Some cell components (such as chlorophyll and phenolic compounds) are inherently fluorescent (autofluorescence). In general, however, fluorescence microscopy involves labelling the molecule or feature of interest with a fluorescent dye or fluorophore. There are many ways of achieving this, but the two commonest are immunolabelling and using fluorescent proteins. Immunolabelling involves raising an antibody to the molecule of interest, and coupling that antibody to a fluorescent dye such as fluorescein, rhodamine or one of the new commercial dyes such as the Alexa series. The antibody latches on to its target molecule, which thereby becomes fluorescent. Fluorescent proteins are actually produced by the cell, expressing introduced DNA coding for the protein typically attached to the gene for the target molecule. The best known such protein is the green fluorescent protein (GFP) from the jellyfish Aequoria victoria, but now a complete spectrum of such proteins is available.
Each fluorochrome has a specific set of spectra so that optimal excitation and detection can only be achieved within a small bandwidth of light wavelengths. When excited, the fluorochrome absorbs photons leading to the shuttling of electrons to higher energy states (see the Jablonski diagram). The electrons quickly return to the ground state and in the process lose energy and emit light. The emitted light is always of a lower energy and longer wavelength compared with the excitation light.
A sample is expressing eGFP, which has excitation (Ex) and Emission (Em) peaks of 488 and 509 nm. The peaks in the spectra indicate the optimal wavelengths to excite the sample and to collect the emitted light. The Jablonski diagram illustrates the energy states of the fluorophore and excitation and emission paths. Note that the blue wavelength light is shorter than that of green light.
The light path and microscope parts
The basic requirements for fluorescence microscopy are the abilities to produce fluorescence from the sample, separate the excitation and emission light, resolve microscopic structures and acquire an image. To achieve these goals, the following microscope parts are necessary.
Lamps available for fluorescence microscopy may emit over a broad spectrum of light and/or produce discrete wavelengths of light. A high-pressure short arc mercury lamp, for example, has excellent lines for excitation in the green, violet and UV range, albeit blue is relatively weak.
The filter turret contains one or more sets of filter cubes that can separate the spectra of various fluorophores or flourescent proteins or dyes. The turret can be rotated in turn when capturing more than one emitted wavelength of light. Each filter cube contains a set of filters known as an excitation filter and a barrier filter, which selects for the transmission of excitation light and emitted light, respectively. A dichroic mirror further separates the excitation from the emission by reflecting shorter wavelengths of light (excitation) and transmitting longer ones (emission). The diagrams below illustrate the orientation of the filters within a filter cube and how they work together in fluorescence microscopy.
In addition to imaging fluorescent entities in the specimen by their emitted fluorescence, the objective lens in epi-illumination is also responsible for the transmission of light to the sample.
There are many camera systems available that can be tailored to the needs of the instrument, whether it may be for rapid acquisition or high-resolution. Most use similar CCD technology to consumer cameras, but the more advanced cameras today utilise EMCCD technology. The advantage of EMCCD cameras is that they have essentially overcome an intrinsic weakness in conventional CCDs where speed and sensitivity are not compatible, and thus have enabled greater sensitivity without compromising speed.
Components and light paths of a fluorescence microscope. The excitation light is filtered by an excitation filter, which allows only a narrow band of wavelengths to enter. The dichroic mirror reflects this light allowing it to pass through the lens onto the specimen. The light emitted by the sample returns via the same path and is transmitted by the dichroic mirror. A barrier filter further eliminates any excitation light allowing only emitted light to reach the detector
Separation of excitation and emission by filters. The arrangement of the filters in the filter cube results in the transmission of a band of excitation light in the 450-500 nm range and transmission of light from 500-550nm to the eyepieces. This set-up neatly segregates the excitation and emission light so that the former reaches the sample and the latter reaches the detector.
Resources and references
A Spectra Viewer courtesy of Invitrogen
Cox, G. Optical Imaging Techniques in Cell Biology, 2nd edition. CRC Press, 2012
Soon L.L., Braet F., Ratinac K., Schuliga M., Chien H-Y., Stewart A., The benefits of microfluidics for imaging cell migration. In: Microscopy: Science, Technology, Applications and Education. Microscopy Book Series, 4. Editors: Antonio Méndez-Vilas and Jesús Díaz Álvarez, Publisher: Formatex Research Center, 2:1146-1154, December 2010