Advantages of Improvements for Laser-Scanning Multiphoton Microscopes
Laser-scanning multiphoton microscopes represent a major advancement in optical microscopy. They are mainly capable of observing deep structures of live cells, fixed cells and tissues, and acquiring sharp, clear multi-layer Z-plane structures, namely optical sections, which can be used to reconstruct the three-dimensional solid structure of specimens.
A confocal microscope uses a laser light source. The expanded laser beam fills the entire back focal plane of the objective lens and then passes through the objective lens system to converge into an extremely small spot on the focal plane of the specimen. Depending on the numerical aperture of the objective lens, the diameter of the illuminated spot is approximately 0.25–0.8 μm, with a depth of about 0.5–1.5 μm. The size of the confocal spot is determined by microscope design, laser wavelength, objective characteristics, scanning unit parameter settings and specimen properties.
Widefield microscopes feature a large illumination area and deep illumination depth, while the illumination of confocal microscopes is concentrated on a tiny focal point on the focal plane. The fundamental advantage of confocal microscopes is that they can perform fine optical sectioning on thick fluorescent specimens (up to 50 μm or thicker), with section thickness ranging from 0.5 to 1.5 μm. A series of optical section images can be obtained by moving the specimen vertically via the microscope Z-axis stepping motor. Image acquisition is confined to the selected plane and will not be disturbed by signals emitted from other regions of the specimen. After eliminating background fluorescence and improving the signal-to-noise ratio, confocal images exhibit markedly higher contrast and resolution compared with conventional widefield fluorescence images.
In many specimens, numerous intricate structural components interweave to form complex systems. Once sufficient optical section images are acquired, three-dimensional reconstruction can be performed using software. This experimental method has been widely applied in biological research to elucidate the complex structural and functional relationships between cells or tissues.
