Extended depth of field microscopy for rapid volumetric two-photon imaging

Abstract

Two-photon fluorescence microscopy is an influential tool in biology, providing valuable information on the activity of cells deep inside the tissue. However, it is limited by its low speed for imaging volume samples. Here we present the design of a two-photon scanning microscope with an extended and adjustable depth of field, which improves the temporal resolution for sampling thick samples. Moreover, this method implies no loss of optical power and resolution, and can be easily integrated into most commercial laser-scanning microscopy systems. We demonstrate experimentally the gain in performance of the system by comparing volumetric scans of neuronal structures with a standard versus an extended depth of field system.

Extended two-photon microscopy in live samples with Bessel beams: steadier focus, faster volume scans, and simpler stereoscopic imaging

“…Axicons are refractive optical elements shaped as a cone (McLeod, 1954), which deviate light toward the optical axis by an angle β simply calculated from Snell's refraction law. The complete details of this method are presented in a previous paper (Thériault et al, 2013). …”

“…The transverse resolution ρ and the DOF L of the two-photon excitation at full-width at half maximum are given by: $\rho =m\frac{1.629\lambda }{2\pi \sin \beta }\text{ and }L=m^2\frac{0.577w}{\tan \beta }$ where λ is the wavelength of the laser beam and m = Ff 1 / f 2 f α is the magnification applied to the Bessel beam while being relayed to the sample, with focal lengths F of the microscope objective, f 1 and f 2 of relay lenses in the scanning system and f α of the lens after the axicon. The advantage of this approach is that it allows adjusting the DOF independently (without changing the resolution) by using a simple telescope (Thériault et al, 2013). …”

“…Different approaches have been recently proposed to shape the distribution of light at the sample into a Bessel-Gauss beam (Botcherby et al, 2006; Dufour et al, 2006; Thériault et al, 2013), which is characterized by an intense central lobe and is nondiffractive, i.e., the central lobe has a constant radius.…”

Three-photon fluorescence microscopy with an axially elongated Bessel focus

“…Several approaches have been demonstrated for generating a Bessel beam under two-photon excitation. The most common approach involves the use of an axicon (conical lens) [8, 9, 10]. Other approaches employed a phase mask [11], or alternatively a spatial light modulator (SLM) [12], with the latter allowing for great flexibility in generating different types of Bessel foci with shaped axial intensity profiles.…”

Optomechatronics for Biomedical Optical Imaging: An Overview

“…Here, the SLM is tuned to provide programmable illumination needed to simultaneously excite all neurons at different focal points while the wavefront coding via the phase mask makes it possible to simultaneously accept light from neurons at all different focal points. Fast axial scanning by changing focal position has been attempted by several methods: adaptive aberration correction with DM [53], extended DOF with a scanning axicon [54,55], and two photon microscopy with diffractive optical elements (DOE) [56]. For high spatiotemporal resolution, a two photon microscope with simultaneously focusing multiple excitation beams at different positions was developed.…”