[Credits: Manoj V Mathew, Centre for Cellular and Molecular Platforms (CCAMP), Bangalore, India.
Experiments were conducted at the Central Imaging and Flow Cytometry Facility (CIFF), National Center for Biological Sciences (NCBS), Bangalore, India.]
Diode lasers are very popular these days as sources of excitation for various fluorescence microscopy applications.
In the earlier days of this technology, it was plagued by problems like lack of reliability, inferior beam quality, and operation at lower powers. This technology has evolved rapidly over the last decade and they have come a long way. The beam quality of the latest diode lasers is very close to the ideal TEM00 mode with M2 values <1.3, which is very good. The diode lasers are now very suited for applications like confocal microscopy that demands near-perfect Gaussian beam profiles.
They have numerous advantages over the conventional gas lasers:
- Available in a large range of wavelengths (all standard fluorophores can be excited using commercially available diode lasers)
- Available at rather high optical powers (10s of milliWatts to few Watts)
- Consumes much less power for compared to gas lasers of same output optical power
- Produces much less heat
- Have much longer lifetimes (upto 10000 hours)
- System and Operation costs are lower
The biggest advantage, however, is that they can be directly modulated. You can control the laser intensity and blank (switch ON and OFF) by directly modulating the laser diode current. The blanking can be performed at very high speeds of the order of few 100 MHz. This means you can avoid the use of an external device like AOTFs for blanking and intensity modulation. This makes laser excitation based microscope (like TIRF, Confocal, SIM etc.) design, especially its laser combiner design much simpler.
In this write-up, I will describe how to configure a 'Vortran Stradus Diode Laser' using Micromanager software and directly modulate it using the software. I used a Stradus® 488-150 laser here. Figure 1848/1 shows the picture of the laser and its controller.
The laser datasheet is shown in Figure 1848/2.
Step 1: Hardware setup
Connect the laser head to the heatsink. Then connect the laser driver cable from the laser controller to the laser input port of at the back of the laser head. This is shown in Figure 1848/3.
Interface the laser controller to the PC using an RS232 cable. The RS232C port is available at the front end of the controller If you use a normal RS232C cable you would need to have a serial port (COM1) available on your PC. The easier would be to use an RS232C to USB converter. This is what I have used. This is shown in Figure 1848/4.
The USB port at the other end of the cable can be connected to any of the available USB port on the computer.
Step 2: Loading the device drivers
Once the system is connected to the serial port (COM1 or USB) the laser controller automatically uploads the drivers and they get installed. You can verify this by checking the 'Device Manager' on the 'Control Panel' of your computer. You will see a new COM port appear in the 'Ports(COMs and LPT)' section of the 'Device Manager'. This is shown in Figure 1848/5.
Note the COM port number. This would depend on your PC and the COM ports already occupied.
Step 3: Installing the Votran Software
You can now install the Votran Laser Software. This is optional. Micromanager should be able to communicate with the Laser without the need for this step. But it is good to test the system out before configuring it in Micromanager. Figure 1848/5 shows the Software Interface.
Once the software is run, it will automatically detect the lasers connected to the computer. If it does not detect select the COM port number to which the laser is connected from the 'Add RS232C Laser' tab. The interface helps you to turn the laser ON/OFF, set the laser power, and enable external triggering of the laser. Also, the laser baseplate temperature and other parameters can be monitored.
Step 4: Configuring Micromanager Software
Before running Micromanager, close the Votran Software, else the COM port will not be available for Micromanager to communicate with the Laser.
Run 'Hardware Configuration Wizard' and add 'VLTStradus|VLTStradus' from the 'Stradus' in 'Available Devices' section. This is shown in Figure 1848/6.
Add the laser and press 'Next'. Now, configure the COM port settings:
- Value: COM port number
Figure 1848/7 shows the COM port settings for Micromanager to properly communicate with the Laser Controller.
Finish the 'Hardware Configuration Wizard' by setting parameters for other devices added if any.
In the front panel of Micromanager select 'Shutter' as 'VLTStradus'. Check the 'Shutter Auto' checkbox. This is shown in Figure 1848/7
This will help the software to blank the laser in sync with the camera acquisition by sending commands to the laser to directly modulate the diode current.
Now press the '+' button on 'Group' to add and configure the laser parameters. The Votran laser parameters can be found under the 'Shutters' section of the 'Group Editor'. This is shown in Figure 1848/8.
Add the required parameters to the front panel. This is shown in Figure 1848/9.
Select 'ON' on the 'Laser Emission' control and set the laser intensity in milliWatts. Then switch the 'Laser Emission' OFF. Leave the rest of the parameters as in Figure C. Now the system is ready for acquisition.
Video 1848/1 shows the laser in action controlled by the Micromanager software.
Logarithmic interval based time-lapse muliD acquisition was used to generate the switching pattern seen in Video 1848/1. The parameters used for the Logarithmic interval is shown in Figure 1848/10.