Comparison of objective lenses for multiphoton microscopy in turbid samples

Abstract

Optimization of illumination and detection optics is pivotal for multiphoton imaging in highly scattering tissue and the objective lens is the central component in both of these pathways. To better understand how basic lens parameters (NA, magnification, field number) affect fluorescence collection and image quality, a two-detector setup was used with a specialized sample cell to separate measurement of total excitation from epifluorescence collection. Our data corroborate earlier findings that lowmag lenses can be superior at collecting scattered photons, and we compare a set of commonly used multiphoton objective lenses in terms of their ability to collect scattered fluorescence, providing guidance for the design of multiphoton imaging systems. For example, our measurements of epifluorescence beam divergence in the presence of scattering reveal minimal beam broadening, indicating that often-advocated over-sized collection optics are not as advantageous as previously thought. These experiments also provide a framework for choosing objective lenses for multiphoton imaging by relating the results of our measurements to various design parameters of the objectives lenses used.

Determination of scattering properties and damage thresholds in tissue using ultrafast laser ablation

“…We then investigated the properties of porcine vocal folds and found l s;ep ¼ 51.0 AE 3.9 μm, F th;ep ¼ 1.78 AE 0.08 J∕cm 2 , l s;SLP ¼ 26.5 AE 1.6 μm, and F th;SLP ¼ 1.14 AE 0.12 J∕cm 2 . The method is a direct measure of l s rather than the reduced scattering length, l 0 s , because unscattered photons form the vast majority of the photons reaching the focal volume to initiate ablation; 29 assumptions about the anisotropy of the tissue do not need to be made. The method has several advantages over current techniques, such as the simultaneous determination of l s and F th , straightforward calculations that do not require Monte Carlo simulations, and applicability to multilayered tissue.…”

Restored interlaced volumetric imaging increases image quality and scanning speed during intravital imaging in living mice

“…In rIVI imaging, the laser density in the Z ‐direction is one‐fourth that of the conventional method, so that laser damage to cells may be reduced accordingly. It is known that the effects of photobleaching and photodamage caused by reactive oxygen species (ROS) produced in cells by laser radiation depend on its dose and power , and it can be considered that our method, which employs interlaced imaging, may significantly better prevent such effects. This benefit makes it possible for signals to be continuously acquired during long‐term imaging and increases the feasibility of imaging living mice using 2‐PE microscopy.…”

Characterization of wavefront errors in mouse cranial bone using second-harmonic generation

“…AO correction using these correction elements will allow us to image fine structures in the bone marrow (e.g., subcellular features, such as mitochondria) that would not be resolvable using a lower NA, lower magnification objective lens to reduce distortions; 57 or by moving to longer wavelength two-photon or three-photon excitation that requires an exotic optical assembly and fluorophores. 6 Our bone characterization approach can also be used to directly estimate wavefront aberrations and be applied to a correction algorithm; however, it should be noted that the illumination wavelength used to estimate aberrations should match the wavelength to be corrected.…”