To get the best results from your SONICC imager, you should fully understand all of the image controls — what they do, how to use them, and how to optimize them.
The angles of the scanning mirrors are controlled to provide continuous zoom while imaging. The laser power is automatically adjusted when the zoom is adjusted to compensate for over-exposure. The scan angles can be adjusted from 1x (2 mm x 2 mm FOV) to 10x (200 µm x 200 µm FOV).
If performing drop location, drops are first located with the visible imager. Then, the coordinates are translated to the SONICC imager and the scan angles automatically adjusted to match the visible FOV.
The SHG and UV-TPEF signal scales with the quadratic of the incident laser power. The laser power is defined as the power at the sample and is calibrated for each system. There is a large dynamic range of signal intensity from one sample. It is recommended to test a few drops with SONICC in manual mode to determine the optimal laser power.
The intensity of the signal generated from protein crystals varies significantly from sample to sample based on the protein, crystal class, orientation, size, and quality. Therefore, it is impossible to give precise imaging parameters to optimize the amount of signal collected. Based on experience, it is suggested to use the following parameters:
Start with 350 mW, 1x zoom, 1x1 binning.
If image is saturated due to too much signal, reduce power to 200 mW and optimize from there.
If the signal is too weak, increase the power to maximum (500 mW) and increase binning to 2x2.
When the zoom is greater than 6x, there is no advantage to having 1x1 binning. At this point, the pixel size is smaller than the laser spot size so it advantageous to increase binning to 2x2 to increase signal.
Start with maximum power of 110 mW, 1x zoom, 1x1 binning.
If the image is saturated due to too much signal, reduce power as necessary.
The green laser is absorbed by plates, so it is important to keep power low for high zooms. The software automatically adjusts the power when zooming, but in manual mode, be careful not to increase it too much. Maximum power at 12x zoom will burn your plates.
When the zoom is greater than 6x, there is no advantage to having 1x1 binning. At this point, the pixel size is smaller than the laser spot size so it advantageous to increase binning to 2x2 to increase signal.
The current electronic package allows 512 x 512 image acquisition for one z-slice in 500 ms. This corresponds to 8 traces of the fast scanning mirror per line. A one drop 96 well plate can be imaged with SHG in 15 minutes with 8 z-slices and 5 minutes for visible imaging.
Signal intensity in UV-TPEF images is dependent upon the number of tryptophans in the protein. A recommended starting point would be around 100 mW. Depending on the system, full power is usually around 200 mW. If needed, full power can be used relatively safely, but do not leave full power focused on a single spot or on for an extended amount of time due to the potential for thermal decomposition to your samples.
SHG signal intensity is dependent on the presence or absence of a chromophore, crystal class, and crystal quality.
For example, proteins with chromophores need only ~100 mW, whereas 350 mW would be an appropriate power for other samples. A good starting point is 300 mW. From there, you can adjust according to your specific sample.
The maximum laser power is ~550 mW (dependent on system), users should exercise caution when using such high power for extended periods on samples. Routine imaging with < 400 mW is optimal.
The minimum exposure time for SONICC images is 447ms, due to the way SONICC images are captured. You can input any value you like for exposure time in SONICC, but the system will always round to the nearest multiple of 447ms. For example, if you input 500ms the system will use 447ms, if you input 1000ms the system will use 894ms, and so on. Usually, exposure times of 447ms or 894ms are sufficient for screening. Higher quality images can be obtained for specific drops with longer acquisitions.
In manual mode, the first time you turn Live Image ON, the frame is mistimed resulting in a segmented image. If Live Image is turned OFF and then back ON (within 15 seconds of each other) then the image will be re-synced.
In automatic imaging, we address this "behind the scenes" — the first slice of the first drop or region imaged is discarded and then repeated.
SHG: The SHG imaging mode images the sample with IR and detects the SHG (532 nm) in transmission. Signal is only generated from chiral crystals.
UV-TPEF: The UV-TPEF imaging mode images the sample with green (532 nm) and detects the fluorescence generated (325-400 nm). Signal is generated from UV-fluorescent molecules like tryptophan.
Imaging Settings: Our technicians set up three imaging settings for each imaging type. The settings are as follows:
| Name | Exposure Time (ms) | Resolution | Laser Power (mW) | Sensor |
|---|---|---|---|---|
| UV-TPEF Low Power | 500 | 1x1 | 20 | UV-TPEF |
| UV-TPEF Imaging | 500 | 1x1 | 125 | UV-TPEF |
| UV TPEF High Power | 1000 | 1x1 | 200 | UV-TPEF |
| SHG Low Power | 500 | 1x1 | 50 | SHG |
| SHG Imaging | 500 | 1x1 | 325 | SHG |
| SHG High Power | 1000 | 1x1 | 400 | SHG |
Important: Urea and tryptophan are good test samples, but should ONLY be imaged with the low-power imaging settings.
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