Determine the best exposure time for your sample
Exposure time is the length of time the camera collects light from your sample, and there are tradeoffs between exposure time, image brightness, and phototoxicity, especially for live-cell imaging.
Now that you’re taking the image
- Use an exposure time that uses as much of the dynamic range of the camera as possible without saturating any of the pixels
- In cases where you will be observing a group of samples, the exposure time you select should be based on the sample with the brightest expected signal
- For live-cell imaging, you may want to sacrifice signal intensity (i.e., use shorter exposure times) in order to maintain cell health
Determining the optimal exposure time can take some trial and error
When you acquire a digital image, one of the parameters you can adjust is the exposure time. The exposure time is how long the camera will be exposed to the light (photons) emitted from your sample. The longer the exposure time, the more photons the detector will receive, resulting in increased pixel intensity and a “brighter” image. Ideally, you want to use an exposure time that takes advantage of the full dynamic range of the camera you are using.
Determining the optimal exposure time can take some trial and error, especially if you have samples that have uneven signal due to uneven staining, or varied reactions to stimuli. It is very important to take some time to determine the best exposure time for your sample. This is one thing that you cannot alter afterwards (unless you take all of the images again).
In order to determine what exposure time you should use, you first need to ask yourself, “What do I want to see?” Here are some examples.
“I want it to be as pretty as possible”
If you are only interested in getting a beautiful picture, you can set the exposure time based on a single sample—the one you want a pretty picture of. The exposure time that will result in the most beautiful image is the one that utilizes the full dynamic range of your camera.
The relationship between dynamic range and exposure time
Ideally, when you acquire an image you want to use the longest possible exposure time, without saturating any of the pixels. This means that for the brightest pixels, the intensity is just below saturation. Many software programs use a red indicator for a saturated pixel. Some software programs also display pixel intensities in a histogram. The histogram shows the range of intensities in the image collected.
Figure 1. Your imaging software program can help you choose a good exposure time by visually flagging saturated pixels and/or by presenting a histogram of intensity values for each pixel in the image. Ideally, you want to use the entire dynamic range of your camera without saturating any pixels.
This sample was imaged using three different exposure times. The camera used has a dynamic range of 0–4,095 (0 = no intensity, 4,095 = the highest pixel intensity value). In the 40 ms exposure (left), the image is dim and the entire dynamic range was not used; the highest pixel intensity is around 2,000. The image on the right is very bright, but some pixels are saturated (indicated in red). The histogram also shows that some pixel intensities are off-scale. The exposure time for this image is too long. The image in the middle is ideal: the intensity values span the entire dynamic range, and there are no saturated pixels.
“I want to compare the intensities between a treated and a control sample”
To do this, determine which sample should have the brightest expected signal and then set the exposure time based on that sample. In some cases the control sample will be brighter than the treated sample (e.g., loss of signal with treatment); in other cases the treated sample will have a brighter signal (e.g., increase in signal with treatment). Whether the control sample is brighter or dimmer than the treated sample will depend on the assay you have chosen. Once you've chosen your control, choose the longest possible exposure time without saturating any of the pixels, to use the full dynamic range of the camera (see Figure 1).
Figure 2. Example of loss of signal upon treatment. Untreated HeLa cells (left) exhibit bright fluorescence intensity when stained with mitochondrial dye TMRM. Cells treated with CCCP (carbonyl cyanide 3-chlorophenylhydrazone, which uncouples oxidative phosphorylation resulting in loss of mitochondrial membrane potential) exhibit dim fluorescence signal (right). In cases like these, use the untreated sample to set exposure time.
Figure 3. Example of increase in signal upon treatment. Untreated HeLa cells (left) stained with NucGreen Dead reagent are dim. Cells treated with a drug to induce cell death, staurosporine (right) are bright green. In cases like these, use the treated sample to set exposure time.
Set the exposure time as described in the previous example (taking a pretty picture) using the sample that has the brightest expected signal. Remember to use the entire dynamic range of your camera without saturating any of the pixels, especially if you are planning on gathering any quantitative data from your images. Remember to take images for both the control and treated samples using the same exposure time. Otherwise you cannot compare their intensities during analysis.
“I’m imaging in live cells”
There are some special exposure time considerations for imaging live cells, since light used to excite the fluorophores in your sample can hurt your cells. When imaging live cells, sometimes you’ll want to sacrifice signal intensity in order to maintain cell health. In this case, you would not set the exposure time to use the entire dynamic range of the camera. You may want to set the exposure time just long enough to be able to see what you need to see. See the section on live-cell imaging for more on this.