Researchers Test Capabilities Of Mobile Phone Microscopy For In Situ Mutation Analysis
By Benjamin Ross
January 30, 2017 | The distance between next-generation DNA sequencing and the everyday patient might be closing significantly. A recent study published in Nature Communications describes how researchers from the Science for Life Laboratory in Sweden, led by Malte Kühnemund, along with engineers from the University of California, Los Angeles, have developed molecular assays and portable optical imaging designs that permit on-site diagnostics with a cost-effective mobile phone-based multimodal microscope. The authors of the study have called this development “a new milestone for telemedicine technologies.”
According to the authors of the Nature Communications-published study (doi:10.1038/ncomms13913), “We show that a cost-effective and compact multimodal microscope integrated on a mobile phone can be used for (i) targeted DNA sequencing and (ii) in situ point mutation analysis that allow integrating molecular analysis with tumor tissue morphology.”
The authors preface their research by stating that molecular diagnostics at the point of care (POC) is by and large an unmet need in in places where resources are limited. Efficient management of a wide range of disease conditions is severely limited by the lack of molecular information. To address this problem the researchers designed a light-weight optomechanical attachment that is integrated with the existing camera of a mobile phone, in this case a Nokia Lumia 1020. “Mobile phones may further accelerate this emerging trend of telemedicine and enable on-site implementation of digital pathology in resource-limited settings by supplying a cost-effective technology infrastructure for imaging and diagnostic analysis,” Kühnemund and his colleagues wrote. “With their rapidly expanding imaging and sensing capabilities, computational power, and connectivity, mobile phones help translating biomedical measurements from lab environments to the POC and field settings.”
Kühnemund and company began production on an optomechanical attachment that would show individual rolling circle amplification (RCA)-amplified single molecules, generated on glass slides and inside preserved cells and tissues. This attachment, which looks reminiscent to a flip phone from the 1990s, integrated with the existing camera module of a mobile phone, and the external lens module used a half-pitch resolution of 0.98 μm and an imaging field of view of ∼0.8 mm2. During the design of this module they established that individual RCA products (RCPs) could be discriminated and precisely quantified by their mobile-phone-based microscope over a 4-log dynamic range (1 fM–10 pM), demonstrating its utility to image and analyze individual RCA-amplified single molecules.
After designing the mobile phone-based microscope, Kühnemund and his colleagues determined that the next step was to find out if it could in fact read NGS reactions. To do this, they imaged and quantified sequencing by ligation (SBL) reactions of RCPs generated on standard microscopy slides. The results showed that nine mutant RCPs with base A-specific stain were detected using the mobile phone microscope among 1,552 wild-type RCPs with G-specific sequencing signal. Researchers were optimistic that a high mutation detection sensitivity of greater than 1% can potentially be achieved at such high sequencing depths.
The next test was to validate the targeted sample preparation and sequencing scheme on genomic DNA extracted from cell lines. The results showed that the measurements gathered from the mobile phone attachment were consistent with those gathered from regular benchtop fluorescence microscopy, combined with a custom-developed sequencing analysis pipeline.
In short, the mobile attachment the researchers constructed passed every test thrown its way, including the important function of imaging and quantifying in situ generated RCPs, and detecting KRAS mutations directly in tumor tissue sections. The mobile phone microscope-based in situ genotyping resulted in 100% concordance to clinical NGS analysis and whole tissue scanning with an automated benchtop fluorescence microscope. “These results provide evidence that our mobile phone multimodal microscopy platform can be used to analyze RCA-based in situ genotyping assays and accurately genotype cancer patient biopsies directly in situ,” the researchers wrote, “which may facilitate integrating molecular diagnostics with tumor morphology directly in pathologists’ offices and POC.”
Kühnemund et al. concluded by hoping that their analysis could lead to applications in many other fields, such as infectious disease diagnostics. “With a simple DNA sequencing library preparation scheme and the capability to image NGS reactions, mobile phone-enabled imaging and sensing tools may soon be used for targeted DNA sequencing in clinical settings and POC offices, with the potential to dramatically decrease the cost of NGS-based diagnostics globally,” stressed the writers. As long as technology keeps advancing, especially in the field of telemedicine, there’s no reason not to think that this idea may in fact become a reality.
