A simple but precise method for quantitative measurement of the quality of the laser focus in a scanning optical microscope

Abstract

SummaryWe report a method for characterizing the focussing laser beam exiting the objective in a laser scanning microscope. This method provides the size of the optical focus, the divergence of the beam, the ellipticity and the astigmatism. We use a microscopic‐scale knife edge in the form of a simple transmission electron microscopy grid attached to a glass microscope slide, and a light‐collecting optical fibre and photodiode underneath the specimen. By scanning the laser spot from a reflective to a transmitting part of the grid, a beam profile in the form of an error function can be obtained and by repeating this with the knife edge at different axial positions relative to the beam waist, the divergence and astigmatism of the postobjective laser beam can be obtained. The measured divergence can be used to quantify how much of the full numerical aperture of the lens is used in practice. We present data of the beam radius, beam divergence, ellipticity and astigmatism obtained with low (0.15, 0.7) and high (1.3) numerical aperture lenses and lasers commonly used in confocal and multiphoton laser scanning microscopy. Our knife‐edge method has several advantages over alternative knife‐edge methods used in microscopy including that the knife edge is easy to prepare, that the beam can be characterized also directly under a cover slip, as necessary to reduce spherical aberrations for objectives designed to be used with a cover slip, and it is suitable for use with commercial laser scanning microscopes where access to the laser beam can be limited.

Application of Two-Photon-Absorption Pulsed Laser for Single-Event-Effects Sensitivity Mapping Technology

“…Practically, there are several ways to measure the spot size, either by sharp blade edge or proper scale of grid [26,27]. By blade edge method, the spot size was measured as 2.7 μm, which is closer to the value by Airy ring theory.…”

High-Fidelity 3D Nanoprinting of Plasmonic Gold Nanoantennas

“…With the molecule density c Ref = 0.3 molecules·nm –3 from the reference solution in ethanol, and the effective height H eff = 19 μm, determined according to refs , , A spot = ω 0 2 ·π/2 is the cross-sectional area of the laser in the focus, with a beam waste of ω 0 = 1.0 ± 0.3 μm (measured using the knife edge method on a 2 × 2 μm square FIB-milled into a 100 nm thick Au layer on a Si wafer); the laser-light-illuminated surface area of the FEBID nanopillar A tip = 1.49 × 10 5 nm 2 one half of the surface area of the FEBID nanoantenna’s tip cone region due to the fact that only one side is illuminated with laser light during Raman measurements; and the surface density of 4-MBT μ S = 5.3 nm –2 , which was taken from ref . With a comparison of the maxima of the peak at around 1100 cm –1 (peak heights measured after background subtraction), I SERS (polarization=0°) = 2067 counts, I SERS (polarization=90°) = 2069 counts, and I Ref = 8780 counts, from Raman measurements of 4-MBT on purified AuC X FEBID nanoantennas (purification TET, 250 s) and reference measurements in ethanol, respectively, …”

Large-scale neuroanatomy using LASSO: Loop-based Automated Serial Sectioning Operation

“…To compare the image quality of the microfabricated films (SiN) used in LASSO with conventional TEM grids (Luxel), we used two standard metrics to quantify the detectability of neuroanatomical structures (e.g., synaptic vesicles). First, the edge spread function (ESF) was computed by manually annotating synaptic vesicles in ITK-Snap on four separate images (2 SiN, 2 Luxel), drawing a line from the vesicle exterior to the interior, and then measuring the change in pixel intensities across the vesicle boundaries [ 26 ]. Using derivative-based change-point detection, the change-points in the ESF were obtained and a line was fit to all points between the identified change points.…”