Elsevier

Radiation Measurements

Volume 40, Issues 2–6, November 2005, Pages 311-315
Radiation Measurements

Development of a high speed imaging microscope and new software for nuclear track detector analysis

https://doi.org/10.1016/j.radmeas.2005.02.013Get rights and content

Abstract

Automated digital imaging optical microscopy is widely used for diagnostic applications in the health care and biology fields and for routine inspection in industrial applications such as semiconductor fabrication. These applications require the imaging of large areas at high speed in order to obtain sufficient data for image processing with good statistics. Track detector analysis also benefits from the rapid acquisition of large areas on the detector surface. We have developed a new microscope system, the HSP-1000, for high speed image acquisition that uses a line sensor camera in place of a traditional CCD camera. Continuous, automatic focusing of the microscope is achieved by means of an optical pick-up system that provides fast feedback for control of distance between the objective and the image surface. Using transmitted light illumination, the microscope is able to digitize a 1cm2 area at 0.35μm/pixel resolution in 20s. As a result of continuous stage motion and continuous focusing, we have attained image acquisition speeds that are 50–100 times faster than conventional CCD-based microscope systems. In this paper, we describe a number of aspects of the microscope system including the use of the line sensor and the automatic focus system.

Introduction

In recent years, automated digital imaging in optical microscopy has achieved widespread application not only for the analysis of nuclear track detectors (Trakowski et al., 1984, Ashaboglu et al., 1989, Price and Krischer, 1985, Fews, 1992, Espinosa et al., 1996, Dolleiser and Hasemi-Nezhad, 2002, Weaver and Westphal, 2002, Boukhair et al., 2000), but for a number of different fields (Inoue and Spring, 1997). Most often, an automated optical microscope system is equipped with a charge couple device (CCD) camera, a computer controlled stage and an autofocus drive. In such systems, the size of each image is limited by the area of CCD element and, although dependent on total magnification, typical image sizes are limited to several hundred micrometers square. In order to digitize substantially larger areas, the total image must be reconstructed out of a composite of many smaller images captured individually by the CCD camera. This method, often referred to as “image tiling,” is illustrated on the left in Fig. 1. A major limitation of this method is the time consumed by the mechanical movement of the stage, followed by auto-focusing, between the capture of each individual image (Dolleiser and Hasemi-Nezhad, 2002).

In this paper, we describe the HSP-1000, a new microscope system for capturing large images (>1cm2) in relatively short periods of time (<1min). To achieve this high rate of image acquisition, the HSP-1000 microscope system makes use of a line sensor in place of the traditionally-used CCD camera and the microscope stage is constantly in motion. In this way, the microscope objective sweeps over the sample, continuously acquiring the image in much the same way as a desktop scanner or fax machine. As illustrated on the right in Fig. 1, the use of a line sensor permits not only the continuous acquisition of image data, but the image can have considerably greater width than that typically acquired using a CCD camera. A complete image of the entire sample surface can then be reconstructed from a relatively small number of long strips, rather than the much larger number of discrete square CCD images. In addition to its use in the analysis of nuclear track detectors, such a microscope system has many potential applications, especially in medicine, biology, high technology industry, or whereever large-scale acquisition of microscopic images is required.

Section snippets

Overview

A dedicated controller is used to coordinate the various subsystems of the microscope (illumination, autofocus, stage movement and image capture) and to interface these subsystems with a personal computer. The microscope has light sources for both transmitted and incident light illumination. Image capture is achieved through use of an optical line sensor at the top of the microscope. For the monochrome system, the resolution is 256 greyscale values. A CCD camera is also included in the system,

Autofocus

In order to verify the autofocus performance, motion of the XY stage was fixed, and the change in value of the autofocus signal AF=[(A+C)-(B+D)]/[A+B+C+D] was measured as a function of Z height (in 0.25μm steps). The AF values, plotted as a function of Z height relative to the image plane, are shown in Fig. 3. The AF value provides information concerning both the direction and distance that the Z-axis motor must move to achieve sharp focus.

The method of continuous focusing is responsible for

Image analysis

Fig. 4 shows an example of a CR-39 nuclear track detector image reconstructed from three long image strips captured by the HSP-1000 microscope. The size of one strip is 1000 pixels (the center 1000 pixels of 4096 pixels/line) ×20,000pixels and 3000pixels×20,000pixels in total.

With the HSP-1000, acquisition of the detector image and the analysis of the image data for the location and measurement of nuclear track etch pits are carried out independently from one another. This approach lends

Conclusions

A new microscope for image digitizing, the HSP-1000, was developed using a monochrome line sensor camera instead of a CCD camera. An optical pick-up system is used in the autofocus unit for surface detection, enabling fast feedback for continuous height control above the image plane. This makes it possible for the microscope stage to always be in motion during the acquisition of the image. In case of transmission illumination, the HSP-1000 is able to digitize an area of 1cm2 within 20 s at 0.35μm

Acknowledgements

We would like to extend our thanks to S. Inagaki, I. Kobayashi and S. Takayama of Matsudo Medical Laboratory for their valuable comments and for preparation of samples. We are grateful to K. Komata and I. Ishizuka in Opcell Inc. for theoretical aspects of optics for the autofocus unit. This work has been supported by the government-private sector joint research project in NIRS and was supported partially by Grants-in-Aid for Scientific Research from the Japan Ministry of Education, Culture,

References (10)

There are more references available in the full text version of this article.

Cited by (90)

View all citing articles on Scopus
View full text