Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, IA, USA.
J Neurosci Methods. 2014 Jan 15;221:15-21. doi: 10.1016/j.jneumeth.2013.08.026. Epub 2013 Sep 8.
Although recent developments in methodologies for light microscopy have enabled imaging of fine biological structures, such imaging is often accompanied by two types of problems. One is a tilting of the specimen with respect to the x-y plane (i.e. rotation around the x- or y-axis) such that the sample is not perpendicular to the optical z-axis, and the other is rotation around the z-axis that precludes optimal orientations for imaging and experimentation. These rotation problems can cause optical aberrations and hamper imaging experiments, even when the angular difference from the ideal position is small.
In order to correct for these practical issues, we have developed a specimen holder with 3-axis (x-y-z) rotation for an inverted light microscope. This allows for full-range rotations of 2-4° for x-, y-axes, ~24° for z-axis, and a small-angle control of <0.1° for either axis.
Using this device, we observed the cultured hippocampal neurons stained by immunofluorescence for a dendritic marker, or the sub-resolution fluorescent beads plated on a glass coverslip. The rotations and associated problems could be manipulated, while viewing the specimens by laser-scanning confocal microscopy.
This tilting/rotation device is easily manufactured and installed on a conventional microscope stage without requiring changes to the existing optical components. Similar devices with full capability have not been available.
It will be useful for imaging experiments with biomedical applications.
尽管最近在显微镜方法学方面取得了进展，能够对精细的生物结构进行成像，但这种成像通常伴随着两种类型的问题。一种是标本相对于 x-y 平面倾斜（即绕 x 或 y 轴旋转），使得样本不垂直于光 z 轴，另一种是绕 z 轴旋转，使得无法获得最佳的成像和实验方向。这些旋转问题会导致光学像差并阻碍成像实验，即使与理想位置的角度差异很小。
为了解决这些实际问题，我们开发了一种用于倒置显微镜的具有 3 轴（x-y-z）旋转的标本架。这允许对 x、y 轴进行全范围旋转 2-4°，对 z 轴进行约 24°的旋转，以及对任何轴的小角度控制 <0.1°。
使用该设备，我们观察了用免疫荧光染色的培养海马神经元的树突标记，或在玻璃盖玻片上接种的亚分辨率荧光珠。可以在激光扫描共聚焦显微镜下观察标本时操纵这些旋转及其相关问题。
这种倾斜/旋转装置易于制造并安装在常规显微镜台上，而无需对现有光学元件进行更改。具有完整功能的类似设备尚未面世。
它将对具有生物医学应用的成像实验有用。
