A fluorescence microscope is basically a conventional light microscope with added features and components that extend its capabilities. A conventional microscope uses light to illuminate the sample and produce a magnified image of the sample. A fluorescence microscope uses a much higher intensity light to illuminate the sample. This light excites fluorescence species in the sample, which then emit light of a longer wavelength.

A fluorescent microscope also produces a magnified image of the sample, but the image is based on the second light source — the light emanating from the fluorescent species — rather than from the light originally used to illuminate, and excite, the sample.

The Basic Requirements of Fluorescence Microscope Optics

Nearly all fluorescence microscopes use the objective lens to perform two functions: The fluorescence microscope focus the illumination (excitation) light on the sample. In order to excite fluorescent species in a sample, the optics of a fluorescent microscope must focus the illumination (excitation) light on the sample to a greater extent than is achieved using the simple condenser lens system found in the illumination light path of a conventional microscope. The fluorescence microscope collects the emitted fluorescence. This type of excitation-emission configuration, in which both the excitation and emission light travel through the objective, is called epifluorescence. The key to the optics in an epifluorescence microscope is the separation of the illumination (excitation) light from the fluorescence emission emanating from the sample. In order to obtain either an image of the emission without excessive background illumination, or a measurement of the fluorescence emission without background “noise”, the optical elements used to separate these two light components must be very efficient.

In a fluorescence microscope, a dichroic mirror is used to separate the excitation and emission light paths. Within the objective, the excitation emissions share the same optics. It shows the dichroic mirror’s position in an inverted fluorescence microscope. This fluorescence microscope is illuminated and imaged from below the stage. In a fluorescence microscope, the dichroic mirror separates the light paths. The excitation light reflects off the surface of the dichroic mirror into the objective. The fluorescence emission passes through the dichroic to the fluorescence microscope eyepiece or detection system. The fluorescence microscope has the dichroic mirror’s special reflective properties allow it to separate the two light paths. Each dichroic mirror for the fluorescence microscope has a set wavelength value called the transition wavelength value which is the wavelength of 50% transmission. The mirror reflects wavelengths of light below the transition wavelength value and transmits wavelengths above this value. This property accounts for the name given to this mirror (dichroic, two color). Ideally, the wavelength of the dichroic mirror is chosen to be between the wavelengths used for excitation and emission.

The dichroic mirror is a key element of the fluorescence microscope, but it is not able to perform all of the required optical functions on its own. Typically, about 90% of the light wavelengths below the transition wavelength value are reflected and about 90% of the light wavelengths above this value are transmitted by the dichroic mirror. When the excitation light illuminates the sample, a small amount of excitation light is reflected off the optical elements within the objective and some excitation light is scattered back into the objective by the sample. Some of this “excitation” light is transmitted through the dichroic mirror along with the longer wavelength light emitted by the sample. This “contaminating” light would otherwise reach the detection system if it were not for another wavelength selective element in the fluorescence microscope.

The fluorescence microscope has an emission filter. The excitation and emission filters in fluorescence microscope with the dichroic mirror. In order to select the excitation wavelength, an excitation filter is placed in the excitation path just prior to the dichroic mirror. In order to more specifically select the emission wavelength of the light emitted from the sample and to remove traces of excitation light, an emission filter is placed beneath the dichroic mirror. In this position, the filter functions to both select the emission wavelength and to eliminate any trace of the wavelengths used for excitation. These filters are usually a special type of filter referred to as an interference filter, because of the way in which it blocks the out of band transmission. Use in fluorescence microscope. Interference filters exhibit an extremely low transmission outside of their characteristic bandpass. Thus, they are very efficient in selecting the desired excitation and emission wavelengths of fluorescence microscope. The dichroic mirror is mounted on an optical block commonly referred to as a filter cube of fluorescence microscope. The excitation and emission filters are usually affixed to the filter cube. This cube provides a convenient means to change the dichroic mirror without direct handling of either the mirror or filters. It shows the light path through the filter cube in a fluorescent microscope. The narrow red line emanating from the objective to the filter cube represents the scattered and reflected emission light that must be removed by these optical elements. Light path through the filter cube in a fluorescence microscope. It is often the case that of specific combination of excitation filter, emission filter and dichroic mirror are needed to visualize or quantitate the fluorescence emission from a particular fluorescent species.

In newer models of fluorescence microscopes, manufacturers have provided a means to change these optical elements in a convenient manner by arranging a set of four or more filter cubes in a circular or linear turret under the objective. With a turret arrangement, a specific filter cube can be selected in a manner similar to that of selecting a specific objective. The need for quick wavelength changes of fluorescence microscope.

The fluorescent probes, which exhibit changes in their wavelength properties, are essential to be able to change excitation and emission wavelengths quickly, for adequate temporal resolution. These newer probes, also known as wavelength-shift probes, exhibit changes in their excitation or emission spectra or both and depending upon their environment. In order to use the fluorescent probes in fluorescence microscope needs to determine the nature of their environment, the changes in spectra must be monitored. This process involves making several measurements of the same sample at different excitation and/or emission wavelengths. In order to determine the extent to which wavelength-shift probes have altered their wavelength characteristics under specific conditions, it is imperative that measurements of these wavelength properties occur in a reasonable time frame. Thus, if the desired quantization requires a measurement of a ratio of intensities at two wavelengths, the time resolution of the measurement is dependent on how quickly the system can change excitation and emission wavelengths. Other popular advanced fluorescence techniques, such as fluorescence resonance energy transfer and fluorescence recovery after photobleaching, as well as spectroscopy, are often combined with total internal reflection to achieve additional information. The result is a very powerful tool for the study of individual fluorophores and fluorescently labeled molecules. The conventional turret method of changing filters of fluorescence microscope is very slow and is, thus, not appropriate in the use of wavelength-shift probes.

A fluorometer has been designed and developed specifically for high-speed multiwavelength applications. In the Dye Fluorometer, the excitation and emission filters normally found in a filter cube are each housed, instead, in a filter wheel. These filter wheels are located outside of the body of the fluorescence microscope. The filter wheels are computer controlled and filter changes can be implemented in milliseconds. Both excitation and emission filters are housed externally from the microscope, permitting vibration free microscopy.