When reading through the manual of automated cell counter you are using, you will most likely come across the terms such as wavelengths, filter set, excitation and emission.
Understanding all of the terms and values might seem overwhelming at first, and we hope this post helps you understand fluorescence filters better.
Fluorescence microscopy and cell counters share similar structure as fluorescence filters. The excitation filter, emission filter, and dichroic filter (mirror) are three important filters that allow spectators to see fluorescently dyed objects. Only certain wavelengths of light can pass through these filters.
Below is the illustration of the fluorescence microscopy structure.
In order to observe an object under fluorescence device, the object must be excited first. The light source brings up the object to its excited state with light passing through a filter called excitation filter. This lets certain wavelengths of light through that are within the excitation range set. While light source with desired wavelengths passes through the excitation filter and illuminates the object, those that do not fall within the range are filtered out.
Once the object has been excited, it then emits light. This light then passes through an emission filter. Similar to excitation filter, an emission filter allows the light that the fluorescent object emits so that the observer can see the image. This filter optimizes the image quality by blocking other unwanted light wavelengths. As the longer wavelength of light is emitted from the object, the light with shorter wavelength is blocked to prevent any interference.
Both excitation and emission lights are directed by a dichroic filter to the desired location where excitation light is directed to the object and emission light to the camera (cell counter) or to the observer’s eye (microscopy). To define the meaning of the term, dichroism is referred to a property of substance that involves two different lights by reflecting one light and absorbing the other light. This filter is set at 45 degrees to guide both excitation light and emission light to their final locations, allowing desired wavelengths to be reflected or pass through.
Below are excitation / emission wavelengths of mostly used fluorescence dyes.
Acridine Orange (AO) | DNA : 502 / 525 nm RNA : 460 / 650 nm |
Propidium Iodide (PI) | w/ DNA : 535 / 617 nm w/o DNA : 493 / 636 nm |
DAPI | w/ DNA : 358 / 461 nm |
