Interferometric imaging typically measures optical path length changes to provide label-free contrast for structural and functional imaging. Since molecular specific imaging remains the bedrock of modern biological research and medical diagnostics, LBRC also aims to extend the scope of interferometric measurements to additionally providing molecule-specific information of biological cells. Spectroscopic interferometric imaging frameworks based on both linear absorption, transient absorption, and photothermal signal will be developed. Successful development of these technologies will have considerable impact on studies aimed at quantifying absolute oxy- and deoxy-hemoglobin concentrations in sickle RBCs during hypoxia and high throughput detection of eumelanin vs pheomelanin distribution in skin cells. Figure 1: Schematic for a structured illumination PP-DPM microscopy.
Pump-Probe digital phase microscopy for wide-field PT, TA, and SRS imaging
Photothermal (PT), transient absorption (TA), and stimulated Raman scattering (SRS) microscopy all relies on pump-probe spectroscopic detection - a spatial modulation of temperature, ground-state depletion, and induced bond vibration for the PT, TA, and SRS cases, respectively [1,2]. While pump-probe microscopy is typically implemented on focused beam geometry, wide-field implementation can significantly improve the throughput. Structured illumination microscopy (SIM) has been used for enhanced resolution and optical sectioning capability mostly for incoherent imaging [3], and in some cases for coherent imaging [4].
However, SIM has not been studied for wide-field coherent pump-probe schemes. We have established the theory and performed simulations to implement three-dimensional (3D) wide-field super-resolution pump-probe digital phase microscopy (PP-DPM) with varying transient grating period inside the specimen (Fig. 1) [5]. It is important to note that quantitative phase and amplitude imaging of the electric field is required for the reconstruction in these coherent image conditions. The structured illumination pump beam serves as a key element that encodes the missing information in both lateral and axial dimensions into the conventional imaging passband.
As seen in Fig. 2, structured illumination enables a lateral resolution improvement by a factor of three and provides 0.5 μm axial optical sectioning. Previous work on coherent SIM has been based on intensity measurements for 2D imaging, and this framework may be applied to 3D pump-probe SIM reconstruction [4]. However, our optical field based pump-probe SIM framework requires much simpler pump beam configuration (30~50 times fewer pump beams) since we can shine the sample with a single grating period and integrate the results with various grating periods in optical field. In contrast, the intensity-based methods must shine the sample with all grating periods at the same time. Moreover, our field-based framework needs 3~5 times less number of images to reconstruct a single 3D image with the same synthetic optical transfer function than the intensity SIM framework which generates more unknown components from the cross terms in intensity. This difference comes from the fact that object information is linearly related to the optical field while its relation with the light intensity is nonlinear for the coherent imaging.