Upgrading a microplate reader for photobiology and all-optical experiments.
Automation can vastly reduce the cost of experimental labor and thus facilitate high experimental throughput, but little off-the-shelf hardware for the automation of illumination experiments is commercially available. Here, we use inexpensive open-source electronics to add programmable illumination capabilities to a multimode microplate reader. We deploy this setup to characterize light-triggered phenomena in three different sensory photoreceptors. First, we study the photoactivation of Arabidopsis thaliana phytochrome B by light of different wavelengths. Second, we investigate the dark-state recovery kinetics of the Synechocystis sp. blue-light sensor Slr1694 at multiple temperatures and imidazole concentrations; while the kinetics of the W91F mutant of Slr1694 are strongly accelerated by imidazole, the wild-type protein is hardly affected. Third, we determine the light response of the Beggiatoa sp. photoactivatable adenylate cyclase bPAC in Chinese hamster ovary cells. bPAC is activated by blue light in dose-dependent manner with a half-maximal intensity of 0.58 mW cm(-2); intracellular cAMP spikes generated upon bPAC activation decay with a half time of about 5 minutes after light switch-off. Taken together, we present a setup which is easily assembled and which thus offers a facile approach to conducting illumination experiments at high throughput, reproducibility and fidelity.
Humboldt-Universität zu Berlin, Institut für Biologie, Biophysikalische Chemie, Berlin, Germany. florian.richter.1@hu-berlin.de andreas.moeglich@hu-berlin.de.
Florian
F
Richter
Ulrike S
US
Scheib
Jennifer
J
Mehlhorn
Roman
R
Schubert
Jonas
J
Wietek
Oliver
O
Gernetzki
Peter
P
Hegemann
Tilo
T
Mathes
Andreas
A
Möglich
D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D017360:Arabidopsis; D029681:Arabidopsis Proteins / Q000737:chemistry; D057205:Automation, Laboratory / Q000295:instrumentation*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D053524:Beggiatoa; D016466:CHO Cells; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D008027:Light; D009154:Mutation; D055096:Optical Devices; D018462:Photobiology / Q000295:instrumentation*; D055668:Photochemical Processes; D051939:Phytochrome B / Q000737:chemistry; D046939:Synechocystis; D013696:Temperature
D029681:Arabidopsis Proteins; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D007093:Imidazoles; C497952:PHYB protein, Arabidopsis; D051939:Phytochrome B; C029899:imidazole; D000242:Cyclic AMP; D000262:Adenylyl Cyclases