A fluorescence microscope is a type of optical microscope. When the specimen being observed is transparent or its internal structures cannot be clearly distinguished by color, a fluorescence microscope is an excellent choice, as it can overcome the limitations of ordinary microscopes in observing transparent materials. The principle behind a fluorescence microscope is to illuminate a specimen stained with a fluorescent dye using light of short wavelengths, which excites the dye molecules and causes them to emit long-wavelength fluorescence. This allows observers to examine the specimen's internal structure. In a fluorescence microscope, it is essential to select specific wavelengths of excitation light from the illumination source to generate fluorescence. Subsequently, the fluorescence must be separated from the mixed light consisting of both excitation and emitted fluorescence so that it can be observed clearly. Therefore, filter systems that selectively transmit specific wavelengths play an extremely important role in this process. Fluorescence microscopes are widely used in fields such as biology and medicine.
A fluorescence microscope consists of the following basic components:
a. Light source: Typically a xenon arc lamp or a mercury lamp, but high-power LEDs have also been used in recent years.
b. Filter (incident light): This filter reduces the wavelength of the incident light to leave only the wavelength needed to excite the sample; interestingly, it’s referred to as an excitation filter.
c. Two-way dichroic mirror or reflector: Reflects the excitation light onto the sample while simultaneously transmitting only the emission light originating from the sample to the detector (as shown in the figure below).
d. Emission filter: It allows only the emission wavelengths originating from the sample to pass through, while blocking all light that has passed through the excitation filter. As you might expect, it is referred to as an emission filter.
e. CCD Camera: If the emitted light cannot be detected, the camera is of no use at all. For fluorescence imaging, the detector is typically a CCD camera, which is usually connected to a computer screen and can display the image for you.
A dichroic beam splitter allows light with longer wavelengths to pass through the filter while reflecting light with shorter wavelengths.
Fluorescence Microscope Classification:
Fluorescence microscopes are generally divided into two types: transmitted-light and reflected-light.
a. Transmission Type: The excitation light originates from beneath the specimen, and the condenser is a dark-field condenser, which prevents the excitation light from entering the objective lens while allowing the fluorescence to pass into it. Under low magnification, the image appears bright; under high magnification, it becomes dark. This type of illumination is relatively difficult to adjust when using oil immersion or centering the microscope, especially in determining the appropriate illumination range at low magnifications. However, it can produce a very dark background for the specimen. The transmission-type illumination method is not suitable for specimens that are not transparent.
b. Epi-illumination: While transmitted-light microscopy has largely been phased out, most modern fluorescence microscopes now employ epi-illumination. In this configuration, the light source is positioned above the specimen, and a beam splitter is incorporated into the optical path, making it suitable for both transparent and opaque specimens. Since the objective lens functions as a condenser, this setup not only simplifies operation but also ensures uniform illumination across the entire field of view—from low to high magnification.
