Ultrafast Widefield Optical Sectioning Microscopy

The need for optical sectioning in bio-imaging has amongst others led to the development of the two-photon scanning microscopy. However, this comes with some intrinsic fundamental limitations in the temporal domain as the focused spot has to be scanned mechanically in the sample plane. Hence for a large number of biological applications where imaging speed is a limiting factor, it would be significantly advantageous to generate widefield excitations with an optical sectioning comparable to the two-photon scanning microscopy. Recently by using the technique of temporal focusing it was shown that high axial resolution widefield excitation can be generated in picosecond time scales without any mechanical moving parts. However, the achievable axial resolution is still well above that of a two-photon scanning microscope. Here we demonstrate a new ultrafast widefield two-photon imaging technique termed Multifocal Temporal Focusing (MUTEF) which relies on the generation of a set of diffraction limited beams produced by an Echelle grating that scan across a second tilted diffraction grating in picosecond time scale, generating a widefield excitation area with an axial resolution comparable to a two-photon scanning microscope. Using this method, we have shown widefield two-photon imaging on fixed biological samples with an axial sectioning with a FWHM of ~0.85 µm.

Comparison of MUTEF to other imaging methods.
Figure: Comparison of MUTEF to other imaging methods. The three-dimensional representation of an image stack over a range of ~20 µm shows clearly the higher optical section capability of the MUTEF imaging compared to conventional temporal focusing and widefield two-photon excitation (left) and comparison of the axial localization of excitation via these schemes a spot size of ~6 µm. While regular two-photon excitation (green trace) with such a beam size only leads to a poor axial confinement (FWHM ~7 µm) and the widefield temporal focusing (blue line) to a confinement of ~1.6 µm, both the line scan temporal focusing (black trace) and the MUTEF imaging (red) curve yield the same axial confinement (~0.85 µm) as a two-photon scanning microscope. However only in MUTEF imaging a wide field of view can be achieved without any mechanical scanning the two other alternative methods.

Relevant Publications:
Vaziri, A., Shank, CV.,
“Ultrafast widefield optical sectioning microscopy by multifocal temporal focusing.”
Optics Express 18(19), 19645-55 (2010).