Young Jae Won

Referencing techniques for the analog mean-delay method in fluorescence lifetime imaging

The analog mean-delay (AMD) method is a new powerful alternative method in determining the lifetime of a fluorescence molecule for high-speed confocal fluorescence lifetime imaging microscopy. Even though the photon economy and the lifetime precision of the AMD method are proven to be as good as those of the state-of-the-art time-correlated single photon counting method, there have been some speculations and concerns about the accuracy of this method with respect to the absolute lifetime value of a fluorescence probe. In the AMD method, the temporal waveform of an emitted fluorescence signal is directly recorded with a slow digitizer whose bandwidth is much lower than the temporal resolution of the lifetime to be measured. We have found that the drifts and the fluctuations of the absolute zero position in a measured temporal waveform are the major problems in the AMD method. We have proposed electrical and optical referencing techniques that may suppress these errors. It is shown that there may exist more than 2 ns drift in a measured temporal waveform during the period of the first 12 min after electronic components are turned on. The standard deviation of a measured lifetime after this warm-up period can be as large as 51 ps without any referencing technique. We have shown that this error can be reduced to 9 ps with our electronic referencing technique. It is demonstrated that this can be further reduced to 4 ps by the optical referencing technique we have introduced.

Fluorescence lifetime imaging of lipids during 3T3-L1 cell differentiation

Obesity is becoming a big health problem in these days. Since increased body weight is due to increased number and size of the triglyceride-storing adipocytes, many researchers are working on differentiation conditions and processes of adipocytes. Adipocytes also work as regulators of whole-body energy homeostasis by secreting several proteins that regulate processes as diverse as haemostasis, blood pressure, immune function, angiogenesis and energy balance. 3T3-L1 cells are widely used cell line for studying adipogenesis because it can differentiate into an adipocyte-like phenotype under appropriate conditions. In this paper, we propose an effective fluorescence lifetime imaging technique which can easily distinguish lipids in membrane and those in lipid droplets. Nile red dyes are attached to lipids in 3T3-L1 cells. Fluorescence lifetime images were taken for 2 week during differentiation procedure of 3T3-L1 cells into adipocytes. We used 488 nm pulsed laser with 5MHz repetition rate and emission wavelength is 520 nm of Nile Red fluorescent dye. Results clearly show that the lifetime of Nile red in lipid droplets are smaller than those in cell membrane. Our results suggest that fluorescence lifetime imaging can be a very powerful tool to monitor lipid droplet formation in adipocytes from 3T3-L1 cells.

Confocal fluorescence lifetime imaging microscopy based on a real-time sampling method

We present a fluorescence lifetime imaging microscope (FLIM) based on a real-time waveform acquisition method. The fluorophores were excited by a 635-nm gain-switched laser diode, which produced short pulses with duration ~50 ps in a 20-MHz repetition rate. The fluorescence signals were detected by a silicon avalanche photo-diode (APD) in addition to a wide-band electric amplifier. The converted electric pulses were sampled by a high-speed digitizer of which sampling rate was 2 GS/s. In order to reduce the sampling interval for analyzing sub-nanosecond lifetimes, an interleaved data acquisition technique was used. The effective sampling rate was increased to 10 GS/s. In addition, the impulse response was measured simultaneously with the lifetime signals by an interleaving manner and was used in calibration of the system. By using these methods, accurate lifetime information was acquired in a short time less than 8 μs.

Generation of optical lattice and trapping particle using DMD-ALP

We describe optical trap lattices, their manipulation, and optical trapping using the digital micromirror device (DMD)-accessory light modulator package (ALP). The proposed device flexibly controls the trap profile, array dimension, hopping over trap lattice, and steering therewithin. In order to generate optical trap lattice with Gaussian intensity profile, desirable input electronic images with LP01mode of single mode fiber to the DMD-ALP was loaded, which formed 2-dimensional optical trap lattice with Gaussian intensity profile. We generated 2-dimensional multiple optical trap lattice, where the individual intensity profile took LP01mode. This technique flexibly controlled the intensity profile, array dimension, and the hopping over trap lattice. We reported a new 2-dimensional optical trapping by means of the proposed system providing superior benefits in flexible digital control. In order to identify the possibility of optical trapping using the proposed device, single optical trapping was proposed. We demonstrated a polystyrene bead was attracted to a focused beam spot when the focused beam was near by the polystyrene bead and trapped bead was fixed by moving the sample stage of microscope up and down or right and left.

Image-Guided Laparoscopic Surgical Tool (IGLaST) Based on the Optical Frequency Domain Imaging (OFDI) to Prevent Bleeding

We present an image-guided laparoscopic surgical tool (IGLaST) to prevent bleeding. By applying optical frequency domain imaging (OFDI) to a specially designed laparoscopic surgical tool, the inside of fatty tissue can be observed before a resection, and the presence and size of blood vessels can be recognized. The optical sensing module on the IGLaST head has a diameter of less than 390 µm and is moved back and forth by a linear servo actuator in the IGLaST body. We proved the feasibility of IGLaST by in vivo imaging inside the fatty tissue of a porcine model. A blood vessel with a diameter of about 2.2 mm was clearly observed. Our proposed scheme can contribute to safe surgery without bleeding by monitoring vessels inside the tissue and can be further expanded to detect invisible nerves of the laparoscopic thyroid during prostate gland surgery.

Design of ultra-compact optical system for disposable epidural spinal endoscope

We have presented the plastic based ultra-compact aspheric lens for disposable epidural spinal endoscope. We have also showed the analysis of the stray light distribution on the image plane using optical illumination system design software (Light Tools). The optical system consists of the aspheric lens with a size of 1.4mm (total track of optical system). The effective length and field of view (FOV) is 0.66mm and 90 degrees. The distortion of the optical system is below 25%. The curves of modulation transfer function (MTF) are higher than 0.3 at 80 line pairs/mm (lps/mm) in image space. For the analysis of stray light, we assumed that the 98 percent of incident light is absorbed inside lens barrel and the rest is scattered on the inner surfaces of the lens barrel. The average value of stray light is 0.16% in the image intensity. The maximum stray light and minimum stray light of the proposed optical system is 0.57% and 0.0005% in the image intensity, respectively. The effective transmission rate of the proposed optical system is 89.6%.

A study on the efficiency improvement of a single fiber guided illumination system for disposable flexible endoscope (DFE)

We have studied the method to improve the illumination efficiency in DFE with optical illumination design software. We have showed the result of illumination efficiency according to the change of geometrical shape in distal tip of plastic optical fiber (POF). We simulated the illumination efficiency in the case of the polished POF distal tip and the unpolished one, respectively using optical illumination system design software. We obtained the illumination efficiency was increased by about 46 percent in the polished distal tip more than in the unpolished distal tip when a light emitting diode (LED) was directly excited to the distal tip of POF. In order to demonstrate the simulated results, we showed the polishing fabrication of the distal tip in POF and have measured the illumination efficiency of the polished POF using LED. The measured results showed that illumination efficiency was increased by about 23 percent in the polished distal tip more than in the unpolished distal tip of POF. We have demonstrated the optimized geometrical shapes of the POF for minimization of the illumination loss. We have suggested the method to improve the illumination efficiency by 69 percent for a single fiber illumination delivery system of DFE.

The study on the color reproduction by illumination source for disposable endoscope

Most of cameras like CCTV cameras or video cameras are shoot in available light or lightings. But the medical cameras like endoscope or laparoscopy are shoot in the situation in vivo. Generally, inside ad body is lightless and moist. So we can shoot the inside with a single light from outside the body. So medical cameras should be set more clearer and vivider in low-light than in high-light. The camera setting of disposable endoscopes is especially very important because those are low price and should be guaranteed against faulty workmanship. In this study, We suggest effective conditions for camera settings of disposable endoscopes, and request value of test measurement item with analysis from the commercial endoscopes.

Multiple labeling fluorescence lifetime analysis in a single shot imaging

We propose new approaches analysis for fluorescence lifetime imaging measurement of multiple labeling at single shot process. This paper suggests analysis concept of which can extract small scale signal ratio simply in the complex environment.