Advancement Benefits of Laser Scanning Multiphoton Microscopes
Laser scanning multiphoton microscope is a significant improvement of optical microscopy, mainly manifested in the ability to observe deep structures of living cells, fixed cells, and tissues, and obtain clear and sharp multi-layer Z-plane structures, namely optical slices, which can be used to construct three-dimensional solid structures of specimens. Confocal microscope uses a laser light source, which is expanded to fill the entire focal plane of the objective lens, and then converged into very small points on the focal plane of the specimen through the lens system of the objective lens. According to the numerical aperture of the objective lens, the diameter of the brightest illumination point is about 0.25-0.8 μ m, and the depth is about 0.5-1.5 μ m. The size of the confocal point is determined by the microscope design, laser wavelength, objective characteristics, scanning unit state settings, and specimen properties. The illumination range and depth of a field microscope are large, while the illumination of a confocal microscope is focused on a safe focal point on the focal plane. The most basic advantage of confocal microscopy is that it can perform fine optical sectioning on thick fluorescent specimens (up to 50 μ m or more), with a thickness of approximately 0.5 to 1.5 μ m. The series of optical slice images can be obtained by moving the specimen up and down using the Z-axis stepper motor of the microscope. The collection of image information is controlled within a safe plane and will not be interfered by signals emitted from other locations on the specimen. After removing the influence of background fluorescence and increasing the signal-to-noise ratio, the contrast and resolution of confocal images are significantly improved compared to traditional field illumination fluorescence images. In many specimens, intricate structural components interweave to form complex systems, but once enough optical sections can be collected, we can use software to reconstruct them in three dimensions. This experimental method has been widely used in biological research to elucidate the complex structural and functional relationships between cells or tissues.
