Multi-line fluorescence scanning microscope for multi-focal imaging with unlimited field of view

Abstract

Confocal scanning microscopy is the de facto standard modality for fluorescence imaging. Point scanning, however, leads to a limited throughput and makes the technique unsuitable for fast multi-focal scanning over large areas. We propose an architecture for multifocal fluorescence imaging that is scalable to large area imaging. The design is based on the concept of line scanning with continuous 'push broom' scanning. Instead of a line sensor, we use an area sensor that is tilted with respect to the optical axis to acquire image data from multiple depths inside the sample simultaneously. A multi-line illumination where the lines span a plane conjugate to the tilted sensor is created by means of a diffractive optics design, implemented on a spatial light modulator. In particular, we describe a design that uses higher order astigmatism to generate focal lines of substantially constant peak intensity along the lines. 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, and provides intrinsic alignment of the simultaneously scanned focal slices. As proof of concept, we have scanned 9 focal slices simultaneously over an area of 36 mm 2 at 0.29 µm pixel size in object space. The projected ultimate throughput that can be realized with the proposed architecture is in excess of 100 Mpixel/s.

Computational imaging modalities for multi-focal whole-slide imaging systems

“…8(i)-8(k) provide a maximum intensity projection of a multi-focal fluorescence image of the same section, for the same areas. This fluorescence image was obtained in previous research [26] using a multi-focal multi-line confocal scanner. A rigid transform is used to register the fluorescence reference image to the phase image, where the optimal transform is found by minimizing the root mean square distance between a series of manually selected landmarks.…”

“…The sensor contains separate "sensorlets," groups of adjacent pixel rows, which can be read-out independently. The sensor is tilted with respect to the optical axis resulting in a tilted object plane [26]. The readout of pixel information is done via two separate, simultaneously obtained, data streams, one obtained from a single sensorlet for providing the primary imaging information, the other obtained from multiple sensorlets for providing autofocus information.…”

A forward reconstruction, holographic method to overcome the lens effect during 3D detection of semi-transparent, non-spherical particles

“…This process typically requires minutes to complete, which results in a limited temporal resolution for fast-changing systems with a shorter time scale of variation. 18–20…”

“…This process typically requires minutes to complete, which results in a limited temporal resolution for fast-changing systems with a shorter time scale of variation. [18][19][20] One possible way to resolve this problem is to adopt imaging methods based on holographic imaging techniques. Digital holographic microscopy (DHM) utilizes a coherent light source (laser) to illuminate the target object.…”

High-speed mosaic imaging using scanner-synchronized stage position sampling

“…Although the high photon throughput of SiPM detectors enables high imaging frame rates, this does not directly translate into proportionally faster mosaic imaging of large specimens because at high imaging rates an increasingly large fraction of the total imaging time is spent translating the specimen between positions. To address this, previous work in light sheet, 18 confocal, 19 , 20 line-scan confocal, 21 fluorescence microscopy, 22 and HiLo microscopy 23 has utilized so-called “strip mode” or “push broom” scanning wherein the specimen is translated at a constant velocity through a fixed line imaging position. By setting the translation speed proportionally to the line imaging time, lines of adjacent pixels along the translation axis can be assembled into seamless frames.…”