EVOS™ 40X Objective, fluorite, LWD

Objectives can hit the vessel holder when they are focused too high in the Z axis (up and down). This is a particularly a problem with the EVOS FL Auto Imaging System during instrument start-up, when the stage moves during system initiation, or when changing objectives. Coverslip-corrected objectives tend to be wider and flatter at the top of the barrel, which means that they are more likely to run into the edges of the vessel holder, particularly if you are imaging at the edges of the sample container. In those cases, use of that objective for those areas of the container may not be possible. If the objective if “jammed” by the vessel holder, then carefully unscrew the thumbscrews of the vessel holder and lift it straight off the stage, then move the objective downward in focus and toward the center of the stage. It is a good idea to have a shut-down procedure in your lab that includes moving the objectives to the lowest magnification and focusing downward with course focus prior to turning off the instrument for the day.
An objective can be damaged by scraping against the vessel holder. If this happens, take out the objective and examine it carefully for damage, particularly on the lens.

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If the lens is running up into a sample, this may be an issue with either focusing too quickly and missing the focal plane (if focusing manually) or a problem with the objective calibration (if using autofocus). It is a good idea to calibrate your objectives using the FL Auto calibration slide that comes with the system. Check to see if your objective is a long-working distance (LWD) or coverslip-corrected objective (CC). If coverslip-corrected, it is only for use with very short working distances for imaging through thin coverslips, but not through the slide or through plastics in microplates or culture dishes). If working with high magnification and oil immersion, by eye, move the objective upwards to touch the bottom of the sample and then only move slowly away from the sample for further focusing. An objective lens can be seriously damaged by scraping against samples. If this happens, check the objective lens for damage.

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Plan Achromat objectives are perfect for general applications where color and focus have standard correction; these are suitable for samples requiring low magnification (2x to 4x). Plan Achromat objectives are recommended for basic brightfield microscopy and simple fluorescence detection.
Plan Fluorite objectives provide the next level of improved resolution for brighter fluorescence signal and high contrast. These objectives are recommended for basic fluorescence imaging and brightfield microscopy at higher magnifications.
Plan Apochromat provides the highest level of resolution, fluorescence brightness, contrast, and chromatic correction. If you are imaging very small structures and require high contrast and brightness, the Plan Apochromat objectives are the best option.

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All the EVOS imaging systems are inverted microscopes. For CC objectives, the coverslip must be face down, facing the objectives as the lenses have a short working distance suitable only for thin glass or plastic coverslips. LWD objectives are designed for viewing from the bottom of microplates, petri dishes, or culture flasks; the longer working distances of the lenses in these objectives accommodate thicker materials such as the plastic bottoms of various vessels.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.