Title: “Label-free chemical nanoscopy by stimulated Raman image scanning microscopy”
Ji-Xin Cheng, PhD – BME/ECE (Advisor, Chair)
Jerome Mertz, PhD – BME
Irving Bigio, PhD – BME
Lei Tian, PhD – ECE
Optical super-resolution microscopy, which can surpass the 180-nm diffraction limit of conventional microscopy, is of great interest in biology since the scale of many cell structures and underlying intracellular processes are below 100 nm. However, current super-resolution techniques mainly rely on fluorescent tags, which do not apply to specific small molecules since labeling may significantly perturb their properties. Stimulated Raman scattering (SRS), which detects signals arising from the inherent molecular vibrations, is an appealing label-free approach to measure specific molecules and to observe cellular states. Up to date, using visible laser sources, SRS has realized a diffraction-limited spatial resolution of 130 nm. However, reaching a spatial resolution below 100 nm for SRS is challenging, as most mechanisms for super-resolution fluorescent imaging, such as fluorescent blinking or ground-state depletion, are not applicable to SRS. Herein, we propose a novel design, which incorporates the idea of image scanning microscopy (ISM) to a visible SRS imaging scheme. We will use a lab-built 32-channel tuned amplifier (TAMP) array, which is tailored for parallel detection of the SRS signal, to replace the original detection with a single-point detector and a lock-in amplifier. Consequently, at each laser-scanned position, we collect a sub-image in which each pixel records the illumination region with a slight spatial shift. By fusing all the sub-images, a high signal level image with a doubled spatial resolution is reconstruction. Moreover, the method does not require high laser power on the sample, which is ideal for live-cell imaging. On completion, a spatial resolution of 65 nm is expected, which will lead to novel label-free biological applications, including real-timing of lipid rafts on the cell membrane domain and virus infection.