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High-speed microscopy of continuously moving cell culture vessels

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Compensation of the delay between required and actual position.The reaction time for the piezo z-stage (nPoint 300 μm) to reach the required position is about 7 ms for a random height profile. By shifting the required positions by the product of the reaction time and the scan velocity, the focus error (required z-position minus actual z-position) can be greatly reduced below the depth of field.
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f6: Compensation of the delay between required and actual position.The reaction time for the piezo z-stage (nPoint 300 μm) to reach the required position is about 7 ms for a random height profile. By shifting the required positions by the product of the reaction time and the scan velocity, the focus error (required z-position minus actual z-position) can be greatly reduced below the depth of field.

Mentions: For fast scanning applications of microtiter plates, it proved useful to carry out a quick, continuous focus scan at the stage’s maximum velocity using the OCT-based focus sensor before the actual imaging scan. In this way the resulting focus values can be filtered, especially at the edges of or between the wells where no valid measurement data is obtained. The high measurement rate permits thousands of focus sampling points to be measured. Averaging these data points attenuates the effect of outliers and results in robust focus values for each field of view. Additionally, the focus correction procedure can be optimized when all focus values are known before the continuous imaging scan is conducted. In our solution we use a highly dynamic piezo z-stage to synchronize the z-axis position with the movement in scan direction. The misregistration that would occur due to the fast lateral object movement and the lapse of time that it takes for the piezo to reach its required positon can be easily compensated by relocating the focal plane positions relative to the scan direction (Fig. 6).


High-speed microscopy of continuously moving cell culture vessels
Compensation of the delay between required and actual position.The reaction time for the piezo z-stage (nPoint 300 μm) to reach the required position is about 7 ms for a random height profile. By shifting the required positions by the product of the reaction time and the scan velocity, the focus error (required z-position minus actual z-position) can be greatly reduced below the depth of field.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036042&req=5

f6: Compensation of the delay between required and actual position.The reaction time for the piezo z-stage (nPoint 300 μm) to reach the required position is about 7 ms for a random height profile. By shifting the required positions by the product of the reaction time and the scan velocity, the focus error (required z-position minus actual z-position) can be greatly reduced below the depth of field.
Mentions: For fast scanning applications of microtiter plates, it proved useful to carry out a quick, continuous focus scan at the stage’s maximum velocity using the OCT-based focus sensor before the actual imaging scan. In this way the resulting focus values can be filtered, especially at the edges of or between the wells where no valid measurement data is obtained. The high measurement rate permits thousands of focus sampling points to be measured. Averaging these data points attenuates the effect of outliers and results in robust focus values for each field of view. Additionally, the focus correction procedure can be optimized when all focus values are known before the continuous imaging scan is conducted. In our solution we use a highly dynamic piezo z-stage to synchronize the z-axis position with the movement in scan direction. The misregistration that would occur due to the fast lateral object movement and the lapse of time that it takes for the piezo to reach its required positon can be easily compensated by relocating the focal plane positions relative to the scan direction (Fig. 6).

View Article: PubMed Central - PubMed

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.

No MeSH data available.