Recently we have demonstrated that spatial frequency modulation imaging can use extended excitation sources in linear and nonlinear image modalities, is compatible with single element detection, and results in enhanced lateral resolution across the excitation beam. In this paper, we will present new methods where the SPIFI platform goes from one-dimensional to two-dimensional imaging while still exhibiting the enhanced resolution across the added dimension. Significantly, we present the physical mechanism responsible for the resolution enhancement for all imaging modalities, we provide computational models that support the physical model for the increased resolution, and finally, present experimental verification of the resolution enhancement.
Through spatial frequency modulated imaging (SPIFI), multimodal, multiphoton microscopy (MPM) benefits from an extended excitation source without compromising the key performance characteristics afforded by point scanning MPM platforms. For example, the introduction of an in-house custom machined mask, which imparts a spatially distinct, temporal amplitude modulation to the extended excitation source, allows one and two-dimensional images to be captured with single element detection. This enables extended source imaging methods to retain a key feature of the point scanning systems; namely, the ability to image within scattering media, at depth.
Further, the range of contrast mechanisms for the extended source techniques presented here are not limited and readily extend to both linear and nonlinear imaging modalities. The SPIFI method developed here enables facile detection of such images with the added benefit of enhanced resolution. Notably, the resolution improvement holds across contrast mechanisms, and is independent of whether the contrast is generated through linear or nonlinear processes. Significantly, phase also comes into play as we present new SPIFI geometries that illustrate the role of phase in strategically controlling the source geometry and/or generating image contrast.
MultiPhoton SPatIal Frequency modulated Imaging (MP-SPIFI) has recently demonstrated the ability to simultaneously obtain super-resolved images in both coherent and incoherent scattering processes — namely, second harmonic generation and two-photon fluorescence, respectively.1 In our previous analysis, we considered image formation produced by the zero and first diffracted orders from the SPIFI modulator. However, the modulator is a binary amplitude mask, and therefore produces multiple diffracted orders. In this work, we extend our analysis to image formation in the presence of higher diffracted orders. We find that tuning the mask duty cycle offers a measure of control over the shape of super-resolved point spread functions in an MP-SPIFI microscope.