Live cell microscopy(live cell imaging) is a very popular method in the field of cell biology. Obtaining correct and high quality information is subject to several specific factors that can significantly affect live cell experiments. For live cell imaging, we recommend a spinning disk confocal microscope ( SDCM ) or a widefield fluorescence microscope with deconvolution software.

In live cellimaging, great care must be taken to reduce the photo-toxic effects of light(bleaching). The illumination power density at the sample plane, compatible with the life of the cells under investigation, ranges from mW/cm2(widefield fluorescence microscopes) to W/cm2( spinning discconfocal microscopes SDCM ). At high light power densities, intense bleaching of fluorophores occurs, generating free radicals and other reaction products that destroy surrounding molecules and alter the environment outside and inside the cells.

Cell cultures and tissues must be maintained under physiological conditions throughout the experiment. Factors such as temperature, humidity, medium composition and pH are very important to obtain meaningful information about the processes under investigation. Depending on the origin of the cultured cells, the above factors need to be set to specific values and kept stable.

Mitochondria in glioma cells. iXon Ultra, 60x, NA: 1.49. A total of 6,478 1Mpx images were taken! Voxel 0.201*0.201*0.293 um, X/Y:1024*1024 pixels, Z:41, T:79, 2 channels. The data was taken during a short (13 min) time-lapse experiment during the demonstration of the Andor Dragonfly system. Samples were provided by Dr. Veronika Huntošová from UPJS/CIB, Košice, Slovakia.

Essential equipment for live cell imaging microscope is listed in the table:

Equipment Contribution Reason Manufacturer
Confocal microscope (SDCM)
 Low bleaching
High scanning speed		 Pinhole kit disk ensures confocality
Illuminates the entire field of view at once
Use of cameras for emission signal detection		 Andor
Deconvolution
 3D reconstruction of widefield fluorescence microscope images
Higher resolution		 Algorithms for removing signal from out-of-focus planes
Signal reorganization according to the parameters of the microscope used		 Bitplane
Cameras
 High sensitivity
High speed
High resolution		 Sophisticated sensor architecture (sCMOS/EMCCD)
Fast signal readout		 Andor
Incubator Temperature, humidity, pH control Isolation of the sample from the surrounding environment and precise control of the conditions during the experiment Okolab
Motorized stage
 High resolution
Repeatability		 Precision compontents
Piezo motors for maximum resolution		 PI
Visualization and analysis
 3D visualization
Analysis of acquired information
Comparison of results		 Colocalization (2D/3D colocalization)
Tracking (2D/3D tracking)
Statistical data acquisition
Creation of graphs		 Bitplane

The Andor Dragonfly confocal microscope meets all the requirements for live cellimaging:

Scanning speed

Sensitivity

Environmental control

Microscope

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