High Resolution Wide Field Stimulated Raman Scattering Microscopy

Abstract

Fluorescent imaging modalities, such as STED, PALM and STORM, has demonstrated the feasibility of super-resolution imaging. However, no comparable super-resolution imaging has been achieved based on non-fluorescent contrast mechanisms. We present a novel super-resolution approach based on incorporating stimulated Raman scattering (SRS) contrast into a standing-wave (SW) total internal reflection microscope. SW-SRS microscopy has the potential to improve the lateral resolution of current SRS microscopy in total internal reflection geometry. There is a critical difficulty to implement SW-SRS microscopy. Stimulated Raman gain, SRG, is a weak modulation of the intensity of the Stokes beam. The ratio of the SRG to Stokes beam intensity is a function of pump beam instantaneous intensity. The need for wide field imaging further reduces pump beam flux resulting in very unfavorable SRG to Stokes beam intensity ratio. As an example, using a standard Ti-Sapphire laser exciting a 100 × 100 micron square region, SRG to Strokes beam intensity can be as low as 10ˆ-9. This low signal to noise ratio is particularly challenging for wide field imaging that requires area detectors, such as CCD cameras, with limited dynamic range. To overcome these difficulties, we show that SRG to Stokes beam ratio can be improved to 10ˆ-4 by utilizing mJ pulses using a regenerative amplifier, optimization of pulse durations and bandwidths, and destructive interference of the Strokes beam background. We gratefully acknowledge funding from the Singapore-MIT Alliance (SMA-2), the Singapore-MIT Alliance for Research and Technology (SMART), and the Samsung Scholarship.