LED Light Source: Major Advance in Fluorescence Microscopy

Abstract

Fluorescence microscopy requires an intense light source at the specifi c wavelength that will excite fl uorescent dyes and proteins. The traditional method employs a white light, typically from a mercury or xenon arc lamp. Although such broad spectrum lamps can generate ample light at desired wavelengths, only a small percentage of the projected light is useful in any particular application. The other wavelengths need to be suppressed to avoid background noise that reduces image contrast and obscures the fl uorescent light emissions. This process of subtraction is complex, expensive, and only partially effective. Even after decades of refi nements, the best fi lters are not 100% successful at blocking the bleedthrough of non-specifi c photons. Some mitigation techniques end up not only suppressing peripheral light, but also signifi cantly diminishing the intensity of the desired wavelengths. To address the root cause of the problem—the presence of non-specifi c photons—a radically different approach is coming to light. In theory, a design that only introduces specifi c photons is preferable to one that requires subsequent mitigation. In other words, an ideal light source would start with a baseline that contributes zero peripheral light and would allow the user to precisely control the addition of only those selective wavelengths of light that match the particular excitation wavelengths of the fl uorochromes. The user should also be able to control the intensity and duration of the light to achieve the highest possible image quality while protecting the sample against bleaching and phototoxicity. Recent advances in high performance Light Emitting Diode (LED) technology have enabled the practical implementation of this theoretical model. High-intensity monochromatic LEDs are now available in a variety of colors that match the excitation bandwidth of many commonly used fl uorescent dyes and proteins. This new LED technology uses specifi c wavelength windows with much less need to suppress unwanted peripheral wavelengths from a white light arc lamp. One such system has up to four LEDs, each individually and instantly controlled by electrical current without any of the mechanical switching devices like fi lterwheels or shutters required by traditional illumination systems. LEDs of different colors can be used in combination, giving users the option of seeing multiple fl uorochromes simultaneously or rapidly capturing sequential images of each fl uorochrome.