Pump-Probe Microscopy for Stimulation Gain and Spontaneous Loss
Subir Das1*, Bo-Wei Ho1, Ankur Gogoi1, Fu-Jen Kao1
1Institute of biophotonics, National Yang-Ming University, Taipei, Taiwan
* presenting author:Subir Das, email:subir009@hotmail.com
Stimulated emission is currently a viable technique for detecting dark fluorophores and lifetime imaging [1-2]. Additionally, the stimulated emission based pump-probe technique is also used for sub-diffraction resolution imaging [3]. In this work, we have established a multimodal stimulated emission based pump-probe configuration for the detection of stimulated gain and spontaneous loss. We used a diode laser at wavelength λpu = 635nm for excitation (pump) and a Ti:sapphire laser with central wavelength λpr = 780 nm for stimulation (probe). The time delay between the two pulses is precisely controlled by varying the length of the trigger cable that synchronizes the pulse diode laser and the Ti:sapphire mode-locked laser. Both beams were modulated at different frequencies with detection with a lock-in amplifier. For stimulated gain, the pump beam was modulated at a frequency, ƒ₁, with only the probe beam allowed to pass through the filter. The transmission light which carries stimulated emission in the forward direction was then detected by a photodiode. The spontaneous loss was measured in reflection mode. In this case, the probe beam was blocked and modulated at a different frequency, ƒ₂. The spontaneous loss signal was detected by a PMT.
Note that the detection of spontaneous loss allows elimination of the large background which commonly exists in stimulated gain configuration. The additional modality would allow unprecedented manipulation and investigation of fluorophores in fluorescence imaging.

[1] W. Min, S. Lu, S. Chong, R. Roy, G.R. Holtom and X. S. Xie, “Imaging chromophores with undetectable fluorescence by stimulated emission microscopy,” Nature (2009).
[2] Po-Yen Lin, Shin-Shian Lee, Chia-Seng Chang and Fu-Jen Kao, “Long working distance fluorescence lifetime imaging with stimulated emission and electronic time delay,” Optics Express (2012).
[3] Jun Miyazaki, Hiromichi Tsurui, Akiko Hayashi-Takagi, Haruo Kasai and Takayoshi Kobayashi, “Sub-diffraction resolution pump-probe microscopy with shot noise limited sensitivity using laser diodes,” Optics Express (2014).

Keywords: Pump-probe microscopy, Stimulated emission, Lock-in detection, Background removal, Bioimaging