KEYWORDS: Confocal microscopy, 3D metrology, Microscopy, 3D image processing, Profiling, High speed imaging, 3D scanning, Sensors, Tissues, Luminescence
We propose a new method for three-dimensional (3-D) imaging without depth scanning that we refer to as the dual-detection confocal microscopy (DDCM). Compared to conventional confocal microscopy, DDCM utilizes two pinholes of different sizes. DDCM generates two axial response curves which have different stiffness according to the pinhole diameters. The two axial response curves can draw the characteristics curve of the system which shows the relationship between the axial position of the sample and the intensity ratio. Utilizing the characteristic curve, the DDCM reconstructs a 3-D surface profile with a single 2-D scanning. The height of each pixel is calculated by the intensity ratio of the pixel and the intensity ratio curve. Since the height information can be obtained directly from the characteristic curve without depth scanning, a major advantage of DDCM over the conventional confocal microscopy is a speed. The 3-D surface profiling time is dramatically reduced. Furthermore, DDCM can measure 3-D images without the influence of the sample condition since the intensity ratio is independent of the quantum yield and reflectance. We present two types of DDCM, such as a fluorescence microscopy and a reflectance microscopy. In addition, we extend the measurement range axially by varying the pupil function. Here, we demonstrate the working principle of DDCM and the feasibility of the proposed methods.
We propose a new method for three-dimensional fluorescence imaging without depth scanning that we refer to as the
dual detection confocal fluorescence microscopy (DDCFM). Compared to conventional beam-scanning confocal
fluorescence microscopy, where the focal spot must be scanned either optically or mechanically to collect a three-dimensional
images, DDCFM is able to obtain depth information without depth scanning. DDCFM utilizes two photo
multiplier tubes (PMTs) in the confocal detection system. The emitted fluorescence is divided by the beam splitter and
received by the two PMTs through pinholes with different size. Each PMT signal generates different axial response
curve according to the pinhole diameter, which decides stiffness of the curve. Since the PMT signal is determined by the
intensity of the fluorescent emitter and the distance from the focal point, we can acquire depth position of a fluorescent
emitter by comparing two intensity signals from the PMTs. Since the depth information can be obtained by a single
excitation without depth scanning, DDCFM has many advantages. The measurement time is dramatically reduced for
volume imaging. Also, photo-bleaching and photo-toxicity can be minimized. The system can be easily miniaturized
because no mechanical depth scan is needed. Here, we demonstrate the feasibility of the proposed method by phantom
study using fluorescent beads.
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