Live Cell Imaging: Tips for Success

The number of researchers performing live cell imaging (also called time-lapse imaging) has increased in recent years. The advantage of this imaging is that it enables observation without fixing target cells in place so that changes in the structure of the cells themselves can be observed. On the other hand, many researchers tell that quite a few observations have resulted in failures due to this imaging having difficulty in targeting live cells and requiring more time than normal observation.

This section introduces the three most common failures along with suggested countermeasures to help improve success rates of live cell imaging.

Common failure #1: The cell is damaged.

In the course of or after a live cell imaging experiment, you may find that the cells are immobile, dead, or otherwise damaged. There are several causes of these problems:

Cause 1: Cells are damaged by phototoxicity.

During fluorescence observations, the shorter the wavelength of the excitation light and the more intense the light, the greater the cells are damaged. This is called phototoxicity*1, which may not only weaken or kill cells but may also affect them in unexpected ways.

Countermeasures

  1. *1 Phototoxicity: This normally refers to the damage caused by the exposure to light, but it also refers to the phenomenon in which a cell subjected to fluorescence generates reactive oxygen, which in turn damages the surrounding cells.
  2. *2 Gain: This is a function that electrically amplifies the signal received by the camera. The higher the degree of amplification (the higher the gain), the brighter the display. There is no change to the number of pixels, but noise occurs.
  3. *3 Binning: This is a function that virtually joins together the surrounding pixels to amplify the received light signal per unit pixel. Less noise occurs compared to when the gain is increased, but the number of pixels is decreased.

Cause 2: The appropriate environment is not maintained.

The majority of cells prefer an environment that mimics standard physiological conditions. Human cells, for example, often require that a temperature of 37°C, a carbon dioxide concentration of 5%, and a humidity of 95% or higher be maintained by the chamber or incubator used for their cultivation. If this environment is not supplied in a stable manner, the cell will weaken and it will be difficult to obtain accurate data.

Countermeasures

Cause 3: Contamination within the sample

Some cells may be affected by germs and contamination affixed to the container. Also, mycoplasma contamination*4 and cross-culture contamination*5 can be caused by the pipette and other equipment that is used.

Countermeasures
  1. *4 Mycoplasma contamination: This refers to contamination by mycoplasma. It is one of the most common phenomena that cause cell strain contamination.
  2. *5 Cross-culture contamination: This refers to the erroneous mixing of other cells into the cell being observed. Most commonly, the other cells are transferred by the equipment.

Common failure #2: The cell drifts out of focus.

Another common failure is discovering that the specimen went out of focus partway through the experiment. This could happen for a couple of reasons:

Cause 1: Temperature drift*7 causes the cell to move or the settings to change.

If you configure the image capture settings immediately after turning on the device, the focus may be shifted while you configure the settings. This is because, for some length of time after the power is turned on, the culture fluid within the container is circulated by convection, which moves the cells. Another reason for the image going out-of-focus is that the observation equipment slightly expands due to changes in the temperature. During observation of either suspended cells or adherent cells, pay attention to the temperature drift from the time when settings are configured to the time when images are actually captured.

Countermeasures
  1. *7 Temperature drift: A phenomenon in which temperature fluctuations cause the suspended cell to move or in which the expansion of the equipment itself displaces the in-focus position.

Cause 2: Changes in cell morphology

Live cells move up and down (in the Z direction) especially during cell division. As a result, an image will go out of focus if you are performing one-shot image capturing at high magnification at the instant of cell division.

Countermeasures

With live cell imaging, you can accurately prevent failures caused by the image going out of focus by using the prevention methods shown above. In this situation, the trick is to only set the auto focus and Z-stack setting range*9 to a range as large as necessary to minimize the length of time that the target is subjected to the excitation light.

  1. *8 Depth of field: This indicates the area within the image that is in focus. Generally speaking, the larger the NA, the shallower the depth of field.
  2. *9 Z-stack setting range: Z-stack is a function for capturing multiple images of the data at different Z-positions. You can obtain more information by capturing images over a wide area and with high detail, but this also damages the cell by a matching amount, so it is best to set the number of images captured to as few as possible.

Common failure #3: The cell leaves the field of view.

Sometimes when you view the data after an experiment, you see that the cell left the field of view partway through the experiment. If the cell moves out of the field of view before the target phenomenon is complete, the entire experiment must be repeated.

Cause: The cell moves.

The longer the shutter is kept open, the greater the chance that the cell will move out of the field of view. So long as the cell is alive, this cell movement cannot be avoided. In a variety of papers, you may have seen data that shows the detailed conditions of cells. In many cases, such data is obtained by repeated image capturing performed at high magnifications, which requires a lot of time and effort.

To reliably prevent failures, the ideal method is to capture images of multiple locations at the same time or to track the moving cell while capturing its image.

Countermeasures

You can accurately capture the cells without missing any of them by observing a wide field of view with a low magnification. However, it is difficult to observe the details of a cell with a low magnification. What’s more, note that the longer the exposure time becomes, the greater the chance of the image becoming blurry due to the target moving during image capturing.*10

  1. *10 Exposure time and image blurring: When you reduce the intensity of the excitation light, the exposure time becomes longer. If the cell moves while the shutter is open, the image will be blurry. The effective way to prevent this problem is to use a camera with a high sensitivity.

Obtaining 99 times the live cell imaging experiment efficiency

Conventionally, to achieve better research results, it has been necessary to repeat experiments to obtain more information. However, with live cell imaging, even a single experiment takes a lot of time, so repeating experiments requires a huge amount of time. Ideally, a single experiment should provide more information.

KEYENCE’s All-in-One Fluorescence Microscope BZ-X800 Series solves the problem with live cell imaging of “the amount of information obtained from a single experiment being limited.”It is possible to obtain the data of up to 99 locations at the same time using completely different conditions such as magnifications of 2x, 4x, or 20x. Still, the BZ-X800 Series can be configured easily and thus reduces the time conventionally required to set up the system and for users to become proficient with using the system.