Confocal Multi-line Scanning Microscope for Efficient 3D Fluorescence Imaging

Abstract

Confocal fluorescent imaging is the de facto standard modality for fluorescence imaging. However, the point-to-point scanning technique leads to a very limited throughput and makes the technique unsuitable for large area and fast multi-focal scanning. We propose an architecture for highly efficient 3D line confocal fluorescence imaging. Our design extends the concept of a line scanning system with continuous ‘push broom’ scanning. Instead of using a line sensor, we use an area sensor that is tilted with respect to the optical axis to acquire image data of multiple depths simultaneously. A multi-line illumination with lines illuminating the specimen at different depths, conjugate to the tilted area sensor, is created by means of a diffractive optical element (DOE). The proposed method is suitable for fast 3D image acquisition with unlimited field of view, it requires no moving components except for the sample scanning stage, has intrinsically low losses, and provides intrinsic alignment of the simultaneously scanned layers of the focal stack.