Dual-detection confocal fluorescence microscopy: fluorescence axial imaging without axial scanning.
We propose a new method for high-speed, three-dimensional (3-D) fluorescence imaging, which we refer to as dual-detection confocal fluorescence microscopy (DDCFM). In contrast to conventional beam-scanning confocal fluorescence microscopy, where the focal spot must be scanned either optically or mechanically over a sample volume to reconstruct a 3-D image, DDCFM can obtain the depth of a fluorescent emitter without depth scanning. DDCFM comprises two photodetectors, each with a pinhole of different size, in the confocal detection system. Axial information on fluorescent emitters can be measured by the axial response curve through the ratio of intensity signals. DDCFM can rapidly acquire a 3-D fluorescent image from a single two-dimensional scan with less phototoxicity and photobleaching than confocal fluorescence microscopy because no mechanical depth scans are needed. We demonstrated the feasibility of the proposed method by phantom studies.
Nano Opto-Mechatronics Laboratory, Department of Mechanical Engineering, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701, South Korea.
Dong-Ryoung
DR
Lee
Young-Duk
YD
Kim
Dae-Gab
DG
Gweon
Hongki
H
Yoo
D017076:Computer-Aided Design; D004867:Equipment Design; D019544:Equipment Failure Analysis; D007089:Image Enhancement / Q000295:instrumentation* / Q000379:methods; D021621:Imaging, Three-Dimensional / Q000295:instrumentation* / Q000379:methods; D007909:Lenses; D018613:Microscopy, Confocal / Q000295:instrumentation* / Q000379:methods*; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation* / Q000379:methods; D057054:Molecular Imaging / Q000295:instrumentation* / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity