The performance of light microscopes is limited by the stochastic nature of light, which exists in discrete packets of energy known as photons. Randomness in the times that photons are detected introduces shot noise, which fundamentally constrains sensitivity, resolution and speed1. Although the long-established solution to this problem is to increase the intensity of the illumination light, this is not always possible when investigating living systems, because bright lasers can severely disturb biological processes2–4. Theory predicts that biological imaging may be improved without increasing light intensity by using quantum photon correlations1,5. Here we experimentally show that quantum correlations allow a signal-to-noise ratio beyond the photodamage limit of conventional microscopy. Our microscope is a coherent Raman microscope that offers subwavelength resolution and incorporates bright quantum correlated illumination. The correlations allow imaging of molecular bonds within a cell with a 35 per cent improved signal-to-noise ratio compared with conventional microscopy, corresponding to a 14 per cent improvement in concentration sensitivity. This enables the observation of biological structures that would not otherwise be resolved. Coherent Raman microscopes allow highly selective biomolecular fingerprinting in unlabelled specimens6,7, but photodamage is a major roadblock for many applications8,9. By showing that the photodamage limit can be overcome, our work will enable order-of-magnitude improvements in the signal-to-noise ratio and the imaging speed. A quantum microscope obtains signal-to-noise beyond the photodamage limits of conventional microscopy, revealing biological structures within cells that would not otherwise be resolved.

Quantum-enhanced nonlinear microscopy

Irradiation of 5β-cholest-1-en-3-one oxime 14 in methanol gave 1α-methoxy-5β-cholestan-3-one oxime 16, arising from the photoaddition of methanol to the double bond of the enone oxime. 1α-Methoxy-5β-cholestan-3-one 17 as well as 10β-methoxy-5(10→1)abeo-1β(H),5β,10α(Me)-cholestan-3-one 15, arising from a skeletal rearrangement of 5β-cholest-1-en-3-one (generated from the oxime), were the accompanying products in this photoreaction. Deuterium-labelling studies confirmed that the formation of 1α-methoxy-5β-cholestan-3-oxime 16 involves a stereospecific syn addition of methanol to the photogenerated, twisted, ground-state double bond of the oximes B from the rear side of the steroidal framework.Irradiation of 1-methyl-5β-cholest-1-en-3-one oxime 23 in methanol, on the other hand, gave almost exclusively 1-methylene-5β-cholestan-3-one oxime 24, arising from photodeconjugation of the α,β-double bond to the β,γ-position. A deuterium-labelling study established that deuterium is stereospecifically introduced at the 2β-position of 1-methyl-5β-cholest-1-en-3-one oxime 23 in the photodeconjugation in methanol[2H]ol while deuterium is stereospecifically introduced at the 2α-position in the photodeconjugation of its 5α-isomer 6. These results are fully consistent with our previously proposed pathway concerning the photodeconjugation of 1-methyl-5α-cholest-1-en-3-one oxime 6; the stereospecific addition of a proton to the photogenerated, twisted double bond of the oxime D from the rear side of the steroidal framework was followed by the loss of a proton from the 1-methyl group of the resulting carbocation intermediate. Neither the isoxazole derivative, a product in the photoreaction of 5α-cholest-1-en-3-one oxime 1, nor the unsaturated lactam from a photo-Beckmann rearrangement was formed in the photoreactions of either 5β-cholest-1-en-3-one oxime 14 or its 1-methyl derivative 23.

Photoinduced molecular transformations. Part 134. Photoinduced stereospecific addition of methanol to 5β-cholest-1-en-3-one oxime and photoinduced deconjugation of its 1-methyl derivative involving stereospecific proton transfer

R. E. Lack

Papers

1193. Steroids and walden inversion. Part LVI. The bromination of 5β-cholestan-3-one

Steroids and Walden inversion. LVI. The bromination of 5-beta-cholestan-3-one.