Taejoong Kim

Chromatic confocal microscopy with a novel wavelength detection method using transmittance.

Chromatic confocal microscopy (CCM) is a promising technology that enables high-speed three-dimensional surface profiling without mechanical depth scanning. However, the spectrometer, which measures depth information encoded by axial color, limits the speed of three-dimensional imaging. We present a novel method for chromatic confocal microscopy with transmittance detection. Depth information can be instantaneously obtained by the ratio of intensity signals from two photomultiplier tubes by detecting a peak wavelength using transmittance of a color filter. This non-destructive and high-speed surface profiling method might be useful in many fields, including the semiconductor and flat panel display industries, and in material science.

Enhancement of fluorescence confocal scanning microscopy lateral resolution by use of structured illumination

Confocal microscopy is an optical imaging technique used to reconstruct three-dimensional images without physical sectioning. As with other optical microscopes, the lateral resolution of the confocal microscope cannot surpass the diffraction limit. This paper presents a novel imaging system, structured illumination confocal scanning microscopy (SICSM), that uses structured illumination to improve the lateral resolution of the confocal microscope. The SICSM can easily be implemented by introducing a structured illumination generating optics to conventional line-scanning fluorescence confocal microscopy. In this paper, we report our analysis of the lateral and axial resolutions of the SICSM by use of mathematical imaging theory. Numerical simulation results show that the lateral resolution of the SICSM is 1.43-fold better than that of the confocal microscope. In the axial direction, however, the resolution of the SICSM is ~15% poorer than that of the confocal microscope. This deterioration arises because of a decrease in the axial cut-off frequency caused by the process of generating structured illumination. We propose the use of imaging conditions under which a compromise between the axial and lateral resolutions is chosen. Finally, we show simulated images of diversely shaped test objects to demonstrate the lateral and axial resolution performance of the SICSM.

Cross structured illumination for high speed high resolution line scanning confocal microscopy

Previous research presented the structured illumination confocal scanning microscope (SICSM) so as to improve the lateral resolution of the confocal microscope. However, the image acquisition speed of the SICSM is very slow and also an alignment error due to the mechanical rotation of a grating and a slit can easily occur. As a theoretical study, in this paper we propose a new SI method, the cross SI method, which improves lateral resolution and image acquisition speed. Performances of the conventional SI and the proposed SI methods are compared by analysis of the modulation transfer function. The proposed SI method shows similar lateral resolution and can shorten the image acquisition time compared to the conventional SI method. The cross structured illumination confocal microscope (CSICM) is combined with the cross SI pattern optics and the line scanning confocal microscope. We have introduced a 2-D diffractive grating, four linear polarizers and four cylindrical lenses in order to create the cross SI pattern. The effects of the cross SI pattern, intensity and visibility, on the system performance are analyzed. The CSICM has double the lateral resolution of the conventional microscope, an optical sectioning ability and a fast image acquisition speed.

Optimum conditions for high-quality 3D reconstruction in confocal scanning microscopy

Confocal Scanning Microscopy (CSM) is very useful to reconstruct 3D image of Bio-cells and the objects that have specification shape in higher axial and lateral resolution and widely used as measurement instrument. A 3D reconstruction is used to visualize confocal images and consists of following processes. The First process is to get 3D data by collecting a series of images at regular focus intervals (Optical Sectioning). The Second process is to fit a curve to a series of 3D data points each pixel. The Third process is to search height information that has maximum value from curve-fitting. However, because of various systematic errors (NOISE) occurred when collecting the information of images through Optical Sectioning and large peak deviation occurred from curve-fitting error, high quality 3D reconstruction is not expected. Also, it takes much time to 3d Reconstruction by using many 3D data in order to acquire high quality and much cost to improve signal-to-noise (SNR) using a higher power laser. So, we are going to define SNR, peak deviation and the order of curve-fitting as important factors and simulate the relation between the factors in order to find a optimum condition for high quality 3D reconstruction in Confoal Scanning Microscopy. If we use optimum condition obtained by this simulation, using a suitable SNR and the suitable number of data and the suitable n-th order curve-fitting, small peak deviation is expected and then, 3D reconstruction of little better quality is expected. Also, it is expected to save.

Design and analysis of a cross-type structured-illumination confocal microscope for high speed and high resolution

There have been many studies about a super resolution microscope for many years. A super resolution microscope can detect the physical phenomena or morphology of a biological sample more precisely than conventional microscopes. The structured-illumination microscope (SIM) is one of the technologies that demonstrate super resolution. However, the conventional SIM requires more time to obtain one resolution-enhanced image than other super resolution microscopes. More specifically, the conventional SIM uses three images with a 120° phase difference for each direction and three different directions are image-processed to make one resolution enhancement by increasing the optical transfer function in three directions. In this paper, we present a novel cross structured-illumination confocal microscope (CSICM) that takes the advantage of the technology of both SIM and the confocal microscope. The CSICM uses only two directions with three phase difference images, for a total of six images. By reducing the number of images that must be obtained, the total image acquisition time and image reconstruction time in obtaining the final output images can be decreased, and the confocal microscope provides axial information of the sample automatically. We demonstrate our method of cross illumination and evaluate the performance of the CSICM and compare it to the conventional SIM and the confocal microscope.

Beam Scanning Confocal Differential Heterodyne Interferometry

This article presents a new approach to the design of confocal differential heterodyne interferometer (CDHI), which is a combination of differential heterodyne interferometer (DHI) with confocal laser scanning microscopy (CLSM). The CDHI can measure a step height over a quarter of wavelength of the light source, which can not be accurately measured by DHI and CLSM, respectively. The approach is that it utilizes a beam-scanning method instead of transporting a sample to be measured. The measurement results carried out for samples having step height are found to be comparable in precision with those measured by commercial scanning electron microscopes.

High Precision Measurement of 3D Profile Using Confocal Differential Heterodyne Interferometer

The differential heterodyne interferometer (DHI) is suitable for precise measurement of step height and line width, since its differential configuration can significantly reduce disturbances from the environment [1,2]. Like most phase measuring interferometers, however, the DHI is limited, in that it can obtain only the phase from 0 to 2π, because of the sinusoidal nature of the optical interference involved. Thus, the measurable step height is limited to one quarter of the wavelength of the light source. This study describes a confocal differential heterodyne interferometer (CDHI) for measuring step heights of several micrometers, with a high resolution and line width with high repeatability. The CDHI has a simple structure and rapid measurement speed.

Line scanning confocal microscopy with the use of cross structured illumination

In this paper, we propose new SI method, the cross SI method that improves the lateral resolution and the image acquisition speed. The cross SI pattern is generated by using the 2-D diffractive grating. The acquisition of a total of 6 raw images shortens the image acquisition time. The cross structured illumination confocal microscope (CSICM) is combined with the cross SI pattern generation optics and the line scanning confocal microscope. Performances of the conventional and the cross SI are compared by the analysis of the modulation transfer function. As a result, the cro ss SI method shows similar resolution to conventional SI method. The CSICM has the two times enhanced lateral resolution than the conventional microscope, the optical sectioning ability and the fast image acquisition speed.

Effect of scanning rate on the image contrast in confocal microscopy for biological application

In this research, the method how to estimate the image quality for different scanning rate is suggested and experimentally shown with the laboratory-built confocal laser scanning microscope. The confocal microscope is designed for in vivo reflectance imaging of a biological tissue, which uses the refractive index mismatch at the boundaries of a tissue to generate an image without any additional staining process. The two-dimensional scanning mechanism is built up with a polygonal mirror and a galvanometric mirror that can be controlled to operate at a specific speed. To examine the effect of scanning rate on the image contrast, confocal scanning images of a biological specimen are acquired with various scanning rate while the other conditions are kept same. The contrast of confocal microscopic image is transformed into the numeric expression to describe the relation between image contrast and scanning rate quantitatively. Results suggest some useful methodology of how to determine the allowable maximum scanning rate for the specific application of confocal microscopy.

Measurement of Sub-micrometer Features Based on The Topographic Contrast Using Reflection Confocal Microscopy

We describe the design and the implementation of video-rate reflection confocal scanning microscopy (CSM) using an acousto-optical deflector (AOD) for the fast horizontal scan and a galvanometer mirror (GM) for the slow vertical scan. Design parameters of the optical system are determined for optimal resolution and contrast. The OSLO simulations show that the performances of CSM are not changed with deflection angle and the wavefront errors of the system are less than 0.012λ. To evaluate the performances of designed CSM, we do a series of tests, measuring lateral and axial resolution, real time image acquisition. Due to a higher axial resolution compared with conventional microscopy, CSM can detect the surface of sub-micrometer features. We detect 138㎚ line shape pattern with a video-rate (30 frm/sec). And 10㎚ axial resolution is archived. The lateral resolution of the topographic images will be further enhanced by differential confocal microscopy (DCM) method and computational algorithms.