| Nonlinear microscopy, in particular two-photon fluorescence microscopy, has become a mainstay in microscopic biological imaging. Given the significant investment that a two-photon microscope represents, we seek to maximize the utility of the instrument by developing techniques that enhance two-photon imaging performance or provide additional information beyond fluorescence with minimal changes required to the imaging system.;Four novel methods to improve two-photon imaging are presented here. Graded- Field Autoconfocal Microscopy provides phase gradient contrast images simultaneously to a two-photon image, providing structural context to fluorescence images. Thermionic-Detection Autoconfocal Microscopy is another implementation of Graded F-Autoconfocal Microscopy using an indirect detection method based on heat. Differential Aberration Imaging is a method for removing out-of-focus background fluorescence due to scattering by intentionally worsening the image by adding image-degrading aberrations, and subtracting the aberrated image from an unaltered one. In Active Illumination Microscopy, rather than directly using detector output to measure fluorescence in a sample, a feedback circuit holds the detector output of a sample to a constant, instead using the varying laser power needed to obtain that constant output to measure sample fluorescence. This results in improved sensitivity to dim signals and improved dynamic range characteristics.;Theoretical computations and imaging results for these techniques demonstrate their usefulness for biomicroscopy applications. |
