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I want to know about the imaging process in helicos platform.
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Helicos imaging
Here is the short version of Helicos imaging from Current Protocols in Molecular Biology, supplement 92, 7.10.10. This was published in 2010 and, alas, nothing has changed since then.
During a standard run, two 25-channel flow cells are used, with each flow cell alternating between the chemistry cycle (cleavage of dye/terminator from previous cycle, rinsing, incorporation of next base, rinsing) and the imaging cycle. During the imaging process, four lasers illuminate 1100 Fields of View (FOV) per channel with pictures taken by four CCD cameras via a confocal microscope (see Fig. 7.10.2). Though single molecules are visualized, multiple photon emissions are registered for each molecule, with the time spent at each FOV dependent on the brightness of the dye in the particular nucleotide as well as camera speed and detection efficiency. At the present time, the imaging process is the rate-determining step, and run time could be reduced at the expense of throughput by reducing the number of FOV per channel.
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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