Assignment 2: fluorescence microscopy
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Overview
The phenomenon of fluorescence has been known since at least the sixteenth century. The earliest observers noticed an unusual tint in water infused with the wood of a Mexican tree called Coatli. Other naturally-occurring fluorescent and phosphorescent materials such as Bologna Stone (baryte) and quinine sparked considerable interest. After hundreds of years of investigation by the likes of Robert Boyle, Galileo Galilei, David Brewster, John Herschel, George Stokes, and many others, Aleksander Jablonski finally explained fluorescence in 1935 as a series electronic state transitions &mdash' the Jablonski diagram. Some naturally fluorescent molecules have important biological applications, but a key breakthrough in the early 1940s paved the way for fluorescence-based methods to become some of the most fundamentally important techniques in all of biological research. The key idea was to attach a fluorescent molecule to an antibody. The technique is called fluorescent immunostaining, and it offers spectacular contrast and specificity in the microscopic study of biological samples.
In this part of the lab, you will add the hardware necessary to make epi-illuminated fluorescent images with your microscope and make a few test images of plastic, fluorescent beads. A typical spectrum is shown on the right. The excitation light source is a 5 mW diode laser with a nominal wavelength of 532 nm — a striking, brilliant green. Emission is in the red/orange range. To make the correction for nonuniform illumination, you will also make images of a uniform fluorescence reference slide and a dark image with the illuminator turned off.
As shown in the block diagram, the major components required for fluorescence imaging are an illuminator (laser, L3, L4, and L5), dichroic mirror (DM), and emission filter (BF). The illuminator provides light in the appropriate wavelength range to excite the fluorescent molecules in the sample. Fluorescence microscopes that use broadand light sources such as arc lamps require an additional filter called an excitation filter to limit the wavelengths in the illumination to the proper range. Because lasers emit light in a very narrow range of wavelengths, an excitation filter is unnecessary.
Excitation light comes from beneath the sample, through the objective lens. A dichroic mirror directs the excitation toward the objective and sample. The mirror must reflect wavelengths in the excitation range and pass the longer wavelengths of the emitted fluorescence. In fluorescence imaging, illumination intensity is typically 5 or 6 orders of magnitude greater than emitted fluorescence, so it is crucial to filter out excitation photons as completely as possible. The dichroic mirror passes a substantial amount of green light, on the order of five percent. The barrier filter does a much better job of removing the green light, attenuating the excitation wavelengths by about 5 orders of magnitude. The barrier filter is essential for making crisp, high-contrast images.
To provide collimated illumination in the sample plane, light from the illuminator is focused at the back focal point of the objective.
Assignment details
This assignment has 2 parts: