"

From 2010.igem.org

Microscopy

Details on microscope

A microscope with motorized x, y and z control, a motorized shutter and a 60× lens is used with appropriate fluorescence filters for the fluorescence signals. Light-emitting diode arrays are installed as light sources for red light (660 nm) and far-red light (748 nm) pulses.

Details on the Flow Channel

Figure 2: The mold for the channel. Has to be cleaned after casting.	Figure 3: The casted raw channel with a cover slip and the razor plate needed for preparing the channel.	Figure 4: The assembled channel, yet not bonded.
Figure 5: Bonding the channel.	Figure 6: The ready to use channel under the microscope.

Details on the red light/ far-red light diodes and the filters

For the experimental setup it is essential to use the right wavelengths of light for the activation and deactivation of straight swimming and tumbling of the E. lemming. Furthermore it is important to only use light of wavelengths for the bright field microscopy (necessary for cell detection) which do not induce the to photoconversion between the Pr and the Pfr form of PhyB. In Figure 9 we plotted the photoconversion cross-sections of PhyB in the Pr and the Pfr forms as published in Kendrick and Kronenberg (1994). The photoconversion cross-section is basically the product of the extinction coefficient and the quantum yield and can be interpreted as a measure for the probability that a transition between the two forms take place. When the photoconversion cross-section is multiplied with the photon flux, one obtains the reaction rate constants for the photoconversion process. As one can see in Figure 9, the photoconversion cross sections of the Pr and the Pfr form overlap, but are displaced from each other. This displacement can be used to change the relative amounts of the Pr and the Pfr forms by utilizing light with a given wavelength.

We use two LEDs as light sources with wavelengths around 660nm (red light) and 740nm (far-red light) produced by [http://www.coolled.com/Life-Sciences-Analytical/Products/LED-Wavelengths/ CoolLED]. Unluckily these LEDs are new product development so that their spectra were not available, yet. We thus had to approximate their spectra by shifting the known spectra of the LEDs with wavelength maximums at 700m and 770nm, which are similar in construction, to the left (see Figure 7). The light of the diodes was send through two band-pass filters from [http://www.semrock.com Semrock] (product IDs F39-651 and F39-769), whose spectra are shown in Figure 8.

Besides checking the configuration, the information in Figures 7-9 was also used to calculate the rate constants for the photoconversion reactions in the molecular model. These constants are crucial since they determine the speed with which an E. lemming reacts on changing inputs set by the controller. For maximal light strength (the diodes can be down-regulated smoothly) we obtained for red light a rate constants of 6.64s^-1 (0.102s^-1 for far-red light) for the photoconversion from the Pr to the Pfr form, and 1.02s^-1 (5.68s^-1 for far-red light) for the reverse reaction. These constants were used for the modeling.

Figure 7: The approximated spectral energy irradiances of the red light (red curve) and far-red light (blue curve) diodes.

Figure 8: The amplifications of the red-light (red curve) and far-red light (blue curve) filters.

Figure 9: The photoconversion cross-sections of PhyB in the Pr (red curve) and Pfr (blue curve) states.