feature of SE is the tolerance of high background lighting which precludes the necessity of a dark room or a photomultiplier in detection, an advantage in practical applications.

In this contribution, we report the successful implementation of the intensity modulated semiconductor lasers for SE microscopy applications. Long working distance fluorescence lifetime imaging was realized by using a pair of synchronized diode lasers operating at wavelengths 635 nm and 700 nm that worked as the pump and probe beams, respectively, for the ATTO647N labelled samples. An electronic time delay control and low NA optics was used to measure the fluorescence lifetime of ATTO647N dye. Notably, lock-in detection was used to select the SE signal, the output of which is connected to the A/D converter of the galvano-mirror based laser scanning system (Olympus FV300) to reconstruct the image. Fluorescence decay curves were obtained by correlating the acquired time resolved images at various time delays at steps of 0.5 ns. The scheme presented here proved to be a highly efficient alternative to measure relatively long fluorescence lifetime (of the order of a few nanosecond). In additional, the optimal lock-in configuration for pump-probe detection will also be discussed.

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Keywords: Pump-probe fluorescence microscopy, stimulated emission microscopy, fluorescence lifetime imaging, lock-in detection, electronic time delay.