Signal-to-noise ratio for confocal microscopy when the intensity-modulated multiple-beam scanning (IMS) technique

The Intensity-modulated Multiple-beam Scanning (IMS) technique involves the use of modulated laser beams for specimen illumination, combined with lock-in amplifiers in the detection channels. In previous studies we have shown that this technique efficiently reduces cross-talk between the channels when simultaneously detecting multiple fluorophores. A drawback with the IMS technique is that it tends to produce images with a lower signal-to-noise ratio (SNR) compared with images recorded without using IMS. Typically a reduction of between 30 and 40% is found. In a previous paper, presenting theoretical derivations of the SNR as well as computer simulations, it was found that the SNR depends on the modulation waveform. In this paper a more comprehensive analysis is made, including non-symmetric waveforms and brief pulses. The effect of a finite contrast ratio for the light modulators is also investigated. It is found that, theoretically, the SNR when using the IMS technique can become equal to that for conventional detection. In practice, however, the SNR is limited by the attainable contrast ratio of the light modulators. Experiments performed are in good agreement with the theoretical results, and by optimizing the modulation waveform we have reached a SNR of nearly 90% of the theoretical maximum.