Fluorescence imaging for whole slide scanning using LED-based color sequential illumination

Abstract

In the field of pathology there is an ongoing transition to the use of Whole Slide Imaging (WSI) systems which scan tissue slides at intermediate resolution (0∼.25 μm) and high throughput (15mm²=min) to digital image files. Most scanners currently on the market are line-sensor based push-broom scanners for three-color (RGB) brightfield imaging. Adding the ability of fluorescence imaging opens up a wide range of possibilities to the field, in particular the use of specific molecular (proteins, genes) imaging techniques. We propose an extension to fluorescence imaging for a highly efficient WSI systems based on a line scanning technique using multi-color led epi-illumination. The use of multi-band dichroics eliminates the need for filter wheels or any other moving parts in the system, the use of color sequential illumination with leds enables imaging of multiple color channels with a single sensor. Our approach offers a solution to fluorescence WSI systems that is technologically robust and cost-effective. We present design details of a four-color led based epi-illumination with a quad-band dichroic filter optimized for leds. We provide a thorough analysis regarding the obtained optical and spectral efficiency. The primary throughput limitation is the minimum Signal-to-Noise-Ratio (SNR) given the available optical power in the illumination etendue, and indicates that a throughput on the order of 1000 lines/sec can be obtained.