Practical Implementation of Log-Scale Active Illumination Microscopy

Active illumination microscopy (AIM) is a method of redistributing dynamic range in a scanning microscope using real-time feedback to control illumination power on a sub-pixel time scale. We describe and demonstrate a fully integrated instrument that performs both feedback and image reconstruction. The image is reconstructed on a logarithmic scale to accommodate the dynamic range benefits of AIM in a single output channel. A theoretical and computational analysis of the influence of noise on active illumination feedback is presented, along with imaging examples illustrating the benefits of AIM. While AIM is applicable to any type of scanning microscope, we apply it here specifically to two-photon microscopy.National Institutes of Healt

New implementation of second harmonic generation microscopy for three-dimensional resolution

We present a fast scanning transmission-mode confocal scanning laser microscope system based on the use of a second harmonic generation (SHG) crystal for signal detection. The quadratic intensity dependence of SHG is exploited to preferentially reveal unscattered signal light and reject out-of-focus scattered background. The SHG crystal plays the role of a virtual pinhole that remains self-aligned without a need for de-scanning. We demonstrate that this new microscope method produces images with higher contrast and less speckle than transmission scanning microscopy with linear detection

Axial range of conjugate adaptive optics in two-photon microscopy

We describe an adaptive optics technique for two-photon microscopy in which the deformable mirror used for aberration compensation is positioned in a plane conjugate to the plane of the aberration. We demonstrate in a proof-of-principle experiment that this technique yields a large field of view advantage in comparison to standard pupil-conjugate adaptive optics. Further, we show that the extended field of view in conjugate AO is maintained over a relatively large axial translation of the deformable mirror with respect to the conjugate plane. We conclude with a discussion of limitations and prospects for the conjugate AO technique in two-photon biological microscopy

Video-rate volumetric neuronal imaging using 3D targeted illumination

Fast volumetric microscopy is required to monitor large-scale neural ensembles with high spatio-temporal resolution. Widefield fluorescence microscopy can image large 2D fields of view at high resolution and speed while remaining simple and costeffective. A focal sweep add-on can further extend the capacity of widefield microscopy by enabling extended-depth-of-field (EDOF) imaging, but suffers from an inability to reject out-of-focus fluorescence background. Here, by using a digital micromirror device to target only in-focus sample features, we perform EDOF imaging with greatly enhanced contrast and signal-to-noise ratio, while reducing the light dosage delivered to the sample. Image quality is further improved by the application of a robust deconvolution algorithm. We demonstrate the advantages of our technique for in vivo calcium imaging in the mouse brain.This work was funded by the National Institutes of Health (R21EY026310) and the National Science Foundation (CBET-1508988). The authors wish to thank E. McCarthy and Prof. M.J. Baum for providing mouse brain slices used in this manuscript, and A. I. Mohammed for providing in vivo mouse brain samples in the early stages of this work. (R21EY026310 - National Institutes of Health; CBET-1508988 - National Science Foundation)Published versio