Fluorescence microscopy and light microscopy are specific imaging techniques used to observe cells or cellular components. Each of them has its situational strengths and weaknesses – areas in which the one is more effective than the other. In fact, fluorescence is really a specialized form of light microscopy that is able to produce better results in certain circumstances – though it would not be appropriate in others.
What is Fluorescence Microscopy?
Over the years, light microscopy has further advanced and more techniques and tools have been developed. Fluorescence microscopy is an excellent example. This specialization images cells or molecules using fluorescent dyes, called fluorophores, which have been injected or soaked into the sample under observation.
he light of the microscope excites these fluorophores, causing them to give off a light of their own. This new light, however, has less energy and is of a longer wavelength. Since it is this new light that actually provides the image, rather than the external light source, we say that fluorescent microscopy uses reflected light, rather than transmitted light.
Green fluorescent protein (GFP), for example, is excited using blue light and emits green light – which has a longer wavelength. Filters can then be used to remove the surrounding light radiation, isolating the fluorescent light for better observation.
Fluorescence microscopy is commonly used to image cell structures and other structural features. Using it, we can image particular cells amongst many others, check the viability of cells, and even observe genetic material (DNA and RNA).
What is Light Microscopy?
Light microscopy uses visible light and magnifying lenses to view very small objects to a degree of detail unavailable to the unaided eye. Light microscopes have been around for five hundred years (though they have improved a great deal over the years of course) and were used for the first observation of cells, leading to new branches of scientific endeavor.
Dyes are often used to increase clear visualization of cells or tissue. Commonly used ones – like hematoxylin and eosin – are used to color the main components, such as nuclei (which it colors violet) and the cytoplasm (which it colors pink). There are other, more specialized dyes as well, that are used when specific structures need to be visually identified or isolated from those around them.
Some methods have been developed to quickly examine certain cells in emergency situations, or when information is needed in a time sensitive manner for some other reason. In hospitals, for example, tissues are often frozen in carbon dioxide and then sectioned with a microtome. Light microscopy is then used to examine the sample. Such a quick turnaround means that doctors can even receive critical, new information while still operating on the subject from whom the sample was taken.
Comparing Light vs Fluorescence
Light microscopes use light in the 400-700nm range – the range through which light is visible to the human eye – but fluorescence microscopy uses much higher intensity light.
Because traditional light microscopy uses visible light, the resolution is more limited. Fluorescence microscopy, on the other hand, uses light produced by the fluorophores in the sample itself, which yields a much more detailed and reliable image.
Traditional light microscopes do still have a prominent place in microscopy however, both in combination with dyes and sometimes without. They are simpler to use in most cases, provide a quick turnaround for certain information, and are therefore very useful in clinical settings for such things as cervical smears or biopsy examination.
Fluorescence microscopy is used in conjunction with other kinds of light microscopy because it creates images from reflected light, rather than direct light. It can therefore be used with techniques like phase contrast microscopy.
Some light microscopy techniques can image both live and fixed samples, but sometimes damage can occur due to the intensity of the light, causing increased noise and a decrease in the clarity of the desired signal. On the other hand, fluorescence microscopy is limited by the tendency of cells to bleach under prolonged observation. If this occurs, the fluorescence will decrease and image quality diminishes.
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