Compact oblique-incidence nonlinear widefield microscopy with paired-pixel balanced imaging

Nonlinear (vibrational) microscopy has emerged as a successful tool for the investigation of molecular systems as it combines label-free chemical characterization with spatial resolution on the sub-micron scale. In addition to the molecular recognition, the physics of the nonlinear interactions allows in principle to obtain structural information on the molecular level such as molecular orientations. Due to technical limitations such as the relatively complex imaging geometry with the required oblique sample irradiation and insufficient sensitivity of the instrument this detailed molecular information is typically not accessible using widefield imaging. Here, we present, what we believe to be, a new microscope design that addresses both challenges. We introduce a simplified imaging geometry that enables the measurement of distortion-free widefield images with free space oblique sample irradiation achieving high spatial resolution (∼1 µm). Furthermore, we present a method based on a paired-pixel balanced detection system for sensitivity improvement. With this technique, we demonstrate a substantial enhancement of the signal-to-noise ratio of up to a factor of 10. While both experimental concepts presented in this work are very general and can, in principle, be applied to various microscopy techniques, we demonstrate their performance for the specific case of heterodyned, sum frequency generation (SFG) microscopy.