“Everyone is skeptical when they first hear our story,” comments Marc Beal, director of corporate development and licensing at Biosearch. Nevertheless, he notes, “The synergy of Stellaris assays with StellarVision provides a paradigm shift in gene expression analysis. Current gene expression systems destroy cellular integrity then rescue messenger RNA with multiple amplification steps to provide an indirect ‘average’ of expression. In contrast, StellarVision directly interrogates single cells and single molecules, and quantifies mRNA, lncRNA and viral RNA while imaging hundreds of cells with statistical significance in a single large field of view.”

 

LightSpeed’s SAO technology is a fundamentally new optical detection paradigm that enables massively parallelized optical detection, Beal claims. The technique combines coherent interferometric illumination (similar to synthetic aperture radar) and computational image reconstruction (similar to computed tomography) to achieve ultra-high resolution that is far beyond the native resolution of the lens and the camera. Unlike conventional optical detection, resolution can be independently improved without affecting the field of view, working distance and depth of field, thereby providing a unique mechanism to build a high-speed scanner for ultra-high density imaging. The SAO-based StellarVision gene expression analysis instrument reportedly can analyze hundreds of cells simultaneously within a single field of view using a 20x air lens, but with resolution comparable to 100x oil immersion lens or higher.

 

The limitation of conventional optical imaging is caused by fundamental lens physics. That is, resolution of a lens system cannot be improved without dramatically sacrificing the field of view, depth of field and working distance, as these imaging performance measures are unfavorably linked through the numerical aperture (NA) of a lens. On the other hand, in LightSpeed’s SAO technology, the resolution is primarily determined by the resolution (or “spatial frequency content”) of a series of illumination patterns, and not by the resolution of a lens and a camera. As a consequence, resolution that is far beyond what is specified by the given lens and camera can be achieved by appropriate design and implementation of a set of SAO illuminations, tests conducted by Biosearch indicate.

 

In the StellarVision system, control of the amplitude and phase of the excitation light produces structured interference patterns that expand sub-pixel features, upon processing, to reveal individual molecules of RNA. Using the prototype instrument, hundreds of cells have been analyzed simultaneously within a single field of view using a 20x/0.45NA lens, but with resolution comparable to 100x/1.4NA oil immersion lens. SAO synergizes with single-molecule FISH to provide a promising new method for quantitative gene expression, intron chromosomal expression and single-nucleotide polymorphism detection using oligonucleotide-based probes, the collaborative partners claim.

 

LightSpeed Genomics was founded in 2006, focused on commercialization of its proprietary SAO technology. Five years after LSG opened its doors, a “Genentech guy” told Beal about the fledging company’s SAO technology which uses specially designed illumination and post-processing software to generate multiple virtual sub-pixels (up to thousands) for each physical pixel of a camera, opening a way to transform, for example, a camera with 1 million pixels into a “super-camera” with 1 billion effective pixels. Soon after, Beal sent samples down to LSG to test their capabilities. Now Biosearch is looking toward a second-generation instrument. “We’re developing a machine that can fit on every benchtop,” he says.