A rapid wavelength changer based on liquid crystal shutters for use in ratiometric microspectrophotometry

Many of the fluorescent indicator molecules most useful for measuring intra cellular concentrations of ions of biological importance, such as Ca2+ or H +, require illumination first at one wavelength, at which the fluorescence depends strongly on the concentration of the ion, and then at another wavel ength (e.g. the isosbestic point), so that a ratio can be obtained. Existin g wavelength changers are mechanical and involve moving filters, mirrors or gratings. These systems have the disadvantage that they introduce mechanic al shocks that can interfere with simultaneous electrophysiological recordi ng, In addition, they require special electrical driving systems and are re latively expensive, especially if they are capable of switching rapidly. We describe a new wavelength changer based on liquid crystal shutters which h as the following advantages: (1) it has no mechanical moving parts; (2) it can switch rapidly ( 1 ms) and in any desired pattern (off - on1 - off - on 2 - off, etc.); (3) it is driven by a low-power 15-V pulse; and (4) it is s ubstantially cheaper than existing wavelength changers. Its limitations are that it does not pass wavelengths shorter than about 400 nm and transmissi on in the range 430-700 nm is only 20-40%.