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. 2014 May 20;8:139.
doi: 10.3389/fncel.2014.00139. eCollection 2014.

Extended Two-Photon Microscopy in Live Samples With Bessel Beams: Steadier Focus, Faster Volume Scans, and Simpler Stereoscopic Imaging

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Extended Two-Photon Microscopy in Live Samples With Bessel Beams: Steadier Focus, Faster Volume Scans, and Simpler Stereoscopic Imaging

Gabrielle Thériault et al. Front Cell Neurosci. .
Free PMC article

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.

Keywords: 3D imaging; axicon; cellular imaging; depth of field; functional calcium imaging; nondiffractive beam; nonlinear microscopy; temporal resolution.

Figures

Figure 1
Figure 1
Illustration of the set-up. A Ti:Sapphire laser generates an ultra-short pulsed laser beam with a Gaussian profile. This beam is expanded with a simple two-lens telescope. Once expanded, the beam passes through an axicon followed by a lens. These two elements transform the laser beam into an annulus of light. This annulus is imaged into the back focal plane of the objective lens, which creates a tightly focused Bessel-Gauss beam in the sample. The scanning system enables a beam tilt in the back focal plane of the objective, leading to an x-y scan of the beam in the sample. Fluorescence light is retro-collected with the objective and directed to a photomultiplier tube with a dichroic mirror.
Figure 2
Figure 2
Two-photon fluorescence distributions for the data presented in this paper. The on-axis intensity (top) and the transverse resolution (bottom) of a standard two-photon set-up (red lines) varies much more rapidly along the optical axis than those of the Bessel extended DOF two-photon microscope (green and blue lines). Green dots are experimental values measured with fluorescent microspheres (Molecular Probes, Fluosphere 505/515, diameter 200 nm) mounted on a coverslip with fluorescent mounting medium (Dako).
Figure 3
Figure 3
Extended DOF for imaging thin samples. With the standard two-photon microscope (left), the focal plane is very thin and small perturbations affect the fluorescence signal. With an extended DOF (right), the focus is much more robust.
Figure 4
Figure 4
Extended DOF microscopy provides a steadier focus. (A) Average fluorescence signal from each frame in a z-stack of fixed neurons, grown on a glass coverslip. The stage holder was translated 1 μm in the z direction between each frame. The signal in the extended DOF set-up (blue) is stable over a larger distance than the signal in the standard set-up (red). Single frames at 5 different positions in the z-stack are presented below, for (B) the conventional set-up and (C) the Bessel extended DOF set-up. (D) The DOF extension does not affect the transverse resolution of the system, as shown by this comparison of average intensities from 10 frames for each method.
Figure 5
Figure 5
Bessel beam 3-D raster scan vs. conventional DOF volumetric imaging in a thick sample. (A) With the standard two-photon microscope, the focal volume is very small and one must acquire a stack of raster-scanned images to image the entire volume of interest. (B) With an extended DOF, the entire volume of interest can be examined in a single x-y scan, which leads to much faster volumetric scans without compromise on transverse resolution.
Figure 6
Figure 6
Extended DOF for fluorescence lifetime imaging of dorsal root ganglion neurons (DRGs). (A) Confocal imaging of DRG neurons expressing GFP. Images obtained at various focal depths, with a DOF of 2.1 μm. The image on the far right is the resulting z-projection of 7 images taken every 6 μm, between z = −18 μm and z = 18 μm (example images on the left). (B) Extended depth of field with 2-photon excitation and fluorescence lifetime imaging. The same sample was imaged with 2-photon excitation. Left, single frame obtained with a DOF of 20 μm. Center, average of 7 frames (same acquisition time as for each confocal image). Right, color-coded lifetime image of DRG neurons obtained from photons accumulated from 7 consecutive frames.
Figure 7
Figure 7
Volumetric imaging of calcium dynamics in mouse cortex. (A) Positions of the ROIs in the specimen, acquired with a standard two-photon stack at the end of the experiment (the summed stack of the raw fluorescence images is shown here), and color-coded to indicate the depth of each feature. (B) Single-cell calcium transients in an acute slice of mouse cortex stained with Fluo-4 AM, imaged with the extended DOF set-up and corresponding to the ROIs in (A). Dotted lines correspond to the extended DOF single frames shown below. (C–G) (F-F0)/F0 single frames from the time-lapse acquisition of calcium dynamics (full video available online: Supplementary Movie S1).
Figure 8
Figure 8
Simple method to produce a stereoscopic pair of images. (A) A small displacement of either the lens after the axicon induces a tilt θ of the focal line at the sample, with respect to the optical axis. We scanned a random distribution of small fluorescent beads (Molecular Probes, Fluosphere 505/515, diameter 500 nm) at different depths, spanning 60 μm, to produce a stack of images. The z-projection of these stack show that when (B) no shift is applied, the point of view is perfectly vertical, and when (C) the lens is slightly shifted, the point of view is tilted with respect to the vertical. (D) Two superimposed cells can be distinguished with this method if they are separated by a distance Δz = d/sinθ.
Figure 9
Figure 9
Stereographic imaging of neurons in only two frames. With the extended DOF microscope, two images were acquired, with different values of Δx: (A) Δx = −100 μm and (B) Δx = −100 μm. The composite image (C) can be viewed with 3-D perception using red-cyan glasses.

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