Fluorophores and Optical Filters in Microscopy

We humans only have aspect to certain visual information when performing our jobs and making decisions. This might cause us to overlook the huge realms of possible experience available to us then, or the huge amount of data that is constantly available. Fortunately, this is something that technology does take full advantage of – whether it’s computer vision applications that rely on a combination of IR and visible light, or whether it’s fluorescence microscopy.

In each of these scenarios, the use of effective filters can drastically increase performance by helping machines to ‘see’ the most useful ranges of the electromagnetic spectrum. Below, we’ll take a look at how this crucial technology is saving lives in the field of fluorescence microscopy.

About Fluorescence Microscopy

In fluorescence microscopy, a type of fluorescent dye is applied which will then mark particular proteins, tissues, and cells with a ‘fluorescent label’. In other words, these become more visible under the microscope, making it very quick and easy to identify them and separate them out from other types of tissue.

Fluorophores work specifically by absorbing a particular wavelength – the ‘excitation range’ – and then re-emitting that light back, usually in a different wavelength. This is referred to as the ‘emission range’.

When you consider the way that humans see colors, it is because a specific wavelength of light is absorbed and only the remainder is reflected back. In this case, the energy is stored and then emitted in a different “format” that we can predict and observe.

There are many different uses for this kind of technology, from identifying cancerous tissues, to looking at systems such as the vascular system, to following the lifecycle of a medicine in pharmacokinetics.

Usually, the fluorophore will be excited by higher frequency illuminations – those being UV and violet lights. They will then emit the energy back at a somewhat lower frequency, which will normally be green, or NIR. The specific regions will depend on the precise type of fluorophore being used. The most efficient range of any fluorophore is the ‘peak excitation’ and ‘peak emission’ range.

As explained, this essentially allows a researcher or medical professional to better see specific types of material and tissue inside a human or animal body, or perhaps to better identify specific types of bacterium within a sample. That in turn makes it much easier to quickly identify the relevant information from an image.

If you have ever looked through a science journal, then likely you will have seen micrographs showing bright colors that indicate the presence of specific important elements. Many regular health checkups used in hospitals now rely on this technology, but it is also important in research, as well as in other activities such as gene doping and more.

Fluorochromes generally come in four different types: tiny conjucated organic molecules, fluorescent nano crystals, fluorescent proteins, and finally tandem fluorochromes – which utilize more than one type.

The Role of Optical Filters

So what is the role of the optical filter in all of this?

One type of filter used is the excitation filter. This is placed in the illumination path of the fluorescence microscope. In other words, it filters the light that reaches the fluorophore, thereby ensuring that only the peak excitation range is making it through. This means that less light will be reflected back and more will be absorbed by the target – reducing noise and unwanted information.

The minimum transmission of the filter is what determines the brightness and brilliance of the image, with an acceptable range being 40-85%. Above 85% is considered ideal. The bandwidth should be restricted specifically to the peak excitation range. This type of filter becomes extremely important if the researcher is using more than one dye in order to look at different types of tissue – this way they can cause only one type of fluorophore to respond.

The emission filter meanwhile is placed in the imaging path. The role of this filter is of course therefore to only allow light from the fluorophore to return, while blocking out the unwanted light.

This application sees the same acceptable ranges for the minimum transmission and bandwidth. There may be certain applications however where this filter might be removed in order to view the broader context.

The dichroic filter, also known as the beamsplitter, is a type of filter that is placed in between the excitation and emission filters. This filter is placed at a 45 degree angle with the role of reflecting the excitation signal toward the fluorophore – ensuring that it will be directly stimulated by the light. At the same time, it will also help to transmit the emission signal toward to detector. This works by refracting light with 95% reflection of the bandwidth of the excitation filter and 90% transmission of the bandwidth of the emission filter.

It’s very important not only to ensure that the right filters are used for the application in hand, but also that the filters be correctly matched in order to work efficiently together.

It is up to the client to search for the right filters based on the peak excitation and emission wavelengths of their chosen fluorophores. From there, they might ask for further guidance from the product providers.

Whichever you choose, these filters play a fundamentally important role in the biological sciences and as they improve, so too does the quality and usefulness of the information we can collect.

If you’re looking for any type of optical filter or coating then get in contact with Evaporated Coatings, Inc. today!

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