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Definite Focus from Carl Zeiss Facilitates Research of Cell Cultures Using a Microscope
Sponsored,vendor-submitted protocol   Published in BioTechniques Protocol Guide 2009 (p.41) DOI: 10.2144/000113026

With Definite Focus, Carl Zeiss offers a system for the Axio Observer.Z1 that can be used not only for the research of cell cultures in many biomedical applications, for example, cell biology, pharmacology, molecular genetics, developmental biology, and the neurosciences, but also in basic research. The system has been specially designed for fluorescence applications and time-lapse experiments under live cell conditions. Definite Focus effectively counteracts drifting in the Z-direction. The microscopic image always remains in focus. Furthermore, preparation and waiting times in time-lapse experiments are markedly reduced.


Definite Focus is coupled directly to the nosepiece carrier and directs infrared light of wavelength 835 nm into the beam path of the Axio Observer.Z1 inverted research microscope. To measure the distance between the objective and the specimen, a grid is projected onto the bottom of the culture vessel, and the reflection of this projection is analyzed. Possible deviations from the nominal value are determined and corrected quickly and very precisely via the Z-drive of the Axio Observer in the case of drifting. Even with high-magnification objectives, the system can be used without limitations for all contrasting techniques.

Definite Focus offers increased experimental reliability. Experiments that can no longer be evaluated due to drifting in the Z-direction are a thing of the past. Furthermore, incubation conditions can now be better structured according to needs. With stage incubation using the PM S1 incubator, for example, the user can quickly change the temperature as required during the experiment. Typical examples of dynamic temperature experiments include the analysis of protein folding mutants or heatshock experiments. Until now, performance of the relevant experiments had not been possible due to pronounced focus fluctuations.

Definite Focus can be operated directly via the TFT touchscreen display of Axio Observer.Z1. Convenient additional functions such as the storage of two nominal values and the possibility of using the system as a “focus finder” after replacement of the culture vessel are integrated into the system. Furthermore, Definite Focus is fully compatible with the Axio Vision and LSM software from Carl Zeiss. The module supports all Axio Observer options such as TIRF, LSM, or multiphoton microscopy. It works with all standard culture vessels, including multiwell dishes with glass or plastic bottoms. The supplied hardware comprises a sensor module on the nosepiece carrier, a beam combiner below the nosepiece and a Definite Focus controller.

Figure 1. Definite Focus. (Click to enlarge)


Address correspondence to Carl Zeiss MicroImaging GmbH, 07740 Jena, Germany. BioSciences, Göttingen Location. Tel.: +49 551 5060 660; Fax: +49 551 5060 464. [email protected],

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