Basic structure of confocal fluorescence microscopy
(1) Scanning module
The scanning module mainly consists of a pinhole light bar (controlling the thickness of optical slices), a spectroscope (changing the direction of light propagation according to wavelength), an emission fluorescence colorimeter (selecting light within a certain wavelength range for detection), and a detector (photomultiplier tube). The mixed fluorescence in the fluorescent sample enters the scanner, and after being selected by the detection pinhole light bar, spectrometer, and colorimeter, it is divided into various monochromatic fluorescence, which are detected in different fluorescence channels and form corresponding confocal images. At the same time, several parallel monochromatic fluorescence images and their composite images can be displayed on the computer screen.
(2) Fluorescence microscopy system
Microscope is the main component of LSCM, which is related to the imaging quality of the system. The microscope optical path can be easily inserted into an infinitely distant optical system with optical options without affecting imaging quality and measurement accuracy. The objective should be a large numerical aperture flat field apochromatic objective, which is beneficial for fluorescence acquisition and clear imaging. The conversion of the objective lens group, selection of the color filter group, movement adjustment of the stage, and memory locking of the focal plane should all be automatically controlled by the computer.
The fluorescence microscope used in laser scanning confocal microscopy is generally the same as a conventional fluorescence microscope, but it has its own characteristics: it needs to be connected to a scanner, so that the laser can enter the microscope objective to irradiate the sample and make the fluorescence emitted by the sample reach the detector; A light path conversion device is required, which converts mercury lamps to lasers, and the light intensity of the mercury lamp can be adjusted.
(3) Common lasers
The laser sources used in laser scanning confocal microscopy include single laser and multi laser systems, and the commonly used lasers include the following three types:
Semiconductor laser: 405nm (near ultraviolet spectral line)
Argon ion laser: 457nm, 477nm, 488nm, 514nm (blue green light)
He-Ne laser: 543nm (green light He-Ne green laser) 633nm (red light He-Ne red laser)
UV laser (UV laser): 351 nm, 364 nm (UV light)
(4) Auxiliary equipment
Air cooled, water-cooled cooling systems and regulated power supplies.
The basic working principle of a laser scanning confocal microscope is to first emit a certain wavelength of excitation light from the laser. After amplification, the light passes through the illuminated pinhole light bar in the scanner to form a point light source. The objective lens focuses on the focal plane of the sample, and the corresponding illuminated points on the sample are excited and emit fluorescence. After passing through the detection hole light bar, it reaches the detector and is imaged on the computer monitoring screen. In this way, a complete confocal image is formed by the fluorescence images of each point of the sample on the focal plane, which is called a light slice.
