Custom settings of RCM to speed up imaging

RCM (Re-Scan Confocal Microscope) is the first commercial optics-only super-resolution confocal microscope developed by The University of Amsterdam and its spin-off Confocal.nl.

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.

In addition to super-resolution by re-scanning and high sensitivity due to the use of sensitive CMOS camera as a detector, the third important feature of RCM is its flexibility.

In the recent paper (in Journal of Microscopy), the research team that invented the RCM, explored and explained some of the possibilities to speed up imaging of RCM for highly dynamic biological processes.

For fixed multi-stained samples, the default mode – sequential frame-by-frame scanning mode – is optimal. Also FRET applications that do not need high imaging rate (e.g. apoptosis of HeLa cells) can be best done in this mode. But for highly dynamic biological processes, time lag between different colors needs be minimised to avoid the colour shift.

One solution to reduce the time lag between the acquisition of different colours is line-by-line multicolour imaging by multiplexing – same line is scanned two (or more) times with different fluorophore-specific excitation wavelengths, and the emitted light is directed to the same camera, but each colour is projected on different areas of the camera chip. This way, the time-lag between the colors is reduced to the order of milliseconds, and shift between the colors is becomes negligible.

The multiplexing mode can further be optimised with the choice of emission filter: a double-band pass filter, or a combined “church-window” emission filter (produced exclusively for RCM), where different parts of the camera chip are covered with different single-band filters, or application-specific single band emission filters. “Church-window” emission filter will reduce the cross-talk between the channels. Also real-time simultaneous scanning of different colours (patented technology) will be possible. For fast FRET measurements with super resolution this new technology will be essential.

The speed of RCM is limited by the speed of scanning mirrors, currently about 400 lines per second, which determines the frame rate. Some applications – e.g. calcium activity, ratio-metric pH measurements, FRAP (fluorescence recovery after photo-bleaching) require much higher imaging speed than 1 frame per second.

In those applications, an option is to give up acquisition of 2-dimensional information and monitor intensity changes along a single line (1-dimensional) at an extremely high imaging rate (400 lines per second, i.e. one scan in every 2.5 ms).

In summary – in addition to the affordable pricing, sensitivity and super-resolution capacity, the flexibility of the RCM related to the independent control of the two pairs of scanning mirrors grants further potential for applications that require high speed or fast multicolour imaging. Join Confocal.nl at FOM2017 to learn more!

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