Noise reduction technique for fluorescence imaging in multifocal multiphoton microscopy

Multifocal multiphoton microscopy (MMM) by forming multifocal created with a lenslet array enables high-speed imaging than single-focal multiphoton microscopy. However, most multifocal multiphoton microscopy has a problem of degrading image quality compared to single-focal microscopes. In order to solve this problem, it is necessary to equalize the intensity of the multifocal incident light. The fluorescence signal emitted from the fluorescence material is determined by the intensity of the incident light, but compared to the single-focal multiphoton microscope, the incident light passing through the lenslet array not only causes problems in acquiring fluorescence images but also limits the field of view ( FOV). This problem was overcome by using a specially designed beam shaper to equalize the multifocal incident light intensity. Another problem with MMM is that typical biopsy samples are opaque, so light scattering occurs when light is irradiated onto the sample. This is a cause of the noise that degrades image quality by generating crosstalk in which fluorescence signals emitted from each focus deviate from the focal plane and interfere with each other. For the minimization of crosstalk, we set up the algorithm technology to correct the sensitivity non-uniformity between channels of multi-channel photomultiplier tubes (PMT). And to minimize crosstalk between PMT circuits, an independent short-channel photon discriminator was designed to expand the number of focal points of the MMM system. Among the problems of MMM, two techniques were introduced, and it was confirmed that the noise of the fluorescence image was reduced.