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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.


Related in: MedlinePlus

Flash illumination during global exposure time window when all pixels are opened thus avoiding the rolling shutter effect.
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f8: Flash illumination during global exposure time window when all pixels are opened thus avoiding the rolling shutter effect.

Mentions: The high-speed microscopy solution, which is based on imaging continuously moving objects, is implemented on an inverse research microscope (Nikon Ti-E). We use it mainly in conjunction with phase contrast objectives with 4x, 10x and 20x optical magnification. Higher magnifications and other contrast modalities like differential interference contrast (DIC) can also be used. The core component of the high-speed microscopy solution developed is a TANGO 4 stage controller from Märzhäuser Wetzlar. It ensures hardware-based synchronization of the components and is operated with a firmware that has been enhanced to provide all necessary functionality. It controls both the SCANplus IM 130 × 85 (also Märzhäuser Wetzlar) scanning stage which has an integrated measuring system and the 300 μm piezo z-stage via analogue output to the piezo controller LC.400 (both from nPoint). It also creates the TTL trigger impulses whose purpose is to synchronize the stage position with the high-speed camera pco.edge 5.5 (pco) and the flash controller RT220F-20 (Gardasoft) which controls an LED based transmitted light illumination (LED 100 from Märzhäuser). It is equipped with a Cree XR-E high-power LED. Flash durations as short as 1 μs can be generated when the illumination source is attached to the Gardasoft controller. To generate enough photons for sufficient exposure, the LED brightness can be increased by driving it with more than the current rating for short pulses (up to tenfold overdriving). In phase contrast microscopy, a significant proportion of the light is blocked by the condenser annulus. In addition to a bright illumination source, a sensitive camera is therefore crucial. The sCMOS camera used, combines sensitivity with fast frame readouts and therefore ideally matches the requirements for the flash based high-speed microscopy solution. It is operated in the rolling shutter mode which offers the lowest readout noise. The rolling shutter effect is avoided16 by shifting the flash illumination to the global exposure time window when all pixel rows are active (10 ms after the exposure trigger signal) (Fig. 8).


High-speed microscopy of continuously moving cell culture vessels
Flash illumination during global exposure time window when all pixels are opened thus avoiding the rolling shutter effect.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f8: Flash illumination during global exposure time window when all pixels are opened thus avoiding the rolling shutter effect.
Mentions: The high-speed microscopy solution, which is based on imaging continuously moving objects, is implemented on an inverse research microscope (Nikon Ti-E). We use it mainly in conjunction with phase contrast objectives with 4x, 10x and 20x optical magnification. Higher magnifications and other contrast modalities like differential interference contrast (DIC) can also be used. The core component of the high-speed microscopy solution developed is a TANGO 4 stage controller from Märzhäuser Wetzlar. It ensures hardware-based synchronization of the components and is operated with a firmware that has been enhanced to provide all necessary functionality. It controls both the SCANplus IM 130 × 85 (also Märzhäuser Wetzlar) scanning stage which has an integrated measuring system and the 300 μm piezo z-stage via analogue output to the piezo controller LC.400 (both from nPoint). It also creates the TTL trigger impulses whose purpose is to synchronize the stage position with the high-speed camera pco.edge 5.5 (pco) and the flash controller RT220F-20 (Gardasoft) which controls an LED based transmitted light illumination (LED 100 from Märzhäuser). It is equipped with a Cree XR-E high-power LED. Flash durations as short as 1 μs can be generated when the illumination source is attached to the Gardasoft controller. To generate enough photons for sufficient exposure, the LED brightness can be increased by driving it with more than the current rating for short pulses (up to tenfold overdriving). In phase contrast microscopy, a significant proportion of the light is blocked by the condenser annulus. In addition to a bright illumination source, a sensitive camera is therefore crucial. The sCMOS camera used, combines sensitivity with fast frame readouts and therefore ideally matches the requirements for the flash based high-speed microscopy solution. It is operated in the rolling shutter mode which offers the lowest readout noise. The rolling shutter effect is avoided16 by shifting the flash illumination to the global exposure time window when all pixel rows are active (10 ms after the exposure trigger signal) (Fig. 8).

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.


Related in: MedlinePlus