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Engineered light-dependent switches provide uniquely powerful opportunities to investigate and control cell regulatory mechanisms. Existing tools offer high spatiotemporal resolution, reversibility and repeatability. Cellular optogenetics applications remain limited with diffusible targets as the response of the actuator is difficult to independently validate. Blue light levels commonly needed for actuation can be cytotoxic, precluding long-term experiments. We describe a simple approach overcoming these obstacles. Resonance energy transfer can be used to constitutively or dynamically modulate actuation sensitivity. This simultaneously offers on-line monitoring of light-dependent switching and precise quantification of activation-relaxation properties in intact living cells. Applying this approach to different LOV2-based switches reveals that flanking sequences can lead to relaxation times up to 11-fold faster than anticipated. In situ–measured parameter values guide the design of target-inhibiting actuation trains with minimal blue-light exposure, and context-based optimisation can increase sensitivity and experimental throughput a further 10-fold without loss of temporal precision.

Resonance energy transfer sensitises and monitors in situ switching of LOV2-based optogenetic actuators