Advances in Confocal Laser Scanning Microscopy
From silicon semiconductor manufacture and corrosion analysis, to even archaeological investigations: Surface metrology is rapidly emerging as a vital analytical technique to determine the materials' topology. This technique requires the use of a microscope able to accurately and repeatedly visualize even the minutest of details: a measuring confocal laser scanning microscope.
Clear and precise images with a high-level of resolution are required for the analysis of surfaces properties. These images resolve successfully minute surface details so that they can be distinguished from other details within close proximity. A number of different technologies exist for achieving these aims. For example, surface profilers literally drag an arm over the surface of the material. Scanning electron microscopes require large samples to be broken up and need to be coated before being placed into the vacuum chamber. Not only do these instruments cause damage to the sample, but the SEM also requires a large amount of sample preparation. Confocal-based optical metrology instruments on the other hand are non-contact and the majority of samples can be placed directly on the stage with no preparation.
As such, Olympus developed an optical metrology tool in the form of a mea-suring confocal laser scanning microscope (mcLSM) which determines layer thickness, roughness and surface structures. The system uses a 405 nm laser with highly tuned optics to provide extremely high resolution (120 nm line and space). The mcLSM combines the ability to accurately image specimens in focus, with the non-contact capabilities of laser scanning technology. Therefore, this technique can successfully produce exceptionally clear and detailed optical images of samples that may have previously been difficult to resolve.
Polarization Instead of Fluorescence
Confocal systems used for optical metrology, have a different optical set-up to life science cLSMs. This is because there is no fluorescence excitation/emission and therefore the wavelength of the light impinging the surface of the sample is the same as that reflected back to the detectors. As a result, a dichroic mirror cannot be used to separate the illumination (excitation) and reflection (emission) pathways.
Instead, the system uses polarization to separate the two pathways: Laser light by its nature is plane polarized and as it reflects of a surface the plane is changed to varying degrees depending on the surface materials and features. As a result, as the reflected light passes back up through the microscope it can be differentiated from the illumination light path. This is best achieved by using a polarizing beam splitter, which lets the illumination light through in one direction, but redirects the reflected light to the photomultiplier tube (PMT). To fulfill the requirements of confocal imaging the system must incorporate a pinhole in front of the PMT to block out of focus light. The PMT is then able to covert the photons of light into a digital image.
As light passes through lenses and prism, or is reflected of mirrors, its properties can change and as a result spherical wavefront aberrations are introduced which cause deterioration to the clarity of the image. The optics within the system is therefore designed to eliminate these aberrations in order to provide a sharp and focused image.
Laser confocal technology using a 405 nm laser provides an exceptional level of detail but lacks color information, which may be important in the interpretation of features on some samples. As a result optical microscopy systems use the same optics to generate reflected brightfield images which can be combined with the confocal image to provide high resolution full color images. That is the reason why the optics also needs to be corrected for chromatic aberrations (aberrations caused by the different transmission properties of different wavelengths through the lenses, prisms and mirrors).
Dual Pinhole Technology
The quality of the image generated in a mcLSM system is not only influenced by the optics, but also by the presence and size of the pinhole. The system is only able to produce digital images from the light that has passed through this tiny hole located directly in-front of the detector. Therefore the confocal effect is altered by changing the size of the pinhole - smaller pinholes exclude more light, whereas larger pinholes let more light and subsequently more out of focus blur pass. The smaller the pinhole though, the thinner the depth of field within each image and therefore if samples have very steep slopes there is not enough information within the optical slice to generate an image of the slope. That is why instruments, such as the Olympus OLS4000 Lext, have been designed in that way that they split the reflected light signal as it passes back through the polarized beam splitter into to equal light paths. One of these is directed towards a larger pinhole and the other towards a small pinhole. Each of the PMTs utilizes a different analogue to digital conversion circuitry suitable to the image information being collected. The resulting output from the two pinholes is combined and displayed by the software and the percentage contribution of each one to the image can be easily adjusted to provide the user with control over the final image. As a result, slopes of up to 85º can be clearly visualized and measured.
MEMS-based Laser Scanner
In order to produce a digital image, the laser light beam is focused in the XY plane and scans across the sample surface, line by line. The reflected light is then de-scanned by the same mirror. Two key properties of the scanner that can be optimized to provide superior system performance are the size and the speed of the scanner drive: The larger the mirror, the bigger the optical zoom possible, providing much more flexibility in imaging, especially when in live scan mode. Many mcLSM systems use galvanic mirror-based scanners which provide the accuracy required to generate the raster scan pattern at all different magnifications. More recently, micro electromechanical system (MEMS) technology has provided the capability to significantly increase the scan speed.
A laser does not touch the surface to obtain roughness measurements and does not get stuck on adhesive or complex surface features, like traditional scanning cantilevers. mcLSM optical metrology instruments can be set to linescan such that the instrument measures the z displacement along a defined line in any xy direction. This gives the same roughness information as the traditional contact systems but in much less time. What is more, the confocal system can perform area roughness scans where the roughness of an entire area of the sample is measured. This is an emerging technique which provides a much better idea of overall surface roughness and corresponding standards have recently been introduced (ISO 25178 - Geometric Product Specifications [GPS] - Surface texture: areal).
Optical metrology is a rapidly emerging technique within materials science. The mcLSM optical metrology systems, such as the Olympus Lext OLS4000, make it easier to image and analyze minute details of a surface. The use of specially designed optics removes the associated issue of wavefront aberrations, ensuring that a clear, precise image is obtained from every sample. Combined with dual pinhole technology, and an advanced high-speed MEMS laser scanner, clear visualization of even the most complex surface topology enables the measurement of ultra-fine surface detail.