Confocal microscopy

Confocal Laser Scanning Microscopy (CLSM) is used as an optical 3-dimensional (3D) surface profiler with a high-resolution. With a high numerical aperture objective lens (up to 0.95 in the air) and a shorter wavelength laser, it provide a high-resolution imaging along both axial and lateral direction.

A confocal pinhole improves the imaging quality by rejecting the noise outside of the focal point. Real time imaging is achieved by a fast scanning module and a signal processing algorithm. It takes a few seconds to get the 3D surface profile of the sample. CLSM is a non-destructive high-resolution optical 3D surface profiler for the micro-structure. It is an ideal solution to measure and inspect the semiconductor, FPD products, MEMS devices, glass surfaces, and material surfaces.

The height measuring ability comes from the confocal setup of a source, a target sample, and a detector. When the sample is located in the focal plane of the objective lens, the light reflected at the surface of the sample is focused at the confocal aperture, and the photo detector collects the signal from the sample. However, when the sample is placed in the out-of-focus position, the light signal is rejected by the confocal aperture, pinhole. Thus only the in-focus signal is collected by the photo detector. It gives the optical sectioning ability to the CLSM.

To make 2D image from this in-focus signal, signals from each single point should be gathered by 2D scanning mechanism which moves the focal points pixel by pixel. This in-plane 2D image is an optically sectioned image. To construct 3D image, the sequential 2D images are gathered by axial scanning mechanism which moves the focal plane step by step. More briefly, the confocal 2D image is made by ‘point-by-point’ scanning, and the confocal 3D image is built by ‘plane-by-plane’ scanning.

To get the 3D surface profile of the sample, optical sections are collected through the z-axis, just like 3D reconstructing plane-by-plane scanning. Due to the confocal aperture, the light intensity becomes maximum when the sample surface is placed in the focal plane. It is just like an ‘optical stylus’, of which the probing tip is a focused beam spot.

CLSM can be used for many applications in the industrial fields because it is a fast, nondestructive, and reliable 3D surface profiler. It can measure 3D shape, step height and volume of the micro structures. CLSM is widely used for the biological researches. With a fluorescence die, it can get a clear image of biological specimens maintaining its own nature. 3D structure and dynamics of the cells also can be observed.