Abstract
Two-photon microscopy has revolutionized functional cellular imaging in tissue, but although the highly confined depth of field (DOF) of standard set-ups yields great optical sectioning, it also limits imaging speed in volume samples and ease of use. For this reason, we recently presented a simple and retrofittable modification to the two-photon laser-scanning microscope which extends the DOF through the use of an axicon (conical lens). Here we demonstrate three significant benefits of this technique using biological samples commonly employed in the field of neuroscience. First, we use a sample of neurons grown in culture and move it along the z-axis, showing that a more stable focus is achieved without compromise on transverse resolution. Second, we monitor 3D population dynamics in an acute slice of live mouse cortex, demonstrating that faster volumetric scans can be conducted. Third, we acquire a stereoscopic image of neurons and their dendrites in a fixed sample of mouse cortex, using only two scans instead of the complete stack and calculations required by standard systems. Taken together, these advantages, combined with the ease of integration into pre-existing systems, make the extended depth-of-field imaging based on Bessel beams a strong asset for the field of microscopy and life sciences in general.
Highlights
Since its invention in 1990, two-photon microscopy (Denk et al, 1990) has become an essential tool for biologists, especially in the field of neuroscience (Zipfel et al, 2003)
Even in thin samples like neurons grown in cultures, fine structures such as dendrites are generally located on the same plane close to the substrate, but they grow around and above thicker cell bodies, often 10–15 μm thick, which means that the total sample thickness is well over the standard depth of field (DOF); the different neurites end up not all located in the same plane and cannot be captured in one scan
In this paper, we have presented a simple modification to the standard two-photon microscope, which consists in extending the DOF of the system without compromising on transverse resolution, by adding two optical elements in the laser beam path: an axicon and a regular lens
Summary
Since its invention in 1990, two-photon microscopy (Denk et al, 1990) has become an essential tool for biologists, especially in the field of neuroscience (Zipfel et al, 2003) It can reveal structures deep inside tissue (Helmchen and Denk, 2005), and fluorescent markers can help track activity in networks of cells (Stosiek et al, 2003; Lütcke and Helmchen, 2011). If the labeling is sparse and the cells are distributed at different depths in an extended volume, optical sectioning forces the use of integrating multiple frames at different depths to recover all the information. This limits the temporal resolution of the measurements. A small depth of field (DOF) can become problematic when the sample moves vertically, as it often occurs during in vivo measurements (Laffray et al, 2011)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
