Breaking the 100-nm resolution barrier with multiphoton microscopy using image scanning microscopy and optical...

Multiphoton fluorescence microscopy is the technique of choice for investigations of thick, highly scattering samples, but is outperformed by single-photon super-resolution techniques in spatial resolving power.

We combine two-photon microscopy with two super-resolution microscopy methods, namely, image scanning microscopy and super-resolution optical fluctuation imaging to overcome the reduction in resolution of laser scanning multiphoton microscopy compared with confocal microscopy. Making use of higher-order cumulants and image deconvolution a resolution better than 100 nm can be achieved.

Two-photon image scanning optical fluctuation imaging is achieved by detecting the descanned signal of fluorescence on a 23-element single photon avalanche detector and analyzing (higher order) cumulants of the temporal evolution of the signals. We test the performance of our method with samples of dispersed quantum dots. We show the applicability of two-photon image scanning optical fluctuation imaging to biological samples with fixed mouse ventral midbrain neurons with quantum dot labeled tubulin.

Combining two photon laser scanning microscopy with image scanning microscopy allows to overcome the reduction in resolution caused by the longer wavelength excitation inherent to multiphoton excitation. Analyzing the temporal fluctuations of the signals by calculating cumulants allows to surpass the resolution achieved with conventional confocal imaging of the same fluorophores, and the use of higher-order cumulants and deconvolution allows to achieve a lateral resolution of 75 nm when imaging quantum dots emitting at 625 nm.

Combining two-photon microscopy with image scanning microscopy and optical fluctuation imaging allows to achieve a 5-fold improvement in resolution over standard two-photon microscopy, and a 3.5-fold improvement over conventional widefield imaging of the same fluorophores. This represents the first time, to our knowledge, that sub-100 nm imaging is achieved using multiphoton laser scanning microscopy.