In vivo one-photon confocal calcium imaging of neuronal activity from the mouse neocortex.

In vivo calcium imaging is a powerful tool used to record neuronal activity from living animals. For this purpose, two-photon excitation laser-scanning microscopy is commonly used because of the optical accessibility of deep tissues. In this study, we report that one-photon confocal scanning laser microscopy, when optimally tuned, is also applicable for in vivo calcium imaging from the superficial layer of the neocortex. By combining a Nipkow-disk confocal unit with a fluorescence stereo zoom microscope and a high numerical aperture objective, we succeeded in recording the fluorescence signal of individual cells at a depth of up to 160 µm in brain tissues, which corresponds to layer II of the mouse neocortex. In fact, we conducted in vivo functional multineuron calcium imaging and simultaneously recorded spontaneous activity from more than 100 neocortical layer II neurons. This one-photon confocal system provides a simple, low-cost experimental platform for time-lapse imaging from living animals.

CINCH (confocal incoherent correlation holography) super resolution fluorescence microscopy based upon FINCH (Fresnel incoherent correlation holography).

FINCH holographic fluorescence microscopy creates high resolution super-resolved images with enhanced depth of focus. The simple addition of a real-time Nipkow disk confocal image scanner in a conjugate plane of this incoherent holographic system is shown to reduce the depth of focus, and the combination of both techniques provides a simple way to enhance the axial resolution of FINCH in a combined method called "CINCH". An important feature of the combined system allows for the simultaneous real-time image capture of widefield and holographic images or confocal and confocal holographic images for ready comparison of each method on the exact same field of view. Additional GPU based complex deconvolution processing of the images further enhances resolution.

Design and Performance of a Multi-Point Scan Confocal Microendoscope.

Confocal fluorescence microendoscopy provides high-resolution cellular-level imaging via a minimally invasive procedure, but requires fast scanning to achieve real-time imaging in vivo. Ideal confocal imaging performance is obtained with a point scanning system, but the scan rates required for in vivo biomedical imaging can be difficult to achieve. By scanning a line of illumination in one direction in conjunction with a stationary confocal slit aperture, very high image acquisition speeds can be achieved, but at the cost of a reduction in image quality. Here, the design, implementation, and experimental verification of a custom multi-point aperture modification to a line-scanning multi-spectral confocal microendoscope is presented. This new design improves the axial resolution of a line-scan system while maintaining high imaging rates. In addition, compared to the line-scanning configuration, previously reported simulations predicted that the multi-point aperture geometry greatly reduces the effects of tissue scatter on image quality. Experimental results confirming this prediction are presented.