High throughput spinning-disk imaging bioassay : from single-cell imaging to whole slide histopathology

Abstract

Accessing detailed spatial information of cells, microscopy allows high-content cell-based phenotypic assay, but at a compromised measurement throughput. Notable example is imaging flow cytometry in which streamlines cell interrogation by imaging single cells in suspension at a throughput of 1,000’s cells/sec is achieved [3-5]. However, this throughput is still at least two-orders-of-magnitude slower than classical non-imaging flow cytometry because of the inherent speed-versus-sensitivity limitation of the camera technologies. This pervasive problem hinders the acceptance of imaging cell-based assay in high-throughput screening (HTS) applications, e.g. phenotypic screening in the early drug discovery pipeline in which tens of thousands compounds per screen are involved; and circulating tumor cell (CTC) screening in which rare cell detection within enormous and heterogeneous population (some millions of blood cells) is mandated. Optical time-stretch imaging has been demonstrated to overcome the speed limitation in conventional imaging by adopting an all-optical ultrafast image encoding concept. The ultrafast continuous line-scan imaging (at a rate of tens of MHz) naturally favors imaging flow cytometry applications at a throughput which is impossible elsewhere, e.g. detecting rare cancer cells at 100,000 cells/sec. Despite this uniquely fast imaging capability, two key challenges remain in the context of cell-based assay. First, time-stretch imaging predominantly generates intrinsic image contrasts from cells based on bright-field or phase-contrast imaging. Hence, the retrieved single-cell information mostly lacks chemical specificity. Second, assay of adherent cells, or fixed cells on planar substrates has been missing in the prior demonstrations of time-stretch imaging, which overwhelmingly focused on the suspension-cell assay format. Yet, planar platform is the prevalent format in cell-based assay, especially in the context of high-content imaging. Cell-culture assay on the planar platform emulates the physiologically-relevant environments better than isolated cells in suspension, and thus are particularly effective in monitoring cell kinetics. In addition, planar platform allows space-multiplexed chemically specific cell-capture assays, e.g. array-based immunoassay. These assay types have yet been explored in time-stretch imaging. To address these challenges, this thesis focuses on the development of a spinning time-stretch imaging assay platform based on the functionalized digital versatile disc (DVD) [1]. We demonstrate that adherent cell culture, biochemically-specific cell-capture, tissue-sections can all be assayed with time-stretch microscopy, thanks to the high-speed DVD spinning motion that naturally enables on-the-fly cellular imaging at an ultrafast line-scan rate of >10MHz. As scanning the whole DVD at such a high speed enables ultra-large field-of-view imaging, it could be favorable for scaling both the assay throughput and content as demanded in many applications, e.g. drug discovery, and rare cancer cell screening.