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We use multiphoton quantum-control spectroscopy to discriminate between unbound and enzyme-bound NADH (reduced nicotinamide adenine dinucleotide) molecules in solution. Shaped ultrafast laser pulses are used to illuminate both forms of NADH, and the ratio of the fluorescence from the bound and unbound molecules for different pulse shapes allows us to measure binding without spectrally resolving the emitted fluorescence or relying on the absolute fluorescence yield. This permits determination of enzyme binding in situations where spectrally resolved measurements and absolute fluorescence yields are difficult to obtain, and makes the approach ideal for multiphoton microscopy with molecular discrimination.

Using shaped ultrafast laser pulses to detect enzyme binding