Revolution® FRAPPA

Photo-Bleach and Activate interact with speed and precision

Revolution® FRAPPA - Photo-Bleach and Activate interact with speed and precision

Fluorescence Recovery After Photo-bleach (FRAP) and Photo Activation (PA) are established imaging protocols in which a computer-steered laser beam is used to photo-bleach or photo-activate a user-defined region in the specimen. Andor?s FRAPPA uses a dual galvanometer scan head to provide a computer-steered laser beam delivery system. It can be configured in-line with a CSU and/or camera or on another port.


Features
Bypass? mode provides 1:1 relay imaging for ?in-line?
FRAPPA? mode performs laser scanning into microscope
Diffraction limited spot size ~0.5 ?m @488 nm FWHM
In-line? operation enables all laser lines for FRAPPA actions
Dichroic coupling allows simultaneous imaging and FRAPPA actions
Mode switching in < 10 ms
Integrated control with iQ software provides ?point-and-shoot? and protocol modes
Arbitrary multi-region scanning of points, rectangles and polygons
Integration with Fast LZ imaging and for 3D FRAPPA monitoring and localisation
FRAPPA Scan Head
Andor?s FRAPPA uses a dual galvanometer scan head to provide a computer-steered laser beam delivery system. It can be configured in line with a CSU and/or camera and operates in two modes (see image below).

Switched mode operation - Bypass provides 1:1 relay imaging while ?FRAP? switches to laser scanning mode
Mode switching in < 10 ms
Integrated control with iQ software provides 'point-and-shoot' and protocol modes
Arbitrary multi-region scanning of points, lines and polygons

Under Andor iQ control, the user commands FRAPPA to bleach or activate regions of interest with user-defined times, laser lines and powers.Laser switching is tightly synchronized with FRAPPA modes of operation, using our proprietary Laser Combiner and Multi-Port Switch (MPS).


The FRAPPA unit optical path is shown here in the two operating modes. On the left we have the bypass mode in which the FRAPPA head acts as a relay optical system with 1:1 imaging. On the RHS we have the active laser scanning mode, where a laser beam entering the optical system from the lower port.

Andor Laser Combiner and Multi-Port Switch (MPS)
In order to use the same laser lines for multiple imaging modes, we have developed a proprietary multi-port switch (MPS), which delivers 100% of laser power to one of three fiber delivery ports. MPS uses a fast galvanometer to switch outputs in approximately 1 ms, with external TTL control. Stability is remarkably good, typically ~ 0.5% over a 12 hour period. This includes SS laser long term intensity fluctuations. The rapid performance of MPS ensures that switching between imaging modes is not limited by the ALC, but other factors in the system. Used with our FRAPPA configuration, switching between CSU imaging and FRAPPA takes <5 ms including delays induced by software latency and camera triggering. This represents best in class performance.


 


The figures above show measured switching delay, rise time and long term stability of the MPS under Andor iQ control. On the top graph the measured latency < 5ms (~ 3ms in this case) and the signal rise time is about 500 us (10-90%), as predicted from the design specifications. Best performance in analytical microscopy demands that coupling to the 2 (or 3) output fibers should be both stable and efficient. On the bottom graph we see a power stability curve for one output plotted overnight (14 hours), where the unit was switched every two minutes and sampled every 30 seconds.

Peak to peak fluctuation was approximately -2% to +4%, which compares well to the SS module stability of +/- 3% over 8 hours. Note that the largest fluctuations correspond to the timing of AC switch off for the night (+2.5 hrs) and switch on for the morning (+12.5 hrs). In a more stable room temperature (4-12 hrs) stability is around +/- 1% peak. The efficiency of the Andor MPS is high. For example, a typical coupling efficiency value for each of the three ports is 80%(+0/-10%) at 488nm.