How to Adjust Laser Intensity of a Laser Scanning Confocal Microscope
① Power control knob: Generally set to 30% of the maximum value. Higher output shortens the service life of the laser and introduces laser noise. Optimize the intensity by adjusting AOTF, PMT and other components first.
② Acousto‑Optic Tunable Filter (AOTF): It controls the excitation light beam and intensity. It can turn the laser on or off within milliseconds or adjust laser intensity continuously from 0 to 100%. This enables different laser beams and selected intensity values to be applied to each pixel of the image.
Working principle: It relies on Bragg diffraction of incident light induced by acoustic waves propagating in the medium. When an RF signal of a specific frequency is input, the AOTF diffracts the incident polychromatic light and selects monochromatic light of wavelength λ. There is a one‑to‑one correspondence between the wavelength of the selected monochromatic light and the radio frequency f. The output wavelength can be rapidly and randomly changed simply by tuning the electrical signal. AOTF enables continuous laser intensity adjustment with fast response for local stimulation, scanning and rapid wavelength switching at the microsecond level.
In a laser scanning confocal microscope, the photomultiplier tube (PMT) cannot increase laser intensity, but it adjusts the photoelectric signal intensity for the final image. Therefore, adjusting the voltage of the photomultiplier tube changes image brightness. In addition, the size of the confocal pinhole affects photon count and can also be used to adjust image brightness. The photomultiplier tube is a special vacuum tube extremely sensitive to ultraviolet, visible and near‑infrared light. It amplifies weak incoming optical signals up to 10⁸ times so that the light signals can be measured.
