Centrifuge the unpacked thawed sample at 10, 000g for two minutes at room temperature. Observe the sample to identify any precipitation, color, and condition. Warm the prepared 1, 536 well plate to 23 degrees Celsius, and then centrifuge it at 150g for five minutes.
Using a brightfield microscope, take pictures of the plate containing the cocktail only to be used as a negative control. Dispense 200 nanoliters of the sample to each well using a liquid handling robot. after centrifuging the plate at 150g, incubate it at the desired temperature for six weeks.
Capture brightfield images of the plates with samples at weekly intervals from day 1 to week 6. Perform sonic imaging with the SHG and UV-TPEF. Automated plate imaging throughout the experiment duration allowed for the brightfield identification of rapidly and slow-growing crystals.
The UV-TPEF imaging indicated the proteinaceous nature, while the SHG imaging confirmed the crystalline nature of the sample. Sonic imaging enabled the identification of crystals obscured by precipitation or film which do not produce an SHG signal, or microcrystals that may be mistaken as precipitate. Nonprotein crystals lacking UV-TPEF signal but displaying strong brightfield and SHG signals were also identified.
