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

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 (∼60µm) 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 complementary 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.

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

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.