Megan Farell, technical sales support specialist, Olympus
One of the factors that contributes to the recent considerable reduction in size and high integration of electronic devices is miniaturisation of the electronic components that make them up. Another is the new, fine functional materials that have been developed and widely used in industrial fields including automobile, aviation, chemical and metals. Higher accuracy and resolving power are required for minute 3D measurement of these components and materials. Although a variety of devices are available to satisfy these requirements, the confocal microscope is becoming one of the preferred choices for easy, non-contact 3D surface profile measurements. This article summarises the basic principles, features and applications of industrial confocal laser scanning microscopes, focusing specifically on the reflection-type confocal laser scanning microscope (hereafter referred to as laser scanning microscope) for high-resolution detection.
Image formation optical system versus confocal optical system
The most basic principle of laser scanning microscopes is the confocal optical system. The image formation optical system in conventional optical microscopes and the confocal optical system are shown in figure 1. The confocal optical system affords better contrast than the image formation optical system, the objective of the latter being to evenly illuminate the sample. The confocal optical system has a circular pinhole opening that conjugates the focal position of the objective lens (image forming position) to only detect light at the focused position. A specific wavelength of laser light, since it travels in a very straight line, is used as the point light source and radiated by the objective lens, resulting in a large amount of light converging at one point on the sample and a reduction in unnecessary scattered light from the environment. The light then reflects off the surface of the sample, goes back along the same optical path, is separated by the beam splitter and converges on the pinhole.
Figure 1
A comparison of the image formation and confocal optical systems.
The confocal optical system obtains only data from the current focal position, since it blocks the reflected light from all places other than the focal position at the pinhole. This means it is able to produce a perfectly focused, clear image that affords high contrast and resolution. An image formation optical system, on the other hand, superimposes data from multiple focal planes. For applications that require higher resolution, such as surface roughness measurements, the confocal optical system is a better choice than the image formation optical system.
In addition, the confocal optical system can act like a height sensor at nanometre-level resolution because it has a narrow depth of focus, but an image formation optical system cannot because it has a large depth of focus that includes unfocused data. Although it is said that the diameter of the pinhole of the confocal optical system should be smaller than the divergence caused by the diffraction of the beam spot, this diameter is determined by balancing the sensitivity of the detector because the amount of light is reduced as the pinhole diameter is narrowed.
Two-dimensional scanning
As already described, the confocal optical system only obtains information in the direction of the optical axis, therefore a 2D scanner system orthogonal to the optical axis is required to convert data into images. A 2D scanner system uses raster scanning to create an image, so its accuracy directly determines imaging performance; accurate 2D scanning is one of the most important technologies used in laser scanning microscopes. 2D scanning systems are generally divided into the sample scan method and the laser scan method.
The sample scan method is performed by scanning the XY stage on which the sample is placed. This method allows the user to create an image of a large area without the need for the optical scanning function, meaning that the confocal optical system has a simpler structure. In addition, the XY stage can be accurately driven relatively easily. However, it takes a very long time to capture data, and, at a high resolving power, it is necessary to drive the XY stage, including the sample for observation, with great precision. The laser scan method is performed by scanning a laser beam onto the sample in two dimensions, X and Y, using two scanning mechanisms. The sample scan method is appropriate for capturing large waves on the sample surface, and the laser scan method is ideal for capturing minute shapes.
Most industrial laser scanning microscopes generally employ the laser scan method to prioritise image acquisition time. For example, an acousto-optic deflector (AOD), a polygon mirror or a resonant galvano mirror is used as the laser scanning mechanism in the X direction because high-speed scanning is required. These scanning mechanisms are briefly summarised below.
Acousto-optic deflector
An AOD is a mechanism that makes use of the diffraction of light. When modulating the frequency of ultrasonic waves and applying it to a material, the change of refractivity in the material functions as the diffraction grid, providing an appropriate light deflection angle. Although an AOD can scan very quickly, the scanning range is limited. In addition, a configuration that leads to descanning (re-incidence of reflected light from the sample) must be avoided as descanning greatly decreases efficiency. For this reason, it is necessary to add a line sensor or other technique. It is also important to exercise sufficient care when using an AOD because the lens effect can cause astigmatism.
Polygon mirror
The polygon mirror mechanism has a very simple configuration and is used in many fields. Mirrors are attached to the faces of a polytope that is rotated at high speed using a motor or other device. The scan speed and angle of the laser is determined based on the number of mirror faces (assuming that the rotation speed is constant). Extremely precise adjustment is required because the rotation accuracy determines the scan accuracy.
Resonant galvano mirror
A resonant galvano mirror is a compact mechanism that supports relatively large oscillation angles. Its speed is determined by mechanical resonant frequency and is therefore limited compared with other scanning mechanisms. However, recent resonant galvano mirrors can acquire several one-megapixel images every second. Mirrors manufactured with microelectromechanical systems (MEMS) technology have also been developed, allowing for a reduction in device size. MEMS scanners are a combination of movable plate, torsion bar and support frame, made by etching a single monocrystalline silicon board. The movable plate has coils driven by a magnetic circuit. Two-dimensional scanning can be accomplished by taking advantage of the characteristics of a high-speed scanning mechanism and combining it with a relatively low-speed scanning mechanism in the Y direction. A non-resonant galvano mirror is often used in the Y direction scanning mechanism, partly as a matter of convenience.
Confocal effect and extended focus image
The effect of the confocal optical system is shown in figure 2. This waveform is generally known as an I-Z curve. The vertical axis shows the output from the detector after the light passes through the pinhole. The output is acquired by moving the sample and objective lens relative to each other in the Z direction, without 2D scanning. The horizontal axis shows the travel distance in the Z direction. Comparing the non-confocal output with the pinhole removed and the confocal output acquired, the confocal optical system reveals a steep I-Z curve.
Figure 2
An I-Z curve.
An image of a stepped sample where all height positions are in focus, known as an extended focus image, can be obtained by scanning the laser light in 2D, moving the sample and objective lens relatively, and saving the brightest light intensity value of each pixel on the image. The process for capturing an extended focus image is shown in figure 3. When the laser light is scanned across and focused on the top face, blurry images are eliminated and the square part is captured. When the laser light is moved in the Z direction and the second face is focused on, the smallest L-shaped part is captured. By sequentially repeating this step and then capturing and overlapping the image of each face, an extended focus image, in which every face of the sample is brought into focus at high resolving power in the horizontal direction, can be created.
Figure 3
Capture of an extended focus image.
Three-dimensional images
In the confocal optical system, the brightest Z position, that is the Z position that affords maximum intensity, reveals the height information of the sample surface at that point. Taking advantage of this fact, the height information of the sample can be captured by recording the Z position that affords maximum intensity.
The process for capturing height information of an image is shown in figure 4. In much the same way as capturing an extended focus image, the sample and objective lens are moved relative to each other from height Z1 to Z2, and both the maximum intensity for each pixel and height information are saved simultaneously in the extended image memory and height image memory, respectively. It is therefore possible to obtain the surface profile of the sample in the image acquisition area, enabling a variety of analyses to be performed. This is the most significant characteristic of confocal microscopes and sets them apart from other microscopes.
Figure 4
Capture of the height information of an image.
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