Laser Confocal Microscopy - Features and Applications
Laser scanning confocal microscopy (LSCM) is a type of microscope designed based on conjugate focus technology, that is, the laser light source, the sample to be measured, and the detector are all in conjugate positions with each other.
Fundamental
In a general microscope, the image plane of observation is isolated from the adjacent axial plane by coinciding the focal plane of the objective lens with the detector, while in a confocal microscope, a diffraction-limited light spot is used to illuminate the sample and A pinhole is used in the collected light path at the conjugate focus of the light spot to filter stray light to create this isolation effect and thereby improve resolution.
Imaging features
Optical section scanning imaging
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 captured directly by the naked eye or a detector, but LSCM uses a beam or Multiple focused beams pass through the sample to scan and image. 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, an actual working method of a modern confocal microscope is as shown in the figure below. The excitation light emitted by the laser passes through a dichroic mirror and is scanned in the x direction and y direction of the sample through a pair of galvanometers. The excitation (or reflection) of the sample is Light enters the PMT detector through the pinhole and is recorded, and the recorded scanned image is reconstructed by a computer to reconstruct the actual sample image.
Generate "z-stack" images on different focal planes
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 significant resolution improvements. 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, images commonly referred to as "z-stacks" can be generated, which illustrate the resolution and contrast gains offered by confocal microscopy and the underlying causes of these gains. It can be seen that examining the image at the top of the stack with the imaging plane above the tissue reveals a large amount of scattered light in the fluorescence image, whereas the confocal microscopy image appears black. This reduction in axial PSF directly results in the resolution difference observed at the optical interface in the middle of the z-stack.
Resolution is greatly improved compared to widefield lighting
In fluorescence microscopy, the light intensity emitted from a single point is described by the point spread function (PSF), and its pattern is an Airy disk. The resolution of the fluorescence system can be described by the radius of the Airy disk, which can be described by The numerical aperture of the objective lens and the wavelength of the excitation light determine
