The problem

Seeing fluorescence where you don’t want it

Background fluorescence, sometimes referred to as noise, is the fluorescent signal that you see but you don’t want. Background fluorescence comes from a variety of sources, but it can be separated into two main categories.

Sources of background fluorescence

  1. Background fluorescence that is due to the instrument setup and imaging parameters—for example, light from the excitation source, camera noise, and ambient light
  2. Background fluorescence that is due to autofluorescence of samples, vessels, and imaging media, or the fluorescence resulting from fluorophores not bound to specific targets

Noise from instruments and light sources can be complicated to define and remove, but it tends to remain fairly constant from day to day. Background arising from autofluorescence or from nonspecific staining can be more easily addressed and remedied—at least in theory! Because there can be so many different ways that your experimental design contributes to background fluorescence, you likely have multiple options to reduce it. Some of the sources of background fluorescence in your fluorescent imaging experiment are:

  • Fluorophores: unbound dye or nonspecific binding of dye in the sample
  • The sample itself could autofluoresce and contribute to background fluorescen
  • Some drugs and inducing agents are (or can become) fluorescent
  • Fluorescence from the vessel or substrate upon which the cells are grown
  • The medium used during imaging can contribute to background fluorescence

diagram of a cell culture dish with the vessel, reagent, media, and sample labeled

Figure 1. Background fluorescence can arise from many sources in your experiment.

What you can do to decrease background fluorescence

Getting the best data from your fluorescent image requires you to do everything you can to make the difference between your signal (what you want to see fluorescently labeled) and the background (what you don’t want to see fluorescently labeled) as big as possible. That difference is sometimes even expressed as a ratio when you report the quantitative results: ∆F/F, where ∆F is the [signal – background] and F is the background. Of course you want to use a very bright fluorescent dye, but for the best results you also need to have as little background or off-target fluorescence as possible. In general, decreasing background will result in greater image contrast and make it easier to see the desired signal. 


Background fluorescence due to unbound dye or nonspecific binding of dye in the sample

  • Wash: after labeling your sample with the fluorescent dye or stain, wash the sample 2–3 times with a buffered saline solution such as PBS. This will remove unbound fluorophores and help to decrease background
  • Optimize fluorescent dye concentration: label your sample with a titration of the fluorescent dye you are using (see example). Use concentrations that are below, at, and above the suggested concentration. Using the optimal dye concentration for your experimental conditions will ensure you get bright, specific signal with minimal excess background signal.

 

A four-panel image series showing how increasing dye concentration in a cell staining experiment can lead to increased background fluorescence.

Figure 2. Different dye concentrations under identical experimental conditions can produce significantly different amounts of background.


Try labeling with a dye that matches a different filter

If your sample itself exhibits fluorescence (autofluorescence) that obscures your signal, try labeling with a dye that matches a different filter set (for example, switch from a fluorophore that uses the green filter set to one that uses the red or far-red filter set). 


Measure the fluorescence intensity from a well that contains only your cells and the drug or treatment

It can be helpful to measure the fluorescence intensity from a well that contains only your cells and the drug or treatment (no fluorescent label) to determine if your treatment is contributing to high background during live-cell imaging. If it is, you may be able to subtract this background value from your results. Alternatively, you may have to choose another treatment or dye that uses a different channel.


Check your media

The medium that your sample is in can also affect your results. This is true for fixed samples as well as for live-cell imaging. For fixed-cell imaging, most commonly using immunolabeling, you may want to explore mounting media. For live-cell imaging, you will get better signal-to-background if you image in an optically clear buffered saline solution instead of a complete medium that contains proteins and vitamins that fluoresce themselves (see live-cell imaging). 


Check your vessel

The vessel that you image in can also contribute to background fluorescence. For example, plastic-bottom dishes typically used in cell culture can fluoresce very brightly. If you have excess background fluorescence and are imaging in plastic-bottom vessels, try switching to a glass-bottom vessel. 

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