Three dimensional sidewall measurements by laser fluorescent confocal microscopy

Abstract

Precise three dimensional (3D) profile measurements of vertical sidewalls of concave micro-structures are impossible by conventional profiling techniques. This paper introduces a simple technique which can obtain 3D sidewall geometry by means of laser fluorescent confocal microscopy and an intensity gradient algorithm. The measurement principle is: when a concave micro-structure is filled up with fluorescent solution, the position where the maximum intensity variation lays represents the profile of the micro-structure in the fluorescent 3D volume image. The physical essence behind this measurement principle is analyzed in this paper in detail. The strengths and limitations of this technique are studied by experiments or by illustrations. The factors that are able to improve the measurement accuracy are discussed. This technique has demonstrated the capability for measuring of 3D geometry of various concave features, such as vertical, buried and other micro channels with sub-mum (RMS) measurement accuracy and repeatability.

Measurement of buried undercut structures in microfluidic devices by laser fluorescent confocal microscopy

“…3(b). According to [46], the profiled surface is at the position where the gradient intensity is the maximum. The 3D matrix in the topography software was thus differentiated along the width direction to achieve the 3D profile of the two sidewalls.…”

“…Laser fluorescent confocal microscopy was found to calculate the volume and surface area [44] or to obtain the surface profile [45] from the 3D volume fluorescent image by using the fixed intensity threshold algorithm. However, this algorithm was proved as unreliable for obtaining accurate results [46]. Guilak [44] also acknowledged that the changes in intensity threshold significantly altered the absolute values obtained, which contradicts with the common knowledge that the volume or area of an object should not be determined by the threshold algorithm in software.…”

“…Guilak [44] also acknowledged that the changes in intensity threshold significantly altered the absolute values obtained, which contradicts with the common knowledge that the volume or area of an object should not be determined by the threshold algorithm in software. In fact, the adaptive intensity threshold has to be applied to extract an accurate result, as discussed in our previous work [46]. Exactly speaking, the profiled surface is at positions of maximum intensity variation.…”

Affine calibration based on invariable extrinsic parameters for stereo light microscope

“…[1][2][3] For most applications, the significant issue is to measure three-dimensional (3-D) geometric information at the microscopic scale. [1][2][3] For most applications, the significant issue is to measure three-dimensional (3-D) geometric information at the microscopic scale.…”

Microscopic vision based on the adaptive positioning of the camera coordinate frame

“…Traditional microscopic measurement mainly based on optomechanics has developed into microscopic vision measurement characterized by automatic imaging in high resolution and accurate processing in real time. The microscopic vision measurement is widely applied in geometric metrology of biological objects and small industrial workpieces, and also visual servo for cell projection and MEMS microoperation (Hasegawa et al,2008; Kim et al,1990; Li et al,2008; Sitti and Hashimoto,2000; Wang et al,2008; Windecker et al,1997; Yu and Nelson,2001; Zhu et al,2006). …”