Laser Fluorescence Microscopy

About

Background: 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. Application: Multi-color single-molecule fluorescence microscopy. Advantages: • 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. Technology: In the existing microscope, two broadband mirrors are used to direct laser excitation beams into and out of the objective lens, thereby separating the excitation light from the fluorescence emissions. 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.