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.
Spatial Frequency Modulated Imaging (SPIFI) with single element detection has previously been demonstrated with a time varying amplitude spatial frequency. This has been shown in a variety of modalities (linear, TPEF, SHG) and also with variations on the base design to provide additional dimensions of information. SPIFI is also capable of providing enhanced resolution images. However, the signal-to-noise is a limiting factor in the quality of the resolution enhancement. We present a microscope design which uses a nematic spatial light modulator to provide a time varying amplitude from an amplitude or phase grating. Twophoton excitation fluorescence images of 10-µm fluorescent polystyrene beads are presented using a phase grating. Additionally, the microscope can provide spatial gratings in polarization which provide an alternative means of imaging in third harmonic generation (THG). THG images are provided using an amplitude and polarization-grating modulation pattern.