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Microscope offers 'super-resolution' performance with optical trick

31 May | By Biophotonics.World
Microscope offers 'super-resolution' performance with optical trick
Improved resolution thanks to the RCM technique. Shown on the left, under conventional 100x illumination, are some neuroblastoma cells. On the right, the same cells viewed using RCM.
Image source: Photometrics/Confocal.nl.
Axial resolution as low as 170 nm made possible with pre-aligned optics and mirror-based re-scanning feature.

Tucson-headquartered scientific camera specialist Photometrics says that thanks to a collaboration with the Netherlands-based microscopy startup Confocal.nl, laboratories are now able to buy a new cost-effective system capable of sub-diffraction-limited resolution.

While the confocal setup does not deliver the kind of state-of-the-art resolving power of more advanced approaches like stimulated emission depletion (STED) fluorescence microscopy and related techniques, it is said to deliver axial resolutions as low as 170 nm.

Back illumination
Fitted with a back-illuminated scientific CMOS camera provided by Photometrics, the Re-scan Confocal Microscope (RCM) improves resolution with an optical “trick”, by first scanning the microscope image and then re-scanning it onto the camera chip using a pair of mirrors.

“By doubling the sweep of the re-scanner, the image written on the camera chip is doubled in size,” says Photometrics. “The size of the re-scanning spot doesn't change, so the image is re-scanned with improved resolution as low as 170 nm.”

The RCM technique is based around a box containing pre-aligned optics that Photometrics says can be connected to any microscope, to convert it from a confocal system to one capable of imaging beyond Abbe’s classical diffraction limit.

Described as a cost-effective solution for researchers wanting to get beyond the diffraction limit without blowing their laboratory budgets, the RCM requires a lower laser power compared to conventional confocal microscopy, making it better suited to imaging live cells.

Some features of Photometrics’ “Prime 95B” CMOS camera are also critical to the offering, with the company explaining: “The large 11x11 µm pixels of the Prime 95B add additional sensitivity to the system to make it even better for imaging live cells.”

The 1200x1200 pixel array on offer also means that the high sensitivity is combined with a relatively large field of view.

Confocal.nl startup
Spun out after four years of research activity at the University of Amsterdam, Confocal.nl was officially established in February 2016 by founders Erik Manders, Peter Drent, and Ronal Breedijk. And after a first public demonstration of the RCM kit at the European Light Microscopy Initiative (ELMI) conference held in Debrecen, Hungary, in May 2016, the company began delivering the first commercial units earlier this year.

In a blog post last month, Confocal.nl claimed that the RCM represented the “first commercial-optics-only super-resolution confocal microscope” on the market.

“RCM is a standard confocal microscope extended with a detection unit, where a pair of scanning mirrors project the emission light directly onto a camera in a scanning manner,” wrote co-founder Manders. “In addition to super-resolution by re-scanning and high sensitivity due to the use of [a] sensitive CMOS camera as a detector, the third important feature of RCM is its flexibility.”

Biology applications
More details of the technique and its potential applications are revealed in a recent scientific paper published by the Confocal.nl team and partners including Nikon in the Journal of Microscopy.

In it, Manders and colleagues write that RCM can be configured to address specific imaging applications in biology – including both FRET (fluorescence resonance energy transfer) and FRAP (fluorescence recovery after photobleaching) techniques.

They add that the signal-to-noise ratio of RCM is four times better than standard confocal microscopy, and confirm that the lateral resolution of the technique is 170 nm, compared with 240 nm for the diffraction-limited approach when using a 488 nm excitation laser and optics with a numerical aperture of 1.49.

Apart from improved sensitivity and resolution, the optical setup of RCM is flexible in its configuration in terms of control of the mirrors, lasers and filters, points out the team.

The RCM technology will be making further appearances at a number of microscopy events this year, including the ELMI 2017 event in Dubrovnik, Croatia, next month, and the Microscience Microscopy Congress taking place in Manchester, UK, in early July.

Confocal.nl corporate video explaining RCM:



Press release of Photometrics.

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