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 high laser power could be damaging to tissue. The warning can be disabled if required.
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 while 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 demanding 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
Please Note: The Blanking Percentage interface does not work with the MIC intensity control system
A Pockels cell is a type 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_coMD.vi, rather than the two VI’s used to run the motorised option. By default pockelscontrol_coMD.vi will output a 0-2V analogue output on the specified Piezo Output Channel. See wiring guide for more information.
Resonant scanners oscillate with a sinusoidal velocity profile, resulting in slower angular velocity at the furthest scan angles compared to the centre of the scan where the mirror is moving quasi-linearly. The tissue at the edges of the field of view is therefore exposed to laser radiation for longer durations than central portions of the image. Under some circumstances this may lead to phototoxicity and photodamage. To overcome this potential problem the edges of the sinusoidal scan can be “blanked” by quickly attenuating the laser using the Pockels Cell.
SciScan includes a module to allow for Pockels Cell blanking, pockelscontrol_w_blanking_1.0_roMD.vi which can be used instead of pockelscontrol_coMD.vi.
Using the pockelscontrol_w_blanking_1.0_roMD.vi enables the user to choose a Blanking Percentage on the front panel (next to the laser power control). Importantly, note that this percentage refers to the percentage of sample area that is being “blanked” (and not the percentage of pixels, time, or mirror phase). Adjust the blanked area until it just encroaches on the field of view.
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 in 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. Once at a minimum 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% will be non-linear as most half-wave plates act non-linearly.
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.
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.
To begin calibration place a power meter in the light path downstream of the Pockels cell and adjust the software laser power control to 0% and click the tick box next to the laser power dial. Adjust the bias voltage on the Pockels cell controller and observe how the transmitted laser power differs. Using the bias voltage set the pockels cell to its minimum transmission. Increase the laser power through software until it reaches maximum transmission. Click the green tick button next to the dial to complete the calibration, mapping the voltage that provided you with minimum transmission to 0% and maximum to 100%.
Please Note: If using pockelscontrol_w_blanking_1.0_roMD.vi you need to press “Focus” in the software in order to adjust the transmitted laser power. This is because when using the blanking feature the pockels cell requires the timing signal from the resonant x-mirror.
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 as you image further into tissue. Full details of this features use is outlined in the Z-Stack controls.