Whole-slide imaging systems can generate full-color image data of tissue slides efficiently, which are needed for digital pathology applications. This paper focuses on a scanner architecture that is based on a multi-line image sensor that is tilted with respect to the optical axis, such that every line of the sensor scans the tissue slide at a different focus level. This scanner platform is designed for imaging with continuous autofocus and inherent color registration at a throughput of the order of 400 MPx/s. Here, single-scan multi-focal whole-slide imaging, enabled by this platform, is explored. In particular, two computational imaging modalities based on multi-focal image data are studied. First, 3D imaging of thick absorption stained slides (∼60 μm) is demonstrated in combination with deconvolution to ameliorate the inherently weak contrast in thick-Tissue imaging. Second, quantitative phase tomography is demonstrated on unstained tissue slides and on fluorescently stained slides, revealing morphological features com-plementary to features made visible with conventional absorption or fluorescence stains. For both computational approaches simplified algorithms are proposed, targeted for straightforward parallel processing implementation at ∼GPx=s throughputs.

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This thesis explores a novel optical architecture for Whole Slide Imaging (WSI). This new architecture allows for multi-focal (3D) image acquisitions in a single scan pass. The multi-focal imaging capability is used to demonstrate 3D phase imaging and 3D imaging of thick tissue sections on a prototype scanner. Further, instrumentation for the extension of WSI to fluorescence imaging is developed: a technologically robust and cost effective method based on LEDs and a highly efficient method based on a multi-line laser illumination source. A WSI system is an optical instrument aimed at creating digital images of bio- logical samples mounted on a microscopy slide at a high throughput. WSI systems image tissues over large fields of view (∼ few cm), in 2D or in 3D (up to hundred layers of μm thickness), and at cellular resolution (∼1 μm). They are applied in high throughput screening in biology, and for novel computer aided medical diagnoses in the field of digital pathology. The optical architecture explored in this thesis is based on a tilted multi-line image sensor concept, originating from Philips. The goal of multi-line image ac- quisition is to enable closed-loop autofocus scanning, but it also allows for multi- focal image acquisitions. The core of this scanner concept lies in a novel design for a multi-line image sensor. The sensor is experimentally characterized for gain and noise, and a system model is developed to find the optimum signal-to-noise ratio (SNR) given the available photo-electron flux. This showed that images with a very high SNR of 292 can be acquired, provided that a sufficiently high photon flux can be realized. Two major issues with the sensor were found in our exper- imental characterization. At high line rates, the sensor showed missing symbols, leading to non-linearities in the read out. Further, the sensor showed high frequent fluctuations in the gain. 3D phase imaging and 3D imaging of thick specimens are two novel contrast modalities based on computational imaging techniques and are enabled by the availability of multi-focal images. For both techniques simplified algorithms were developed compatible with parallel processing at very high speeds. For 3D imaging of thick specimens a deconvolution technique is developed for improving the in- herently low axial contrast. 3D phase imaging is realized by a simplified algorithm for Quantitative Phase Tomography (QPT). QPT imaging is found to be able to im- age the sites labeled for Fluorescence in situ Hybridization (FISH) imaging, and provide additional structural information on unlabeled tissues or tissues stained for immunofluorescence. A system design study is presented showing that the in- plane transfer function has the character of a band-pass spatial frequency filter. The major opportunity for WSI systems to become compatible with fluores- cence imaging is addressed by the development of two imaging modalities. First, a widefield fluorescence WSI system with an LED illumination source is developed and built. A color sequential illumination strategy in combination with multi-band dichroics is used for multi-color imaging using a single monochromatic sensor. The main speed limitation is formed by the exposure time required to capture enough photo-electrons for a decent SNR. Based on the experimental results, a system with 96 Time Delayed Integration (TDI) lines is estimated to achieve a rea- sonable throughput of about 130 kPixel/s. This makes scanning possible of an area of 15 × 15 mm2 in three colors in about 23 min. Second, a novel optical architecture for multi-focal fluorescence image acqui- sitions based on a laser illumination source is proposed and realized in a proto- type. Illumination PSF engineering using diffractive optics is applied to generate a set of parallel scan lines in object space, that span a plane conjugate to a tilted im- age sensor. An important new element in the design is the use of higher order astig- matism to improve the uniformity of peak intensity and line width along the scan lines. Focusing the illumination on the sample provides a very high illumination efficiency and a confocal suppression of background. This optical architecture is projected to ultimately achieve a throughput of several hundreds MPixel/s, which would enable scanning an area of 15 × 15 mm2 in 8 layers in less than a minute. This thesis is concluded with an outlook to opportunities for future research in WSI systems. The challenges and some potential solutions for using a general purpose scientific CMOS (sCMOS) camera for multi-line scanning of a tilted ob- ject plane, and some opportunities for extension of WSI techniques to Light Sheet Microscopy (LSM) and Structured Illumination Microscopy (SIM) are discussed. In summary, this thesis investigates the imaging qualities and extension to computational imaging modalities of a brightfield WSI system and describes two approaches for fluorescence WSI. @en

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@en

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.@en

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.

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