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

Leon Van Der Graaff,Geert J L H Van Leenders,Sjoerd Stallinga,Fanny Boyaval

doi:10.1364/boe.10.006313

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 multi-focal 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 mm2 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.

Highlights

In the current era of big data analysis, instrumentation to generate massive amounts of image data is in high demand
In this paper we show how to extend the use of diffractive optical elements to first add defocus such that the set of spots scan the sample at equidistant focal layers, and to second modify the spots to lines orthogonal to the plane spanned by the optical (z) axis and scan (y) axis
The sample is scanned with respect to a mechanically fixed optical setup, thereby providing simple means for an unlimited field of view

Summary

Introduction

In the current era of big data analysis, instrumentation to generate massive amounts of image data is in high demand. For high throughput screening in biology, or for novel computer aided medical diagnoses in the field of digital pathology, there is a need for fluorescence imaging of tissues over large fields of view (∼few cm), in 3D (up to hundred layers of μm thickness), and at cellular resolution (∼1 μm) [1,2,3]. The necessary beam splitters used in these approaches result in a loss in collected fluorescence signal strength by a factor equal to the number of scanned layers Another throughput enhancing technique is the use of a line illumination instead of a spot illumination in combination with a line sensor [12,13,14]. Line scanning can be combined with multi-focus scanning [16], but the proposed method suffers from the same signal losses induced by splitting the beam in the detection path as the point scanning based multi-focus systems

Methods

Results

Conclusion