In 1957, Marvin Minsky applied for a patent in which the basic principle of confocal microscopy is described for the first time. But it took another 30 years and the development of the laser as a light source until confocal microscopy became a standard microscopy technique. The following is a brief overview of confocal microscopy as it is used today in laboratories and industrial applications.
Conventional light microscopes allow a detailed view of an object through a two-stage, magnifying image. In this image, the microscope's optics have a finite depth of field, similar to the (smaller) image with a camera. This means that the image of the object is a superimposition of a sharp image of the points in the focal plane and a blurred image of points outside the focal plane, which are still recognized as "sharp" by the detector (eye, camera line). This depth of field prevents the resolution of object details in axial direction. The confocal image reduces this depth-of-field range extremely and enables virtual optical intersections through the object in axial direction with corresponding detailed information. The trick is a point-to-point to point mapping as showed in the following figure.
The light from a point source (light source plus a pinhole diaphragm) is optically reflected into a point in the focal plane of the lens on or in the target. The light emanating from this illuminated object point (fluorescent light, reflected light) is represented by the same optics and a beam splitter on a pinhole screen in front of the detector.
The detection focus is therefore in the plane conjugated to the focal plane of the lens, i. e. both focuses are superimposed (= confocal). Light that does not come from the focal plane is suppressed because it is not focused on the pinhole, but appears there as a slice, so that it is almost completely blocked.
In the figure on the right, this is represented by the dotted beam paths for one level above and one below the focal plane. Scattered light is also almost completely blocked by this pinhole. This significantly increases contrast and improves lateral resolution. With the confocal imaging, the lateral resolution (still diffraction limited) can be better by a factor of 1.4 compared to conventional microscopy, depending on the size of the pinhole aperture. It should be noted that due to diffraction, the light spot on the sample is actually a spot of finite size (Airy disk).
Therefore, the size of the pinhole must be optimized to the size of this spot. A pinhole aperture that is too small also reduces the amount of light that can be detected, a aperture that is too large allows too much light from outside the focal plane and too much diffuse light.
How is an image of the entire object created?
Since with this point-to-point to point imaging it is only possible to obtain light from one point of the sample, it is necessary to scan the sample and to put the image together on the computer. There are two basic types of confocal microscopes that differ in the type of scanning in the x-y plane: confocal laser scanning microscopes and confocal microscopes with rotating disk.
Confocal laser scanning microscopes
The most commonly used confocal microscope type, the confocal laser scanning microscope (or CLSM for confocal laser scanning microscope), uses a laser for illumination that illuminates the object at the focal plane and excites fluorescent molecules at each point. The fluorescent light is imaged on an aperture in the image plane, whereby only the light coming directly from the focal plane is detected with a photomultiplier or an avalanche photodiode.
The complete focal plane is mapped in x and y direction by means of a raster head. The laser beam is guided over the object with a movable mirror system.
The laser is used to excite fluorescence in certain molecules. Fluorescence refers to the process of absorption of short-wave (high energy) light and the subsequent spontaneous emission of long-wave (low energy) light. Through this process and with the aid of a dichroic mirror, optical excitation is decoupled from optical detection. This makes it possible, for example, by using different fluorescent dyes and excitation sources, to examine different areas on or in the same sample.
The diameter of the detector aperture, together with the microscope lens and its numerical aperture, determines the thickness of the optical section. This lies in the range of 2 to 2.5 times the lateral resolution. A three-dimensional reconstruction of the depicted object is obtained by recording several sections in different focal planes.
Through the detection with a photomultiplier, even very low intensities can still be detected.
Due to the relatively slow line-by-line construction, the confocal image can only be viewed on the screen. However, the measurement rate of confocal scanning laser microscopes has been improved by the use of acousto-optical deflectors and MEMS-based mirrors down to video rates.
Using fluorescence means a certain amount of sample preparation. As a result, these microscopes are less suitable for use in production, manufacturing and quality assurance, and are used more in the biological and medical fields.
The image size depends on the magnification of the lens, but can be adapted to the individual requirements of different examinations by stitching several adjacent individual images.
Rotating disk confocal microscopes
Instead of a laser, a more broadband light source can also be used in scanning microscopes. Usually, however, more broadband light sources are used, mostly LEDs, in connection with simultaneous illumination and observation of several points on the object. The light spots are created here by a rotating disc with helically arranged square or round holes, the Nipkow disc (multipinhole filter), which is known from black and white television technology. The aperture apertures in the lens also serve as a detector aperture and block the light outside the focal plane and diffuse light.
The cross sections are recorded with a CCD video camera. This type of confocal microscopy does not use fluorescence, but detects the light reflected by the object in the focal plane. This also makes it possible to be used in production, manufacturing and quality assurance where technical surfaces are examined and complex sample preparation is undesirable.
Due to the fast image construction with video rates, direct observation is possible on the one hand and very fast processes can be followed on the other. The versatility of this measuring system allows the measurement of geometries and roughness in the range from a few micrometers to several nanometers. This is achieved by precisely shifting the focal plane with piezo actuators.
The image size also depends on the magnification of the lens, but can be adapted to the individual requirements of different examinations by stitching several adjacent individual images.