Advanced Imaging

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Advanced Imaging Magazine

Updated: January 12th, 2011 10:01 AM CDT

LED Light Source Advances Fluorescence Microscopy

Technology is capable of detecting single molecules and sub-microscopic structures too small to be resolved by other conventional techniques
High-intensity monochromatic LEDs
© Carl Zeiss MicroImaging
High-intensity monochromatic LEDs are available in a variety of colors that match the excitation bandwidth of many commonly used fluorescent dyes and proteins.
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© Carl Zeiss MicroImaging
Time lapse image 1
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© Carl Zeiss MicroImaging
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© Carl Zeiss MicroImaging
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© Carl Zeiss MicroImaging
Due to the speed of changing excitation wavelengths and attenuating intensity, LEDs are ideally suited to image living cells. Shown above are 4 time-lapse images of Hela cells labeled with DS Red and expressing mutant YFP.
imaging system
© Carl Zeiss MicroImaging
The performance of an imaging system depends not only on the performance of each individual component but on the sum of all factors making up the system.
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By Becky Hohman, Carl Zeiss MicroImaging

Broad spectrum lamps, such as mercury or xenon arc lamps, can generate the desired amount of light at the specific wavelengths needed to excite fluorescent dyes and proteins. However, this traditional method of fluorescence microscopy, when used in any particular application, is only able to utilize a small percentage of the projected light. 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 percent successful at blocking the bleed through of non-specific photons. Some mitigation techniques end up not only suppressing peripheral light, but also significantly diminishing the intensity of the desired wavelengths.

Recent advances in high-performance light emitting diode (LED) technology have begun to address the root cause of this problem—the presence of non-specific photons. High-intensity monochromatic LEDs now are available in a variety of colors that match the excitation bandwidth of many commonly used fluorescent dyes and proteins.

Carl Zeiss MicroImaging has incorporated this new LED technology in the Colibri illumination system. This new light-source system for wide-field fluorescence microscopy uses specific wavelength windows with much less need to suppress unwanted peripheral wavelengths from a white light arc lamp. The modular Colibri system does not rely on any of the mechanical switching devices, like filterwheels or shutters that traditional illumination systems require.

Instead, the Colibri system employs up to four LEDs, each of which is controlled individually and instantly by electrical current. 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.

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