High-throughput microscopy must re-invent the microscope rather than speed up its functions

Abstract

Knowledge gained from the revolutions in genomics and proteomics has helped to identify many of the key molecules involved in cellular signalling. Researchers, both in academia and in the pharmaceutical industry, now screen, at a sub-cellular level, where and when these proteins interact. Fluorescence imaging and molecular labelling combine to provide a powerful tool for real-time functional biochemistry with molecular resolution. However, they traditionally have been work-intensive, required trained personnel, and suffered from low through-put due to sample preparation, loading and handling. The need for speeding up microscopy is apparent from the tremendous complexity of cellular signalling pathways, the inherent biological variability, as well as the possibility that the same molecule plays different roles in different sub-cellular compartments. Research institutes and companies have teamed up to develop imaging cytometers of ever-increasing complexity. However, to truly go high-speed, sub-cellular imaging must free itself from the rigid framework of current microscopes.

Figure 1

Early high-throughput microscopy. A scientific conversazione with microscopes provided to view microscopic objects. Such events attracted a large public. Engraving from Illustrated London News 28 April 1855. With permission, © The Shrewsbury Museum.

Figure 2

Novel geometries for high-content, high-throughput microscopy. (a) Optofluidic microscopy (OFM). The major component of OFM is a metal-coated CMOS sensor array on which a linear array of subwavelength nanoapertures is patterned. This device is hermetically sealed on one side of a microfluidic delivery channel. The nanoaperture array is laid down in a slanted manner under microfluidic channel so that the illumination spot is scanned across the cell as the cell moves along with the fluid. Spatial resolution is defined by nanoaperture size and alleviates the constraints by the pixel width and pitch size of the underlying linear array charge-coupled device. OT, optical trap; NA, nanoaperture. (b) The cell rotator uses a DFC to trap and spatially position individual, non-adherent living cells suspended in low-conductance media. Periodic phase and intensity differences between the microelectrodes arranged in space to form a 3-D octode cage permit the generation of pN forces that rotate the cell around the focal plane so as to acquire a tomographic image. Combined with confocal detection, the method permits tomography-like multi-viewpoint 3-D imaging.