Abstract
We report a method of high-speed phase contrast and bright field microscopy which permits large cell culture vessels to be scanned at much higher speed (up to 30 times faster) than when conventional methods are used without compromising image quality. The object under investigation moves continuously and is captured using a flash illumination which creates an exposure time short enough to prevent motion blur. During the scan the object always stays in focus due to a novel hardware-autofocus system.
Highlights
Due to the microscope’s small field of view an enormous number of single images have to be taken in order to image large objects, like whole microtiter plates (MTPs)
To keep the frame rate high most of the time, the number of stops is reduced to a minimum by scanning in a meander-like motion along the whole MTP instead of imaging it well by well
For a whole microtiter plate, the scanning time is reduced to approximately 10%, 4% or 3% when using a 4x, 10x or 20x objective compared to the conventional stop-and-go scanning process implemented on the same microscope (Nikon Ti-E)
Summary
Due to the microscope’s small field of view (depending mainly on the objective lens’s magnification) an enormous number of single images have to be taken in order to image large objects, like whole microtiter plates (MTPs). Conventional automated microscopes use an incremental scanning routine to perform this task. This involves repeatedly positioning the object, adjusting the focus and capturing a segment of the stationary object. The imaging rate can be increased dramatically by omitting to stop for each acquisition and imaging the object during a continuous movement. This acquisition method, termed continuous scanning, can be used in conjunction with line and area scan cameras and was first reported in the literature a few decades ago[1,2]. The main challenges related to this acquisition concept are the need to prevent motion blur and to ensure that the object is kept continually in focus
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