The size of CFP-expressing S. flexneri infection foci formed in HT-29 cell monolayers was determined in 384-well plate format as described previously [20] (link). After fixation and DAPI staining, the plates were imaged using a TE2000 automated microscope (Nikon) equipped with a motorized stage (Prior), motorized filter wheels (Sutter Instrument, Inc.) and a 10× objective (Nikon) mounted on a piezo focus drive system (Physik Instrumente). Image acquisition was conducted using the Metamorph 7.1 Software (Molecular Devices, Inc.). For image analysis, high intensity pixels corresponding to the bacteria were selected against the low intensity pixels corresponding to the background levels by using the threshold function of the MetaMorph 7.1 imaging software. Selected pixels were clustered by proximity using the “close” morphological filter in order to create a single object that represented a given focus of infection. The integrated morphometry analysis (IMA) module was used to determine the area of each infection focus in a given image. Image analyses were conducted on at least 100 infection foci in three independent experiments.
Piezo focus drive system
Manufactured by Physik Instrumente
The Piezo focus drive system is a high-precision positioning device designed for laboratory applications. It utilizes piezoelectric actuators to provide accurate and repeatable focus control. The core function of this system is to enable precise positioning and adjustment of the focal point or area of interest within an experimental setup.
Automatically generated - may contain errors
2 protocols using piezo focus drive system
1
Quantifying Shigella Infection Foci in HT-29 Cells
2
Automated High-Throughput Microscopy Imaging
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