In this article, we report a new type of fluorescence confocal microscope: frequency division multiplexed multichannel fluorescence confocal microscope, in which we encode the spatial location information into the frequency domain. In this microscope, the exciting laser beam is first split into multiple beams and each beam is modulated at a different frequency. These multiple beams are focused at different locations of the target to form multiple focal points, which further generate multiple fluorescent emission spots. The fluorescent emissions from different focal points are also modulated at different frequencies, because the exciting beams are modulated at different frequencies (or difference carrier frequency). Then, all the fluorescent emissions (modulated at different frequencies) are collected together and detected by a highly sensitive, large-dynamic-range photomultiplier tube. By demodulating the detected signal (i.e., via the Fourier transform), we can distinguish the fluorescent light emitted from the different locations by the corresponding carrier frequencies. The major advantage of this unique fluorescence confocal microscope is that it not only has a high sensitivity because of the use of photomultiplier tube but also can get multiple-point data simultaneously, which is crucial to study the dynamic behavior of many biological process. As an initial step, to verify the feasibility of the proposed multichannel confocal microscope, we have developed a two-channel confocal fluorescence microscope and applied it to study the dynamic behavior of the changes of the calcium ion concentration during the single cardiac myocyte contraction. Our preliminary experimental results demonstrated that we could indeed realize multichannel confocal fluorescence microscopy by utilizing the frequency division multiplexed microscope, which could become an effective tool to study the dynamic behavior of many biological processes.
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