Explanation | Confocal.nl

The working principle of re-scan confocal microscope

Re-scan Confocal Microscopy (RCM) is a new super-resolution technique based on standard confocal microscopy extended with an optical (re-scanning) unit that projects the image directly on a CCD-camera. This new microscope has improved lateral resolution (170 nm at 488 nm light), and strongly improved sensitivity, while maintaining the sectioning capability of a standard confocal microscope. This technology is useful for biological applications, where the combination of high-resolution and high-sensitivity is required (but not very high imaging speed).

The excitation lasers (blue and yellow lines) are directed via a dichroic mirror towards the first scanning unit SM1. As in a standard confocal microscope, the scanning unit scans the laser light in the sample and de-scans the emission light, directing it at the pinhole PH (green and red lines). After the pinhole, a second re-scan unit SM2 directs the light onto a camera chip.

During scanning, re-scan mirrors (SM2) move faster than the first scan mirrors (SM1), which magnifies the image on the camera chip compared to the sample, and eventually results in the higher resolution of the image. The resolution of the system can be improved with the re-scan step by a factor of √2 (i.e. 1.41 times), compared to Abbe’s resolution limit by changing the angular amplitude of the re-scanner (SM2). Reduction of pinhole is no longer necessary to increase resolution – in fact, closing down the pinhole will only limit the amount of light passing through, and decrease the signal to noise ratio due weaker signal. Since the re-scan is purely optical method, and no further image processing is required, there is also no time cost of improving the resolution.

Because of the sensitive camera as a detector, instead of the photo-multiplier tube as in most confocal microscopes (except the spinning disc systems), the signal-to-noise ratio of the RCM is 4 times higher than in standard confocal microscopy.

To fully understand the principle of rescanning, resolution improvement and the optical layout of the RCM, please watch the video below that explains the components and the light path of the RCM (animation credits to StudioFlip). Additional technical details and test images can be found in De Luca et al (2013).