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miniSOG integration using blue LEDS

Goal: Building a custom blue LED box to use with the miniSOG worms to integrate. We will use the 7-LED star LEDs and see if we can observe background mutations.

Blue light illumination

from the paper: We used a blue LED (UHP-MIC-LED-460) light source (460 ± 5 nm, spectrum half width 27 nm, Prizmatix, Givat Shmuel, Israel) for illumination as described. Briefly, the irradiance at the specimen was 2.01 mW/mm2 as measured using a D10MM power sensor connected to a PM50 Optical Power Meter (Thorlabs, Newton, New Jersey, US). Pulse frequency was controlled by a digital function generator/amplifier (PI-9587C, PASCO, Roseville, CA). The light source was ~10 cm above the plate surface. To illuminate animals, we transferred 15–20 young adults to a 3 cm unseeded NGM plate poured with 60 μl of 100 mM CuCl2 on the rim of the plate. The blue light illumination covers the entire plate surface. Overall, the irradiance of our LED setup is approximately 3.5× higher than that of the epifluorescence setup tested previously and has a slightly different excitation spectrum. After illumination, animals were kept in the dark until analysis.

from the paper: We used a blue Ultra High Power LED (UHP-LED-460) light source (460±5 nm, Prizmatix, Southfield, MI) connected to a digital function generator/amplifier (PI-9587C, PASCO, Roseville, CA) with a TTL cable. The light source was fixed approximately 10 cm above the plate using a custom-made stage. The sine wave of light was illuminated at 4 Hz with 65% of the maximum power (>2.3 W). To prepare animals for optogenetic mutagenesis, a filter paper with a 25 × 25 mm hole was soaked with 100 mM CuCl2 and placed onto a 60 mm NGM plate without bacteria to restrict animals within the illuminated area. Dozens of L4, non-gravid (3–7 h post-L4), or gravid (8–12 h post-L4) young adult worms as P0 were picked and transferred to new plates. The blue light intensity on the plate was measured by a photometer to be 2.0 mW/mm2 under continuous illumination.

Transgene integration protocol

from the paper: Six to eighteen hours after microinjection, we illuminated worms with blue light at 2 mW/mm2 and 4 Hz for 30 min as previously described (Noma and Jin 2015; Noma and Jin 2016). These P0 worms were recovered on new seeded plates after light illumination. A few days later, all transgenic F1 worms were put on individual plates (F1 plates). Among these F1 plates, we selected the plates with >50% F2 worms carrying transgenes and recovered single transgenic F2 onto five individual plates. A few days later, we examined for 100% transgene-positive F3 worms.

Our specifications

Blue light (λ = 475 ± 15 nm) was supplied by a Luxeon Star 7-LED assembly with a diffused optic array driven by a 700 mA FlexBlock driver. minisog.jpg

Measuring the output power of our LED with the diffuser. Measured power was lower for the LED without the diffuser lens, which might be due to power issues or because of the lack of focussing. Note: The decay does not follow the 1/r^2 law, since we do not have a point source and so more of the 7 LEDS are seen by the sensor as we move farther away.

We can achieve the desired power of ca. 2.0  mW/mm2 at approximately 8 cm distance between LED and plate. The diameter with diffuser is ca 3 cm. At any distance we can illuminate all of a 3 cm plate and some of a 6 cm plate.

wiki/documentation/monika/minisog_integration_using_blue_leds.txt · Last modified: 2020/07/20 06:11 by mscholz