Confocal technology
The confocal includes a number of optical methods of measurement ( distance measurement , imaging, profilometry ), based on the Konfokalprinzip: Two optical systems or optical paths are confocal , if they have a common focal point own.
Confocal technology usually uses very small light and face field diaphragms , only a few micrometers in size , also known as pinholes . They limit the illuminated area on the object and the field of view of the observation optics to a spot, the size of which is ideally determined by the diffraction-related resolution limit of the image. The illuminating beam path and the observation beam path are thus confocal .
The confocal technique is a point-by-point measuring method. If this measuring point is guided through a measuring volume in all three spatial dimensions, a three-dimensional image of the volume with sub-micrometer resolution is obtained. In the case of transparent samples, as they are often examined in biology, a three-dimensional image of the tissue structure is obtained. In the case of non-transparent and reflective samples, a high-resolution representation of the surface can be calculated from this volume image.
Confocal principles
There are various confocal measurement principles that differ significantly in terms of their optical structure. In the following, the individual techniques are presented and the associated scanning methods are described.
Confocal point sensor
The simplest confocal process is certainly the confocal point sensor, as it was already patented by Marvin Minsky . It is the best way to explain the basic confocal principle: It consists of a light source that illuminates a very small pinhole . The image of the pinhole is imaged diffraction-limited on the object in the form of an Airy disc . The reflected and scattered light from the sample is imaged via a beam splitter onto a second pinhole, behind which a detector is located. This arrangement ensures that scattered light that is reflected by the sample outside the focal plane is blocked out. As a result, the sensor measures an increased light intensity when the object is in focus, but does not detect any intensity when the object is out of focus. A point sensor can be set up with both white light and a laser .
The point sensor must be moved over the object in all three spatial directions in order to obtain a complete 3D image. The movement along the optical axis can be done by moving the sample or the sensor, but also by moving the objective or a rapidly oscillating mirror in the beam path. The latter method in particular allows very fast measurements with a point sensor. The fastest single-channel systems currently achieve measurement rates of 70,000 distance values per second and channel. The fastest multi-channel systems with up to 128 simultaneously recorded channels - and thus currently the fastest confocal sensors in the world (as of 2013) - achieve up to one million distance values per second.
Chromatic confocal sensor
The chromatic confocal sensor uses the property of dispersive optics to focus white light not in one point, but separated by wavelength at different distances. The blue focus is closer to the optics, the red one is further away. With this principle you can map a surface at different distances at the same time. As a result, a chromatic confocal sensor does not require any scanning movement along the optical axis.
Confocal laser scanning microscope
The confocal laser scanning microscope is in principle a confocal point sensor, in which the scanning is carried out in the focal plane with movable deflecting mirrors. The measuring point can thus be moved relatively quickly over the object. The scanning in the direction of the optical axis is typically carried out by moving the objective or the object. The targeted guidance of the confocal observation point over the object field allows flexible adaptation of the scanning density to the actual optical resolution , but is relatively slow. A few slice images per second are the typical measuring rate of a laser scanning microscope.
Confocal white light microscope
With the confocal white light microscope, the lateral deflection occurs, for example, by a rapidly rotating Nipkow disk or by micromirror actuators . This type of microscope is therefore able to record several measuring points at the same time. A CCD sensor is therefore usually used as the image sensor. Within one revolution of the Nipkow disk or within one cycle of the micromirrors, an entire image can be captured confocally. Because of the high speed of the Nipkow disk of up to 100 revolutions per second, this type of microscope achieves very high measuring rates of up to around 100 slice images per second.
Applications
The applications of confocal technology can be found today mainly in the areas of life sciences and materials research . While in the life sciences the focus is mostly on high-resolution volume mapping of transparent objects such as animal or plant cells , in materials research mainly profilometry , i.e. the three-dimensional measurement of surfaces, is carried out. In addition to geometric issues, roughness measurement is the main area of application.
Volume mapping
In volume imaging, the property of confocal imaging is used to hide scattered light from outside the focal plane in order to be able to see to a certain extent also behind non-transparent objects. This is possible because, thanks to the large numerical aperture from the edge of the lens , the light also shines past small objects to the side.
Profilometry
Confocal profilometry uses the common feature of all confocal measurement methods that, ideally, with a clearly defined object surface, they generate the response function shown on the right above the object height. This function is therefore also called the confocal curve . Its half-value width (engl. Full width half maximum , FWHM) is essentially on the numerical aperture of the objective dependent. The object height results from the location of the maximum on the z-axis. In the simplest case, an arithmetic mean of the z-position weighted with the intensity values is used to determine the maximum . This makes it possible to achieve an accuracy of position determination that is a few nanometers. This is many times better than the optical resolution along the z-axis, which roughly corresponds to the half-width of the confocal curve and corresponds to at least 500 nanometers in visible light.
history
H. Naora already described an early, non-imaging confocal microscope in 1951. He used it for the spectroscopy of nucleic acids .
The imaging confocal technique was developed by Marvin Minsky in the 1950s and a patent has been applied for. The process became practically applicable for the first time, primarily through the development of laser technology. With the emergence of high-performance CCD cameras, confocal white light microscopy could also be implemented in high-performance devices in the 1990s.
Web links
Individual evidence
- ↑ a b Patent US3013467 : Microscopy Apparatus. Applied on November 7, 1957 , published December 19, 1961 , inventor: Marvin Minsky .
- ↑ µsprint Technologie , NanoFocus AG
- ^ Electronic eagle eyes ( Memento of February 12, 2008 in the Internet Archive ) , Pictures of the Future, Fall 2004.
- ↑ Patent DE10125885 : Sensor device for fast optical distance measurement according to the confocal optical imaging principle.
- ↑ Chromatic Confocal Sensing (CCS) ( Memento of the original of May 8, 2009 in the Internet Archive ) Info: The archive link was automatically inserted and not yet checked. Please check the original and archive link according to the instructions and then remove this notice.
- ↑ H. Naora: Microspectrophotometry and cytochemical analysis of nucleic acids In: Science. 14, volume. 114, No. 2959, 1951, pp. 279-280.
