Simultaneous dual-color fluorescence microscope: a characterization study.
BACKGROUND
High spatial resolution and geometric accuracy is crucial for chromosomal analysis of clinical cytogenetic applications. High resolution and rapid simultaneous acquisition of multiple fluorescent wavelengths can be achieved by utilizing concurrent imaging with multiple detectors. However, such class of microscopic systems functions differently from traditional fluorescence microscopes.


OBJECTIVE
To develop a practical characterization framework to assess and optimize the performance of a high resolution and dual-color fluorescence microscope designed for clinical chromosomal analysis.


METHODS
A dual-band microscopic imaging system utilizes a dichroic mirror, two sets of specially selected optical filters, and two detectors to simultaneously acquire two fluorescent wavelengths. The system's geometric distortion, linearity, the modulation transfer function, and the dual detectors' alignment were characterized.


RESULTS
Experiment results show that the geometric distortion at lens periphery is less than 1%. Both fluorescent channels show linear signal responses, but there exists discrepancy between the two due to the detectors' non-uniform response ratio to different wavelengths. In terms of the spatial resolution, the two contrast transfer function curves trend agreeably with the spatial frequency. The alignment measurement allows quantitatively assessing the cameras' alignment. A result image of adjusted alignment is demonstrated to show the reduced discrepancy by using the alignment measurement method.


CONCLUSIONS
In this paper, we present a system characterization study and its methods for a specially designed imaging system for clinical cytogenetic applications. The presented characterization methods are not only unique to this dual-color imaging system but also applicable to evaluation and optimization of other similar multi-color microscopic image systems for improving their clinical utilities for future cytogenetic applications.
School of Electrical and Computer Engineering, University of Oklahoma, Norman, OK, USA.
Zheng
Z
Li
College of Precision Instruments and Opto-electronics Engineering, Tianjin University, Tianjin, China.
Xiaodong
X
Chen
School of Electrical and Computer Engineering, University of Oklahoma, Norman, OK, USA.
Liqiang
L
Ren
School of Electrical and Computer Engineering, University of Oklahoma, Norman, OK, USA Department of Biology, Mudanjiang Medical University, Mudanjiang City, China.
Jie
J
Song
School of Electrical and Computer Engineering, University of Oklahoma, Norman, OK, USA.
Yuhua
Y
Li
School of Electrical and Computer Engineering, University of Oklahoma, Norman, OK, USA.
Bin
B
Zheng
School of Electrical and Computer Engineering, University of Oklahoma, Norman, OK, USA.
Hong
H
Liu
D002877:Chromosomes, Human / Q000235:genetics; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*
Fluorescence microscopy; imaging systems; medical imaging