Improvement Advantages of Laser Scanning Multiphoton Microscopy
Laser scanning multiphoton microscopy represents a major advancement in optical microscopy. It enables observation of deep structures in living cells, fixed cells and tissues, while capturing clear and sharp multi-layered Z-plane structures-namely optical sections-which can be further used to reconstruct the three-dimensional solid structure of specimens.
A confocal microscope adopts a laser light source. The laser beam is expanded to fully illuminate the rear focal plane of the objective lens, then passes through the objective lens system and converges into an extremely tiny spot on the focal plane of the specimen. Depending on the numerical aperture of the objective lens, the diameter of the illuminated spot ranges approximately from 0.25 to 0.8 μm, with an axial depth of about 0.5 to 1.5 μm. The size of the confocal spot is determined by the microscope design, laser wavelength, objective characteristics, scanning unit settings and specimen properties.
Wide-field microscopes feature a large illumination range and deep illumination depth, whereas confocal microscopy concentrates illumination into a precise focal point on the focal plane. The core advantage of confocal microscopy lies in its capability to perform fine optical sectioning on thick fluorescent specimens (up to 50 μm or more), with each section thickness ranging from 0.5 to 1.5 μm.
A series of optical section images are acquired by moving the specimen vertically via the precision Z-axis stepping motor of the microscope. Image signal collection is confined strictly to the targeted focal plane and free from interference by signals originating from other positions within the specimen. After eliminating background fluorescence and improving the signal-to-noise ratio, confocal images exhibit significantly higher contrast and resolution compared with traditional wide-field fluorescence images.
In most specimens, intricate structural components interweave to form complex systems. Once a sufficient number of optical sections are collected, three-dimensional reconstruction can be completed using professional software. This experimental method has been widely applied in biological research to clarify the complex structural and functional relationships between cells and tissues.
