High sensitivity stimulated Raman scattering microscopy with electronic resonance enhancement.

Raman microscopy is an important tool for labelfree microscopy. However, spontaneous Raman microscopy suffers from slow image acquisition rates and susceptibility to fluorescence background. Coherent Raman microsocopy techniques such as coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy, by contrast, offer fast imaging capability and robustness against sample fluorescence. Yet, their rather low sensitivity impedes their broader application. This review discusses sensitivity enhancement of SRS microscopy to µM detection levels by using electronically pre-resonant excitation. We present the foundations of this approach, discuss its technological implementation, and show first successful applications. A special emphasis is given to outlining new experimental developments allowing novel types of investigations.

Double modulation SRS and SREF microscopy: signal contributions under pre-resonance conditions.

Pre-electronic resonance enhancement can increase the sensitivity of non-linear Raman microscopy to the single molecule detection limit. A major problem, however, is the generation of background signal due to unwanted linear and non-linear photophysical processes. In this work, we report the setup of a novel detection scheme for stimulated Raman scattering microspectroscopy based on the simultaneous modulation of pump and Stokes beam. Apart from allowing the parallel detection of stimulated Raman loss and gain (SRL and SRG), the setup gives access to the quantitative analysis of different sources of background signal. We report spectrally and temporally resolved measurements on three exemplary rhodamine dyes and derive the contributions of two-photon absorption and stimulated emission to their SRL, SRG, and stimulated Raman excited fluorescence signals. These results give guidelines for the further improvement of the sensitivity of non-linear Raman micospectroscopy under electronic pre-resonance conditions.

Electronically Preresonant Stimulated Raman Scattering Microscopy of Weakly Fluorescing Chromophores.

Stimulated Raman Scattering microscopy is an important imaging technique. Its broader application, however, is hampered by its comparatively low sensitivity. Using organic fluorophores, it has recently been demonstrated that, similar to spontaneous Raman microscopy, the sensitivity of stimulated Raman microscopy is increased by orders of magnitudes if electronic preresonances are exploited. In this Article, we show that this approach also works with low quantum yield chromophores. We investigate the relevant photophysics and discuss the background arising from preresonant excitation conditions. Applications of preresonant stimulated Raman scattering microscopy for the imaging of weakly fluorescing labels in fixed and live cells are demonstrated.