Features and Applications of Confocal Laser Microscopy
Laser scanning confocal microscope (LSCM) is a type of microscope designed based on conjugate focus technology, that is, to make the laser light source, the sample under test and the detector all in the conjugate position of each other.
Fundamental
In general microscopes, the focal plane of the objective lens coincides with the detector so that the observed image plane is isolated from the adjacent axial plane, while in confocal microscopes, the sample is illuminated by using a diffraction-limited light spot, and in the This isolation effect is achieved by using a pinhole in the collection light path at the focal point of the spot conjugate to filter stray light to improve resolution.
Imaging Features
Only the light reflected back from the conjugated sample layer can pass through the small hole in the collection light path, and the remaining irrelevant sample layer reflections are blocked by the small hole. This can result in a significant increase in resolution. Shown below is a side-by-side comparison of multidimensional fluorescence microscopy and confocal microscopy of the same thick sample. When a series of images are taken at different focal planes, what is commonly referred to as a "z-stack" can be generated, and this image shows both the resolution and contrast gains offered by confocal microscopy and the underlying causes of these gains. Examining the image at the top of the stack with the imaging plane above the tissue reveals a lot of scattered light in the fluorescence image, whereas the confocal image appears black. This reduction in PSF in the axial direction is directly responsible for the difference in resolution observed at the optical interface in the middle of the z-stack.
Another feature of laser scanning confocal microscopy is that it is a scanning imaging technology. The traditional wide-field illumination technology illuminates the entire sample, so the image can be directly captured by the naked eye or a detector, but LSCM uses a beam or Multiple focused beams pass through the sample for scanning and imaging, and the resulting image is called an optical section. The following shows the difference between the traditional wide-field illumination method and the laser scanning confocal illumination method.
Therefore, a practical working method of a modern confocal microscope is shown in the figure below. The excitation light emitted by the laser passes through a dichroic mirror and scans in the x and y directions of the sample through a pair of vibrating mirrors. The excitation (or reflection) of the sample The light enters the PMT detector through the pinhole and is recorded, and the recorded scanning image is reconstructed by the computer to obtain the actual sample image.
