What is laser scanning microscopy used for?

Laser scanning microscopy is a type of light microscopy in which the sample is scanned by a laser beam. Today, confocal microscopes [Confocal Laser Scanning Microscope (CLSM)] are generally used for this purpose. Laser scanning microscopy therefore refers to microscopy techniques in which a laser beam is used to scan a sample point by point. Key areas of application for laser scanning microscopy are surface topography and advanced imaging techniques.
It is used, for example, to characterize topographical structures and evaluate surface roughness - without any contact whatsoever. This provides a detailed insight into the structure of materials. For example, laser scanning microscopy can be used to non-destructively determine the thickness of coatings and thin films and comprehensively characterize materialographic samples. This is made possible by creating 3D profiles, which works as follows.

Laser scanning microscopy with system
at Quality Analysis

Characterization of surface topographies

The laser scanning microscope is used to analyze and measure surface topographies of 3D microstructures down to the nanometer range. Metals, polymers, semiconductors and multilayer systems and many other materials can be visualized in three dimensions and the surface roughness can be evaluated without contact.

  • Visualization and measurement of material wear and volume measurements of indentations
  • Examination of surfaces of additively manufactured components or ceramic components
  • Measurement of layer thicknesses and layers of composite polymers
  • Evaluation of the quality and functionality of surfaces
  • Measurement and analysis of 3D profiles and surface roughness

How does laser scanning microscopy work?

Laser scanning microscopy uses laser light in a confocal beam path, whereby the beams only originate from a specific area. A pinhole diaphragm is used to prevent light from sample layers in front of or behind the focal plane from being reflected and entering the objective. This method ensures that only information from the focus area is processed, resulting in optically high-quality images with the best resolution. Interference from stray light is therefore eliminated. This process produces defined optical sections of the sample, from which a three-dimensional image stack is then created. 3D stacks are created to enable comprehensive analysis of the sample in three dimensions.

Analysing surface roughness with the laser scanning microscope

Entroduction of the image in laser scanning microscopy

In a laser scanning microscope, the image is not created by taking a complete image, but by scanning the specimen line by line. This process, which is measured in Hertz, varies in speed. In modern confocal laser scanning microscopy, speeds of 200 to 2,000 image lines per second are common. While the laser moves continuously within an image line, a scanned point on the sample surface is assigned to a specific pixel of the complete image via the so-called pixel dwell time. This means that a point is assigned to a specific pixel within a defined period of time. This precise control not only enables the creation of two-dimensional images, but also forms the basis for the generation of detailed and high-quality 3D images in laser scanning microscopy.

What is special about laser scanning microscopes?

One of the special features is the ability to create three-dimensional images, which enables precise analysis of the spatial structure of cells, tissues or other sample structures. The exceptionally high resolution allows the finest details to be made visible on the microscale, while at the same time high-precision measurements can be carried out on the nanometer scale.
A further advantage lies in the non-contact analysis, which ensures that sensitive samples have no physical contact with the microscope during the imaging process. The fast analysis speed, combined with the versatility in applicability, makes it possible to examine a wide variety of samples in real time. In addition, laser scanning microscopes are able to analyze objects with large height differences, which is particularly important in the study of uneven surfaces. Overall, laser scanning microscopes offer a comprehensive range of advanced imaging capabilities that provide valuable insights and knowledge in various scientific disciplines.

What other laser scanning microscopes are available?

In addition to the confocal laser scanning microscopes discussed here, there are other models. Only of historical importance are the flying spot microscopes, which also focus the light in a small spot over the sample, but are not equipped with a pinhole aperture. Today, there are several further developments of the conventional CLSM, which often do not exist as separate devices but are used as additional functions in CLS microscopes. STED and 4Pi microscopes should be mentioned here in particular. Both methods provide a better resolution than conventional laser scanning. The multiphoton microscope, on the other hand, works without pinhole apertures, without any loss of sharpness due to stray light.

The laser scanning microscopes in practical use

Damage analysis:

Measurement of wear marks

LSM is used in damage analysis for example to measure the depth of wear marks. Laser scanning microscopy can also be used to determine the surface roughness of the smallest tooth flank surfaces.

Material properties:

Optimization of materials

Laser scanning microscopy is also used in the investigation of surface topographies in the characterization of materials. By precisely capturing surface structures in 3D, detailed and high-resolution images can be generated that provide insights into the microscopic features of material surfaces.

Micro roughness:

Quality assurance of components

One example is the analysis of micro-roughness on metallic components. LSM enables the visual representation of roughness as well as the quantitative measurement of surface profiles. This allows the roughness value to be determined. This is important for the quality assurance of components, as the precise characterization of the surface topography at a microscopic level provides information about the manufacturing process and the material properties. By creating precise 3D images, laser scanning microscopy thus supports the development and optimization of materials for industrial applications.

Briefly summarized: Laser scanning microscopy

Laser scanning microscopy is a light microscopy method in which an object is scanned line by line by a laser. This and the use of pinhole apertures result in images with a particularly high resolution.

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