At the point of interrogation, the entire width of the detection channel is illuminated by a line of laser light, oriented perpendicularly to the direction of flow. The interrogation area is a defined region of the channel cross-section, determined by the channel width and the optical characteristics of the objective. The excitation beam pathways consists of lasers, beam shaping optics, and a high performance objective. As the labeled RCP traverse the illuminated line, each object will emit a short pulse of light. By use of image analysis in two both fluorescence channels, RCPs are discriminated from noise and non-specific objects. The RCP identified in each image are then counted, and results are presented as the number of RCP found in the specific volume passed through the interrogation area for each analysis
Optimized for quantification of Rolling Circle Products
The Aquila 400 design is optimized for quantification of Rolling Circle Products (RCP), i.e. concatemers of short single-stranded DNA sequences, by the use of fluorescently labeled detection oligonucleotides specific to a region of the original sequence. An aqueous solution of labeled RCPs is injected using a high-precision syringe pump into a microfluidic detection channel.
The instrument is supplied with a dedicated computer system. This system comes pre-installed with the software required to control the instrument and manage sample data.
The instrument is controlled through a GUI where procedures such as startup, run sample, shutdown, etc. are accessed through menu commands or toolbar buttons. After analysis of a sample, or set of samples, the object counts from each detection channel are presented in both table and graph form. Results from previously run samples can be retrieved and studied later if desired. Results (object counts) from multiple samples can be exported to a file format readable by standard spreadsheet software such as Excel.
Configuration of frequently used instrument parameters such as sample volume, flow rates, image analysis threshold levels, are defined in a settings file, accessed through the GUI, while more infrequently used functions are set by editing a set of configuration text files. Optionally, functionality could be included to change these parameters through the user interface and allow them to be changed between different samples in an experiment.