Volumetric HiLo microscopy employing an electrically tunable lens

Abstract

Electrically tunable lenses exhibit strong potential for fast motion-free axial scanning in a variety of microscopes. However, they also lead to a degradation of the achievable resolution because of aberrations and misalignment between illumination and detection optics that are induced by the scan itself. Additionally, the typically nonlinear relation between actuation voltage and axial displacement leads to over- or under-sampled frame acquisition in most microscopic techniques because of their static depth-of-field. To overcome these limitations, we present an Adaptive-Lens-High-and-Low-frequency (AL-HiLo) microscope that enables volumetric measurements employing an electrically tunable lens. By using speckle-patterned illumination, we ensure stability against aberrations of the electrically tunable lens. Its depth-of-field can be adjusted a-posteriori and hence enables to create flexible scans, which compensates for irregular axial measurement positions. The adaptive HiLo microscope provides an axial scanning range of 1 mm with an axial resolution of about 4 μm and sub-micron lateral resolution over the full scanning range. Proof of concept measurements at home-built specimens as well as zebrafish embryos with reporter gene-driven fluorescence in the thyroid gland are shown.

Speckle illumination microscopy enables slide-free and non-destructive pathology of human lung adenocarcinoma

“…However, this will collide with the filtering process in HiLo microscopy which achieves optical sectioning by evaluating the speckle contrast over a sampling window containing several imaged grains. Alternatively, we quantify HiLo's axial resolution by imaging 10-µm-diameter fluorescent microspheres 29 , and the resulting axial resolution can be calculated as:…”

Geometry Measurement of Submerged Metallic Micro-Parts Using Confocal Fluorescence Microscopy

“…Established micro-topography measurement techniques can be separated into interferometric methods (e.g., displacement interferometry [3,4], digital holography, etc.) and other techniques [4], such as conventional laser-scanning confocal microscopy, light sheet microscopy or HiLo microscopy [11]. However, these techniques are still hindered by the in situ conditions, such as processinduced currents, thermal gradients and refractive index fluctuations for the case of interferometric methods [3,18], or high surface angles for the case of conventional confocal microscopy [5].…”

3D-scanning microscopy with adaptive lenses and prisms for zebrafish studies

“…Several approaches employing adaptive lenses 11,12 have been introduced to realize axial scanning without the need for any mechanically moving parts in a variety of microscopes, such as confocal microscopy, 2,13 two-photon microscopy, 14,15 light-sheet microscopy, [16][17][18] structured illumination microscopy, 19,20 and standard wide-field microscopy. 21,22 More sophisticated adaptive lenses with 2 degrees of freedom enabled simultaneously tuning the focal position and correcting the spherical aberrations.…”