Wednesday, November 13, 2013

Adapting to your Cells at a Moment’s Notice

Famed photographer and photojournalist Robert Capa once said, "If your pictures aren't good enough, you’re not close enough".  By this definition, digital fluorescence microscopy must yield amazing images since you can’t get much closer than a few hundred microns from your subject!  However, when it comes to the real world, simply being close to a subject obviously does not ensure acquisition of a great digital fluorescent image. There are a number of variables to consider; one of the most critical is the focal position, which determines whether the sample is in or out of focus. For manual fluorescent microscopes, focal position is simply obtained by manually adjusting fine and course focus knobs; for automated imagers, there are several ways that focus position can be automatically found. Two common methods employed are software autofocus and laser autofocus, with each having advantages and disadvantages.  

BioTek’s Cytation 3 utilizes software autofocus, which has some significant advantages over laser autofocus when it comes to obtaining a great image of a sample. Aside from providing cost savings since the method uses software algorithms rather than relying on expensive hardware like laser autofocus, the big benefit is that software autofocus can dynamically adapt to shifting biology.  Laser autofocus uses a laser to find the bottom of the well, and then uses an offset value from the bottom to set the imaging plane of focus for samples; the same offset value is applied to all the wells in a microplate. This means that the focal plane is not based on the actual samples in each well, but on the physical wells themselves.  At higher magnifications, you run the risk of missing the real biology if the sample shifts in height in the z-axis. In contrast, software autofocus tracks the actual sample as it changes in the z-axis, ensuring that an image in the proper focal position is acquired. Some examples where this becomes critical are assays looking at apoptosis, loosely adherent cells, live-cells, spheroids, or organisms such as C. Elegans or Zebrafish embryos. Below is an illustration of this concept as it relates to an Apoptosis assay where the blue channel (DAPI-stained nuclei) is used for focus. 

Laser autofocus

As depicted in the diagram above, as a cell moves from a normal state to an apoptotic state, it begins to detach from the well bottom and ball up, which causes it to move upward in the Z-direction. With software autofocus, the objective adjusts as the cell moves upward in the z-axis, so the image that is acquired remains focused on the true biology. In contrast, laser autofocus uses the same offset from the well bottom which results in a focal plane below the apoptotic cell. 

Physicist Stephen Hawking said "Intelligence is the ability to adapt to change", which seems fitting with the above example. In microscopy, despite an effort to hold variables constant in experiments, there are always changes that occur that can impact an automated fluorescent imager's ability to acquire a good image. After all the work you put into sample preparation, it's nice to know the Cytation 3 is intelligent enough to acquire images of what you expect, even if your cells decide to throw a party and shift around a bit in your microplate.

By: BioTek Instruments, Caleb Foster, Product Manager, Development

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