Method for Introducing Multiple Wavelength Laser Excitations in Fluorescence Microscopy
Fluorescence Microscope for Single-Molecule Studies
The Situation: Fluorescence microscopes typically use dichroic mirrors to separate incident laser excitation from fluorescent sample emissions. However, dichroic mirrors appropriate for use with multiple excitation lasers introduce significant losses in the detected sample fluorescence. Because of these losses, it is extremely difficult to perform single molecule experiments that involve more than two excitation lasers.
Our Solution: Our newly designed fluorescence microscope separates the excitation and emission paths by employing small broadband mirrors in place of the wavelength-specific dichroic mirror. This enables single molecule fluorescence microscopy using multiple dye colors and improved signal-to-noise ratios.
Fluorescence microscopes typically use a wavelength sensitive dichroic mirror to separate the incident laser excitation from the low light imaging path. However, to detect multiple fluorescence dyes simultaneously, a dichroic optical setup becomes unacceptable due to the significant loss of fluorescence signals as the emissions pass through the dichroic mirror—a direct consequence of compromises in designing an optical filter that can separate multiple wavelengths. To circumvent this problem, we employ broadband mirrors to separate the illumination (excitation) light path from the detection (emission) light path in our new microscope setup. This permits us to employ any visible wavelength laser excitation and in addition results in very low background fluorescence compatible with single dye detection.
- Multi-color single-molecule fluorescence microscopy.
- Low background fluorescence and high signal detection sensitivity.
- Broadband mirror works with all visible wavelengths, eliminating the need to switch dichroic filters in order to work with different color dyes.
- The technology allows simultaneous imaging of multiple fluorescence dyes.
- A TIRFM microscope incorporated with this technology has been built and successfully used in multi-color single-molecule fluorescence experiments.
In the existing microscope, two broadband mirrors (MIN and MOUT) are used to direct laser excitation beams (dotted color lines) into and out of the objective lens, thereby separating the excitation light from the fluorescence emissions (black lines). With this novel design, autofluorescence from the objective lens is significantly reduced, thus improving the signal-to-noise level and resulting in high fluorescence detection sensitivity.
- Patent pending in United States