MULTI-VIEW THREE-DIMENSIONAL IMAGE MONTAGING & SIGNAL ATTENUATION CORRECTION FOR MAXIMIZING THE IMAGING DEPTH AND LATERAL EXTENT OF CONFOCAL MICROSCOPES

Confocal Microscopy is valuable for its ability to image thick sections of intact tissue. A compelling need is to image biological structures (e.g. neurons) that are much larger than the conventional three-dimensional (3-D) field of view. We describe a combination of specimen preparation, imaging, and image processing methods that extend not only the lateral dimensions, but also the axial depth. We’re also addressing the compelling need of improving the quantitative accuracy, and minimizing the possibility of missing important structures/phenomena due to non-uniform attenuation of the optical signal within the specimen. Our method is designed to work with existing instrumentation. The axial extent of the image is increased by mounting the specimen in a symmetric manner between a pair of coverslips, and collecting two images. The first is collected in the routine way and the second after flipping the sample upside-down. These two views are separated roughly, but not exactly, by 180o in the axial direction. The image structures in these views are registered precisely using fully automated and robust algorithms. Then, signal-processing algorithms based on modeling of the optical propagation of the epi-illumination and fluorescence signals through the specimen are used to compensate for the depth-dependent signal attenuation in the 3-D volume[2]. Previous methods for depth attenuation correction work by amplifying the signal from deeper parts of the specimen using an estimate of the signal extinction factor, and may need additional instrument setup [11]. In these methods, amplification of noise is an unavoidable artifact. Our approach has the advantage of exploiting data from multiple views to provably improve th