The LED Light Source: A Major Advance in Fluorescence Microscopy

Fluorescence microscopy requires an intense light source at the specific wavelength that will excite fluorescent 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 fluorescent light emissions.

This process of suppressing extraneous light is complex, expensive, and only partially effective: Even after decades of refinements, the best filters are not 100% successful at blocking the bleed-through of nonspecific photons. Some mitigation techniques end up not only suppressing peripheral light, but also significantly diminishing the intensity of the desired wavelengths. To address the root cause of the problem—the presence of nonspecific photons—a radically different approach is needed.

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 fluorescent dyes and proteins.

Figure 1 - Time-lapse images of Hela cells labeled with DS Red and expressing mutant YFP.

Carl Zeiss MicroImaging (Thornwood, NY) has incorporated this LED technology in the Colibri illumination system, a light source system for widefield fluorescence microscopy that uses specific wavelength windows with much less need to suppress unwanted peripheral wavelengths from a white light arc lamp. The modular system employs up to four LEDs, each individually and instantly controlled by electrical current without any of the mechanical switching devices such as filterwheels or shutters required by traditional illumination systems. LEDs of different colors can be used in combination, giving users the option of seeing multiple fluorochromes simultaneously or rapidly capturing sequential images of each fluorochrome (Figure 1).

Expanding use of fluorescence microscopy

Fluorescence microscopy utilizes optical filters to separate excitation light from the emitted fluorescence, which is observed visually or detected by a camera equipped with a charge-coupled device (CCD) or other detectors. Fluorescence microscopy is an increasingly widespread medical and biological laboratory technique that provides highly sensitive detection for medical diagnostics and allows for the detection of cellular components and inter- and intracellular communication. Fluorescence microscopy is capable of detecting single molecules and submicroscopic structures that are too small to be resolved by other conventional microscope techniques.

The availability of (green) fluorescent proteins (GFPs) derived from jellyfish, corals, etc., and its color-shifted genetic derivatives has greatly expanded the use of fluorescence microscopy during the past 10 years. Fluorescent proteins such as GFP are less phototoxic than the small fluorescent molecules in most chemical dyes such as fluorescein isothiocyanate (FITC), which can harm the specimen when illuminated during live cell fluorescence microscopy. This attribute of fluorescent proteins has inspired the development of highly automated time-lapse live cell imaging systems.

Even more important is the fact that fluorescent proteins can be expressed by the cells, while other fluorescent dyes usually cannot penetrate the membranes of living cells. This limits their use to mainly fixed cells or the need for microinjection, electroporation, etc.

Light source requirements

The most basic requirement of a fluorescence microscopy light source is closely matching the excitation wavelength of the fluorochrome to achieve a high-contrast image, i.e., an image with a high signal-to-noise ratio. Wavelengths that match the fluorochrome strengthen the signal, but any peripheral wavelengths produce background noise that can overshadow the signal emitted by the object of interest.

A second and related requirement is illumination intensity, i.e., the number of photons specific to the excitation wavelength that reach the specimen. The human eye is less sensitive than most automated detection systems, and therefore applications involving visual observation typically require higher levels of illumination intensity. On the other hand, lower intensity is necessary for live cell imaging applications to protect against photobleaching and phototoxicity.

Intensity of illumination and signal intensity do not just follow a linear correlation. Saturation effects and dark states become more and more important with increasing illumination intensity.

An additional consideration is protection of the specimen, especially in live cell imaging applications. The dangers of overexposure to light have already been mentioned: phototoxicity of the specimen and photobleaching of the fluorescent dye or protein. Overexposure can be avoided by attenuating the light intensity and by limiting the duration of the illumination to exactly the exposure time of the sensor. The cells also need to be protected from the heat generated by light source lamps and from the vibrations caused by mechanical filtering and switching devices.

Another factor to consider when evaluating light sources is the lifetime and stability of the lamp. Some light sources exhibit short-term intensity fluctuations and a substantial deterioration of performance over time. Limited life span results in more frequent bulb replacements, and poor stability diminishes the reproducibility of illumination conditions.

Advantages of LED in fluorescence microscopy

Now that high-performance LEDs provide sufficient intensity at the specific wavelengths required for many applications, fluorescence microscopy is able to take advantage of the benefits of LEDs, including their compact size, low power consumption, minimal heat output, fast switching and adjusting properties, high emission stability, and extremely long life span.

Figure 2 - The Colibri LED light source system employs a flexible modular design and up to four LED modules that can be used simultaneously in Colibri.

Figure 3 - Ten different LED modules that can be easily exchanged are currently available for Colibri, from UV to dark red.

The Colibri LED light source system employs a flexible modular design and up to four LED modules that can be used simultaneously in the system (Figure 2). The beam paths from each module are steered into the microscope with a series of beam combiners, and 10 different LED modules are currently available for Colibri, from UV to dark red (Table 1 and Figure 3).

Figure 4 - The modular design of Colibri makes it possible to easily implement and exploit further developments in LED technology in the future.

The modules can be easily exchanged by the user, depending on the experimental design of the day (Figure 4). The intensity of each module can be adjusted independently, precisely, and reproducibly in percentage steps, so that for every fluorescent dye, the output emitted is precisely what is needed to achieve the best possible compromise between the required excitation intensity and maximum sample protection. The modular design makes it possible to easily implement and exploit further developments in LED technology in the future.

An advantage of LEDs is that they instantly illuminate at full intensity as soon as electrical current is applied. Unlike arc lamps that are turned on continuously, LEDs can be switched on or off instantly when needed, with no deleterious effects to their life span. Additionally, with no moving parts, the all-electronic system is vibration free.

The Colibri system is particularly well-suited for imaging applications requiring fast switching between wavelengths. Only about 300 μsec are needed to switch between the LED modules.

The intensity of every LED module can be adjusted in percentage steps, enabling equidistant multichannel images to be easily realized in time-lapse-series. Instead of adapting the integration times of the camera to the illumination intensity, LED technology makes it possible to simply set the illumination intensity for the required integration time. The LED illumination intensity is also highly stable over time, making quantitative analyses easier and more reliable.

The performance of an imaging system depends not only on the performance of each individual component, such as the light source, but on the sum of all factors making up such a system. The system software is a critical, but often neglected, component. In order to take full advantage of the Colibri LED light source technology, Colibri has been integrated with the AxioVision imaging platform (Carl Zeiss).

For live cell imaging, LED technology is ideally suited to the fast acquisition framework of AxioVision, which forms the backbone of the Cell Observer HS system (Carl Zeiss). The integration of Colibri with AxioVision results in extremely fast switching times, with precise control of the illumination intensity to protect the sample.

Summary

While the intensity of LEDs has evolved significantly over the past few years, their intensity is still not as high as conventional arc lamps. However, in most live cell imaging environments, the intensity from conventional light sources is typically reduced to minimize phototoxic effects to cells and tissues.

For applications requiring higher illumination intensity, or for applications requiring excitation wavelengths not currently supported by existing LED technology, the company offers a combination system that pairs a Colibri with an externally coupled metal halide (HXP) white light source. The Colibri control panel or the AxioVision software is used to switch over to and control the shutter of the HXP 120.

White light sources have been in use for decades, and much expertise has been developed in the ways of reducing the problems associated with peripheral light. Many laboratories have invested a great deal in filters used to suppress nonspecific light. In the past, it may have been difficult to imagine that any other light source method would ever be viable. Fortunately, a new type of light source is available. With LED technology, users can now take advantage of an effective alternative for live cell imaging, high-speed or multichannel fluorescence microscopy, and many other applications.

Ms. Hohman is Product Manager, Light Microscopy, Carl Zeiss MicroImaging, Inc., One Zeiss Dr., Thornwood, NY 10594, U.S.A.; tel.: 800-233-2343; e-mail: bhohman@zeiss.com.

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