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Stimulated Emission Microscopy

ABSTRACT

Fluorescence labeling and detection is a powerful method in numerous applications, due to its high sensitivity and specificity. Fluorescence emission is a spontaneous process and the emitted photons are distributed among all solid angles (4Pi). Therefore, fluorescence imaging is commonly performed with high numerical aperture (NA) optics, to achieve both a high spatial resolution and effective collection of emitted photons. To detect weak fluorescence signals from dark fluorophores, a highly sensitive detector with a large gain is frequently used. Stimulated emission (SE) was demonstrated as an advantageous method in detecting dark fluorophores. The working principle of SE detection is to induce the electronic transition of an excited fluorophore into SE before its energy dissipates into other radiative or non-radiative decay processes. Therefore, the SE signals depend on both the population of excited state and the intensity of the stimulation beam. Without any saturation effects, the SE signal scales linearly with both excitation and stimulation beams, which renders its overall quadratic power dependence and the 3-D optical sectioning in imaging as a two-photon process. Additionally, spatial coherence of the stimulated fluorescence emission is maintained, leading to the emission in a narrow cone in the forward direction, similar to the case of second harmonic generation or other coherent optical processes. We have exploited the resulting spatial coherence for long working distance imaging of fluorescently labeled specimens.

principle 

Principle of coherent long working distance signal detection using stimulated emission

experimental setup 


APPLICATION 

Long distance stimulated emission (a) and fluorescence (b) images of samples of random Rhodamine 6G distributions. The distance of the sample from the collimating lens was 50 mm. Note that no spontaneous fluorescence signal is detected. For details please read this paper.

Long distance fluorescence lifetime imaging with pulsed diode laser enabled stimulated emission. For details please read this paper.

RELATED PUBLICATIONS

  1. Jianhong Ge, Cuifang Kuang, Shin-Shian Lee, and Fu-Jen Kao, Fluorescence lifetime imaging with pulsed diode laser enabled stimulated emission,Optics Express, Vol. 20, Iss. 27, pp. 28216–28221 (2012). Also selected for the Virtual Journal for Biomedical Optics (VJBO), Editor Andrew Dunn and Anthony Durkin, Vol. 8, Iss. 1, February 4, 2013.
  2. Thilo Dellwig, Po-Yen Lin and Fu-Jen Kao*, Long-distance Fluorescence Lifetime Imaging Using Stimulated Emission, J. Biomed. Opt. 17, 011009 (2012). http://dx.doi.org/10.1117/1.JBO.17.1.011009.
  3. 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, Vol. 20, Issue 10, pp. 11445-11450 (2012)http://dx.doi.org/10.1364/OE.20.011445 Also selected for the Virtual Journal for Biomedical Optics (VJBO), Editor Andrew Dunn and Anthony Durkin, Vol. 7, Iss. 7, June 25, 2012.
  4. Dellwig, Thilo, Matthew R. Foreman, and Fu-Jen Kao. "Coherent long-distance signal detection using stimulated emission: a feasibility study." Chinese Journal of Physics 48.6 (2010): 873-884.