The intensity control area of the user interface allows you to modulate the power of the scanning beam on the fly. Power can be adjusted either by dragging the needle on the dial or by adjusting the numerical control.
A warning will appear if you attempt to increase the laser power over 75% as this level of laser power can be damaging to tissue. The warning can be disabled if required.
SciScan can control up to two Pockels cells simultaneously using the tabs labelled 1 or 2 on the right hand side of the controller.
Please note: laser power is indicated as a percentage between a calibrated minimum and maximum transmission values and should not be considered a measurement of absolute laser power and incorrect calibration can result in over exposure of the sample to the beam.
Motorised Half-Wave Plate (MIC)
The combination of a Half-Wave Plate and Polarizing Beam Splitting cube in series can be used to attenuate the pulsed laser system required for multiphoton imaging. The Half-Wave Plate will polarize the incoming beam and the Polarizing Beam Splitting cube will split the beam with the relative intensities of each path dependent on the incoming polarization. By rotating the Half-Wave Plate you can actively attenuate the beam during imaging. This type of attenuation system is described as a motorised intensity control (MIC) system in SciScan.
This method of beam attenuation may be too slow for some applications. It is suitable for modulating power with depth and for changing power while imaging, however it is not suitable for modulation during volume imaging or turn-around blanking where the beam needs to be modulated very quickly.
Scientifica offers a stepper motor driven system to rotate the Half-Wave Plate which is driven by MIC communication_coMD.vi while MIC_coMD.vi runs the interface between the front panel and the driver.
By editing MIC communication_coMD.vi users can customise the driver and use their own motorised systems. More information can be found in the programming guide.
A Pockels cell is an Electro-Optic Modulator that is commonly used to quickly and accurately modulate laser power in multiphoton imaging systems. The Pockels cell makes use of the Pockels electro-optic effect in which the polarization of an incoming beam can be altered by modulating an electric field within a crystal through which the beam passes.
Pockels cells are able to modulate the beam much more quickly than half-wave plate based attenuation systems and can be used to modulate the laser power with depth during normal stack acquisition as well as volume scanning.
The Pockels cell system is controlled by a single vi, pockelscontrol_goMD.vi, rather than the two VI’s used to run the motorised option. By default pockelscontrol_goMD.vi will output a 0-2V analogue output on DEV2/ao0 and DEV2/ao1 which can be used to modulate the Pockels cell through its entire modulatory range. Both output channels can be specified in the .ini file.
Please note: the PCIe card used to control the Pockels cell and/or a Piezo drive only has two available analogue outputs, ao0 and ao1. If using a pizeo drive only a single Pockels cell can be controlled.
Setup and Calibration
The MIC controller needs to be set up in the .ini file as well as calibrated through the user interface. Once the COM port has been specified and both MIC_coMD.vi and MIC communication_coMD.vi have been specified in the optional modules section of the .ini file, the device should communicate with the front panel controls.
To calibrate the motor simply click on the tick box next to the laser power controls, this should bring up a small pop-up window asking you to use hardware controls to drive the motor until the laser power is at a minimum. Position a laser power meter in the light path, downstream from the MIC, and observe the change in laser power while slowly rotating the half wave plate through 360 degrees. Find the position at which laser transmission is minimal, then click OK. Clicking OK saves the current motor position and maps it to the 0 position in the power indicator as well as launching a second pop-up which asks you to move the motor until laser transmission is at a maximum. Once there clicking OK will map the motor position to the 100% position. The scale between 0 and 100% is a linear interpolation of the positions of minimum and maximum laser power, which is only an approximation of the non-linear relationship between rotational position and laser attenuation.
The motor positions are saved in the MIC Settings section of the .ini file. If the motor coordinates are ever re-set the system will need to be re-calibrated. Similarly, laser output polarization differs with wavelength so calibration might need to be adjusted between wavelengths.
Pockels Cell calibration is very straight forward. As default the VI will scale a 0-2V analogue control voltage between 0 and 100% which is more than enough to drive most commercial Pockels cells through their attenuation cycle. You will find that with the default settings 0% will correspond to the minimum laser power however a maximum will be reached at about 60%.
To map the actual maximum to 100% click on the tick box next to the laser power dial to begin calibration, this will cause the power to drop back to 0. Now use the dial to increase the power until you reach the maximum transmitted laser power. Once a maximum is reached click on the green tick button next to the dial. The power will again drop to 0 but now your maximum power will be mapped to the 100% position instead of where it was previously. The scale between 0 and 100% is a linear interpolation of voltages for minimum and maximum laser power, which is only an approximation of the non-linear relationship between voltage and laser attenuation.
Both the MIC and Pockels cell systems are able to take advantage of SciScan’s built in depth compensation. This feature allows you to optimise your image at deeper imaging planes by varying the laser power through a stack or volume scan. Full details of this features use is outlined in the Z-Stack controls