反射式显微镜物镜在多光子显微镜中的应用。

Application of a reflective microscope objective for multiphoton microscopy.

作者信息

Kabir Mohammad M, Choubal Aakash M, Toussaint Kimani C

机构信息

Department of Electrical and Computer Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.

Department of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.

出版信息

J Microsc. 2018 Apr 20. doi: 10.1111/jmi.12702.

DOI:10.1111/jmi.12702

PMID:29676795

Abstract

Reflective objectives (ROs) mitigate chromatic aberration across a broad wavelength range. Yet, a systematic performance characterisation of ROs has not been done. In this paper, we compare the performance of a 0.5 numerical-aperture (NA) reflective objective (RO) with a 0.55 NA standard glass objective (SO), using two-photon fluorescence (TPF) and second-harmonic generation (SHG). For experiments spanning ∼1 octave in the visible and NIR wavelengths, the SO leads to defocusing errors of 25-40% for TPF images of subdiffraction fluorescent beads and 10-12% for SHG images of collagen fibres. The corresponding error for the RO is ∼4% for both imaging modalities. This work emphasises the potential utility of ROs for multimodal multiphoton microscopy applications.

摘要

反射物镜（ROs）可在很宽的波长范围内减轻色差。然而，尚未对ROs进行系统的性能表征。在本文中，我们使用双光子荧光（TPF）和二次谐波产生（SHG），将数值孔径（NA）为0.5的反射物镜（RO）与NA为0.55的标准玻璃物镜（SO）的性能进行了比较。对于在可见光和近红外波长范围内跨越约1个倍频程的实验，对于亚衍射荧光珠的TPF图像，SO会导致25%-40%的离焦误差，对于胶原纤维的SHG图像，会导致10%-12%的离焦误差。对于这两种成像方式，RO的相应误差约为4%。这项工作强调了ROs在多模态多光子显微镜应用中的潜在效用。