POWRE: An Extended-Depth-of-Focus, Differential Interference Contrast (DIC) Microscope for Improved Observations of Living Cells

一种新的差分干涉对比显微镜（DIC）将允许生物学家观察活细胞制剂与所有的高分辨率的优势，传统的DIC，但与更多的特点，整个标本体积将出现在一个单一的图像焦点的附加优势，将被开发。目前，高分辨率DIC技术受到固有的窄聚焦深度的限制。有时被称为“光学切片”（不要与共聚焦显微镜混淆），DIC倾向于仅从最接近焦点平面的特征中成像最高空间频率（即最精细的细节）。该平面以上或以下的特征迅速失去对比度，呈现为低空间频率模糊。任何使用DIC成像厚层制剂的生物学家都知道，通过快速上下聚焦，可以获得标本的3D形态。不幸的是，目前还没有一种机制可以在一张图像中同时获取这些深度信息。对于许多需要随时间跟踪移动细胞成分的生物学应用，DIC所显示的有限焦深意味着特征可能会迅速移出焦点，这严重限制了获得有用的、可解释的图像的能力。这种新型显微镜将被称为扩展焦深微分干涉对比（EDF-DIC）系统。它将基于一种新颖的“波前编码”光学技术，这种技术最近被用于将标准荧光显微镜转换为一种能够以比共聚焦或宽视场显微镜更快的速度从更大的深度获得清晰图像的显微镜。具体来说，EDF-DIC系统的创建只需要在标准DIC显微镜的成像路径中插入一个定制设计的相位板，然后对记录的图像应用一个非常快速的单次迭代数字滤波器。这也有优点，它可以很容易地适应几乎任何现有的显微镜设计。预计新的EDF-DIC显微镜将为生物学家提供一个有前途的新工具，用于广泛研究活细胞随时间的动态过程。举几个例子，新的EDF-DIC系统应该为生物学家提供以下能力：观察微管的三维空间分布和动态不稳定性（例如在出芽酵母中），或者在整个动物和植物王国的广泛应用中，在整个有丝分裂周期中一次性观察所有焦点染色体。

A new kind of differential interference contrast (DIC) microscope that will allow biologists to observe live-cell preparations with all the high-resolution advantages of conventional DIC, but with the added advantage that many more features throughout the specimen volume will appear in focus in a single image, will be developed. At present, high resolution DIC techniques are hampered by an inherent narrow depth-of-focus. Sometimes called "optical sectioning" (not to be confused with confocal microscopy), DIC tends to image the highest spatial frequencies (i.e. the finest detail) only from features that are nearest the plane of focus. Features above or below this plane rapidly lose contrast and appear as low spatial frequency blur. Any biologist who has used DIC for imaging thick preparations appreciates that by focusing up and down rapidly, one can get a sense of the 3D morphology of a specimen. Unfortunately, there is no current mechanism for acquiring this depth information all at one time in a single image. For many biological applications requiring tracking of moving cell components over time, the limited focal depth exhibited by DIC means that features may rapidly move out of focus, which severely limits the ability to obtain useful, interpretable images.The new microscope will be called an extended-depth-of-focus, differential interference contrast (EDF-DIC) system. It will be based on novel "wavefront coding" optical techniques that recently have been used to convert a standard fluorescence microscope into one that acquires sharp images from extended depths at much faster rates than are possible with confocal or widefield microscopes. Specifically, the creation of an EDF-DIC system requires simply inserting a custom-designed phase plate into the imaging path of a standard DIC microscope and then applying a very fast, single-iteration digital filter to the recorded image. This also has the advantage that it can be easily adapted to virtually any existing microscope design.It is anticipated that the new EDF-DIC microscope will supply biologists with a promising new tool for a wide range of investigations of the dynamical processes of living cells over time. To name a few examples, the new EDF-DIC system should provide biologists with the capability to perform the following: observe the 3D spatial distribution and dynamic instability of microtubules (e.g. in budding yeast) or view all the chromosomes in focus at once throughout the mitotic cycle in a broad range of applications throughout the animal and plant kingdoms.