Confocal microscopy

컨포컬 현미경(confocal microscope)기술은 가장 진보된 방식의 광학 현미경 기술입니다. 컨포컬 현미경은 일반 광학 현미경의 특성을 유지하면서도 높은 해상도로 3 차원 형상을 측정할 수 있기 때문에, 다양한 마이크로미터 스케일의 3 차원 측정이 필요한 대부분의 분야에서 활용할 수 있습니다. 컨포컬 현미경의 원리는 1950년대 중반에 최초로 고안이 되었으나, 충분한 컨포컬 현미경의 성능 구현을 위해서는 레이저와 디지털 이미징 소프트웨어를 필요로 하였기 때문에 당시에는 그 가능성의 일부만을 구현할 수 있었습니다. 현대의 컨포컬 현미경과 기계적으로 유사한 형태는 관련된 컴퓨터 기술과 레이저 기술이 가능했던 1970년 후반에 이르러 구현될 수 있었으며, 1980년대에 이르러서는 생물학 분야를 중점으로 본격적인 보급이 되었는데, 특히 컨포컬 현미경의 형광 영상화 기술은 많은 생물학 연구자들에게 한 단계 높은 시야를 제공해주어 생물 분야의 발전을 촉진시키는데 지대한 역할을 하였습니다.

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.

Extracting depth (or height) information directly

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.