epi-fluorescence with the microscope

Epi-fluorescence microscopy is a technique which is widely used by biologists. In some cases it involves auto or primary fluorescence which occurs with some substances such as chlorophyll. More often it involves staining with a special type of histological stain called a fluorochrome which is only taken up by specific material or hybridization of fluorescently labeled complementary DNA sequences. High energy, short wavelength (eg. UV or blue) light is directed onto the preparation and lower energy, longer wavelength (eg. green or red) light is emitted, according to Stokes' Law. The Jablonski Diagram, seen below, shows a number of possible routes by which an excited molecule can return to its ground or room temperature state via unstable triplet states. A rapid return results in fluorescence and a delayed return results in phosphorescence.

For example, excitation using invisible ultra-violet light results in the emission of visible blue fluorescence light from a label such as DAPI. Another common example involves blue excitation resulting in a brighter yellow-green emission from FITC.

Traditional fluorescence microscopy used transmitted light, at first with carbon arc sources and then later with 200 watt super-pressure mercury lamps. It is now usual to use much more efficient epi-illuminators with small 50 or 100 watt super pressure mercury or xenon lamps. Using a matched optical set that consists of an exciter filter, a dichroic beamsplitter and a barrier filter, the epi-illuminator allows only light of the correct short, exciting wavelength to strike the preparation and only light of the longer, emitted wavelength to reach the eyepiece, detector or the camera.

To use the Zeiss Universal with epi-fluorescence turn on the left-hand transformer for the mercury lamp and the right-hand one for the transmitted light tungsten lamp. You will first need to search your slide using brightfield or phase contrast. Because fluorescence is quite faint it is usual to work in dim light with the door closed. On finding the correct material, cut off the visible light by putting a lens cap in the light path of the microscope and open the blocking slide in the epi-illuminator. Position I on the epi-fluorescence condenser contains the #02 filter set with maximum transmission for the mercury line at 365/366 nm. and emission beyond 420 nm. Position II contains the #09 filter set used for FITC (fluorescein isothiocyanate), transmitting from 450 to 490 nm and allowing emission beyond 520 nm. Position III contains the #15 filter set used for Rhodamine, transmitting at 546 nm and allowing emission beyond 580 nm. Do not use position IV because it is blocked off.

The next figure shows the spectra of the HBO light bulb used for epi-fluorescence. It show a spectra with very strong lines at particular wavelength, such as the one at 365 nm or the one at 436 nm. Some regions of the spectrum are very weak, such as the region between 450 nm and 530nm and excitation at those wavelengths is problematic.

DAPI excites at 359 nm and emits at 461 nm. Rhodamine excites at 570 nm and emits at 590 nm. The figure below shows the FITC excitation at 494 nm and fluorescence emission at 518 nm