The Aurox Clarity laser free confocal unit (Clarity LFC) uses Aurox's patented optical system, based on a new design of spinning disc with a grid-like structured illumination pattern. This structured illumination pattern is used to both modulate the illumination field and demodulate the light emerging from the sample. The unique optical system inside the Clarity LFC allows capturing of the images both transmitted (T) and reflected (R) by the disc to easily differentiate between in-focus and outdof-focus information. Computer subtraction of these images (T-R) creates a sectioned image, i.e. an image where all out-of-focus blur is removed and only the in-focus, sharp image of the sample is retained. At the same time a conventional image is readily obtained by adding the two images (T+R).
Structured illumination has been used in a number of instruments, but until now it has not been combined with a spinning disc system. The Clarity LFC does this to produce an impressive combination of speed and image quality: it is worth mentioning that the optical sectioning properties of Clarity LFC is on a par with the more traditional (and significantly more expensive) point-scanning confocal microscope. Generating a structured illumination pattern by means of a spinning disc removes problems related to image processing artefacts; this is one of the many benefits of the Clarity LFC approach.
The optical paths within the Clarity LFC can be seen in the diagram. The excitation beam (shown in green) originates in the white-light widefield light source, such as a metal-halide lamp, passes through one of the fluorescence filter cubes and strikes the disc. This generates the SIP that is projected onto the specimen. The fluorescence light that is emitted within the sample and collected by the microscope objective lens (shown in red) is then projected along the same optical path back onto the disc. It is at this point that two distinct separate transmitted and reflected images are created. These continue along individual optical parts and are eventually captured by either two individual cameras or, projected side-by-side, by a single large-area sCMOS detector. An internal optical calibration system (shown in yellow) ensures that both images are perfectly registered during the processing.
The use of both transmitted and reflection images in the Clarity LFC image processing pipeline confers a number of advantages. It reduces the waste of fluorescence photons originating from the sample and effectively doubles the available sectioned (confocal) signal. This, in turn, could be used to either increase the image acquisition rate or reduce the sample exposure. The fact that both T and R images are captured simultaneously ensures the virtually complete absence of motion artefacts and, again, facilitates high-speed imaging. All-in-all the Clarity LFC could be said to combine the best qualities of spinning-disc (speed, ease of use) and structured-illumination (white-light illumination, low-cost) designs.