Note: you MUST NOT TOUCH the optical components; doing so could misalign the laser and make the experiment difficult to complete on time.
Turning on the detection system
On the laser power supply, make sure the key is in the “1” position and press the green “laser on” button. If the laser head does not emit blue light within a few seconds, notify the TA or instructor. Visually inspect that the laser spot is centered on the back aperture of the microscope. Turn on the PMT power supply and increase the reading to 0.500 by turning the dial clockwise. Turn on the preamplifier. Also, you will need to close the window blinds to reduce background light interference.
Log in to the data acquisition computer. Double click the “523 Microchip Lab” icon on the desktop to open LabView and the VI that will record your electropherograms. Click on the right-facing arrow in the upper left corner to start the VI. You will be prompted for the number of data points to be recorded per second; a good initial value is 20. The VI will start displaying data from the PMT, but will not write it to a file unless you tell it to. Thus, create a folder for your team’s data and enter that path in the box in the VI, along with a filename. To write your electrophoretic data to the file, press the “record” button. When your separation is done, press “stop”. Be sure to change the filename before you hit “record” again.
Initial alignment of the microchip in the optical detection system
Verify that the front knob on the right side of the microscope is in the “eye” position. Then, turn on the green switch on the white box to the right of the microscope; visible light should shine onto the microscope stage. The microscope has 4X and 20X objectives directly under the stage. Rotate the objective ring until the 20X objective is in position directly beneath the microchip. The stage can be moved in the X- and Y-directions using the wand on the right side; focusing is accomplished using the knob on the microscope body. Adjust the position of your microchip using X-Y translation until you find the injection channel, which should cross the viewing area vertically. Then, adjust the Z position so the bottom of the channel is in focus. Note the number on the microscope focusing knob. Turn off the green switch that controls the stage illumination. You should see a faint greenish spot where the laser is focused. Adjust the X-Y stage so the laser spot is in the middle of the channel. Have the TA or instructor verify that you are correctly positioned before you continue. Next, adjust the microscope stage about 30 units higher—this is approximately where the maximum signal will reach the PMT. Check that the green switch to control stage illumination is off, and turn the front knob on the right side of the microscope from “eye” to “side” to direct 80% of the light collected from the objective to the PMT attached on the left port. Look at the trace from the LabView VI (you may want to rotate the computer monitor so you can view it from the microscope); the overall signal should be about 0.5. If the signal is more than a factor of 2 higher, consult the TA or instructor before continuing.
Trial injection and fine alignment
Before you turn on the high-voltage power supplies, make sure that the electrodes are still in the reservoirs. Push in the on/off switch on the bottom right of the power supplies to turn them on. The settings should be 1000 V for the separation (top) power supply and 400 V for the injection (bottom) power supply. Move the switch on the inject box to the “inject” position. Turn on the voltage for both power supplies by pressing the top half of the black switch on the left of the power supplies (to turn off the voltage, press the bottom half of the black switch). The voltage on the display should increase to the set value. The voltage will drive your sample electrophoretically through the injection channel, and you should see a gradually increasing signal at the detector. When the signal levels off, align the pinhole by turning the black knobs on the side and top to maximize signal, then optimize the Z-position by changing the focus to maximize signal; note the value on the focus knob relative to where you were focused on the channel bottom. You will need to adjust the focus knob (after focusing on the channel bottom) by this difference to maximize signal. Any time you are not actively detecting, make sure that you rotate the right knob on the microscope to the “eye” position.
Carrying out separations
Once you have moved the laser spot to the desired position in the separation channel and adjusted the Z position, turn off the stage illumination light and turn the right knob to the “side” position. Switch the inject box to the “inject” position, turn on the high voltage on both power supplies, and wait for the desired time period (40 seconds is a good trial value). At the end of the injection time, switch the inject box to the “run” position, and at the same time press the “record” button on the VI on the computer. When your run is over, press the stop button and make sure to change the filename before you hit “record” again. When you are done with a set of separations, turn off the high voltage and shut off the power supplies. Then, switch the side knob to the “eye” position.
Switching samples in the microchip
After you have shut off the power supplies and switched the side knob to the “eye” position, carefully remove the electrodes from the reservoirs and pull off the tape securing your device. If your device was working fine, you can just pipet old buffer from each reservoir and replace it with new buffer. Then, pipet your old sample from the reservoir, rinse the reservoir with buffer, and pipet the new sample into the reservoir. Your device is now ready for additional separations.
If your device was having problems, it is best to flush it out and refill with buffer. Spray each reservoir with a wash bottle and then pipet the liquid out. Repeat as needed. Apply vacuum to each reservoir to remove the liquid from the channels. Then, follow the process in steps 3 and 5 of the write-up to fill the microchip with buffer and load sample.
When you are finished for the day, switch off the high voltage, turn off the power supplies, and follow the procedure listed above for removing the microchip and flushing out the channels. You will want to rinse your microchip and channels with water and then store it with the channels dry.
Switch the right knob to the “eye” position, turn the dial on the PMT power supply counterclockwise until the voltage is as low as it goes (~0.24), and then switch off the power button. Turn off the preamplifier, and press the red ‘laser off’ button on the laser power supply. Close the LabView program (do NOT say “yes” to save changes), transfer your data from the computer, and then shut it down. Rotate the microscope objectives so they are not directly under the microchip viewing region. Turn off the stage illumination light on the microscope and put away any samples, supplies or materials you used during the lab period.