Youngjae Won

High-speed confocal fluorescence lifetime imaging microscopy (FLIM) with the analog mean delay (AMD) method

We demonstrate a high-speed confocal fluorescence lifetime imaging microscopy (FLIM) whose accuracy and photon economy are as good as that of a time-correlated single photon counting (TCSPC). It is based on a new lifetime determination scheme, the analog mean delay (AMD) method. Due to the technical advantages of multiple fluorescence photon detection capability, accurate lifetime determination scheme and high photon detection efficiency, the AMD method can be the most effective method for high-speed confocal FLIM. The feasibility of real-time confocal FLIM with the AMD method has been demonstrated by observing the dynamic reaction of calcium channels in a RBL-2H3 cell with respect to 4αPDD stimulus. We have achieved the photon detection rate of 125 times faster than a conventional TCSPC based system in this experiment.

Analog mean-delay method for high-speed fluorescence lifetime measurement.

We present a new high-speed lifetime measurement scheme of analog mean-delay (AMD) method which is suitable for studying dynamical time-resolved spectroscopy and high-speed fluorescence lifetime imaging microscopy (FLIM). In our lifetime measurement method, the time-domain intensity signal of a fluorescence decay is acquired as an analog waveform. And the lifetime information is extracted from the mean temporal delay of the acquired signal. Since this method does not rely on the single-photon counting technique, the signals of multiple fluorescence photons can be acquired simultaneously. The measurement speed can be increased easily by raising the fluorescence intensity without a photon-rate limit. We have investigated various characteristics of our method in lifetime accuracy and precision as well as measurement speed. It has been found that our method can provide excellent measurement performances in various aspects. We hav demonstrated a high-speed measurement with a high photon detection rate of approximately 108 photons per second with a nearly shot noise-limited photon economy. A fluorescence lifetime of 3.2 ns was accurately determined with a standard deviation of 3% from the data acquired within 17.8 micros at a rate of 56,300 lifetime determinations per second.

Reciprocal activation of hypoxia-inducible factor (HIF)-2α and the zinc-ZIP8-MTF1 axis amplifies catabolic signaling in osteoarthritis

OBJECTIVE Hypoxia-inducible factor (HIF)-2α and the zinc-ZIP8-MTF1 axis in chondrocytes serve as catabolic regulators of osteoarthritic cartilage destruction by regulating the expression of catabolic factor genes. We explored possible crosstalk between these signaling pathways and its biological significance in osteoarthritis (OA). METHODS Microarray analysis, various mRNA and protein assays were conducted using primary cultured mouse articular chondrocytes and experimental OA cartilage to reveal molecular mechanisms underlying the crosstalk between HIF-2α and the zinc-ZIP8-MTF1 axis. Experimental OA in mice was induced by intra-articular (IA) injection of adenovirus expressing HIF-2α (Ad-Epas1), ZIP8 (Ad-Zip8), or MTF1 (Ad-Mtf1) in wild-type mice or mice with cartilage-specific conditional knockout of HIF-2α (Epas1(fl/fl);Col2a1-Cre), ZIP8 (Zip8(fl/fl);Col2a1-Cre), or MTF1 (Mtf1(fl/fl);Col2a1-Cre). RESULTS HIF-2α activated the zinc-ZIP8-MTF1 axis in chondrocytes by upregulating the Zn(2+) transporter ZIP8, thereby increasing Zn(2+) influx and activating the downstream transcription factor MTF1. The zinc-ZIP8-MTF1 axis, in turn, acted as a novel transcriptional regulator of HIF-2α. HIF-2α-induced activation of the zinc-ZIP8-MTF1 axis amplified HIF-2α regulation of OA cartilage destruction by synergistically promoting expression of matrix-degrading enzymes. Thus, HIF-2α-induced activation of the zinc-ZIP8-MTF1 axis, together with zinc-ZIP8-MTF1 regulation of HIF-2α, acted collectively to synergistically promote expression of matrix-degrading enzymes and OA cartilage destruction. CONCLUSION Our findings identify a reciprocal activation mechanism involving HIF-2α and the zinc-ZIP8-MTF1 axis during OA pathogenesis that amplifies catabolic signaling and cartilage destruction.

A Digital Shade-Matching Device for Dental Color Determination Using the Support Vector Machine Algorithm

In this study, we developed a digital shade-matching device for dental color determination using the support vector machine (SVM) algorithm. Shade-matching was performed using shade tabs. For the hardware, the typically used intraoral camera was modified to apply the cross-polarization scheme and block the light from outside, which can lead to shade-matching errors. For reliable experiments, a precise robot arm with ±0.1 mm position repeatability and a specially designed jig to fix the position of the VITA 3D-master (3D) shade tabs were used. For consistent color performance, color calibration was performed with five standard colors having color values as the mean color values of the five shade tabs of the 3D. By using the SVM algorithm, hyperplanes and support vectors for 3D shade tabs were obtained with a database organized using five developed devices. Subsequently, shade matching was performed by measuring 3D shade tabs, as opposed to real teeth, with three additional devices. On average, more than 90% matching accuracy and a less than 1% failure rate were achieved with all devices for 10 measurements. In addition, we compared the classification algorithm with other classification algorithms, such as logistic regression, random forest, and k-nearest neighbors, using the leave-pair-out cross-validation method to verify the classification performance of the SVM algorithm. Our proposed scheme can be an optimum solution for the quantitative measurement of tooth color with high accuracy.

Fluorescence lifetime microscopy for monitoring cell adhesion using metal induced energy transfer

A precise control and a reliable monitoring tool for the adhesion properties of a cell are very important in atherosclerosis studies. If endothelial cells in contact with the intracellular membrane are not attached securely, low-density lipoprotein (LDL) particles can enter into the inner membrane. It is therefore necessary to measure conditions under which endothelial cell detachment occurs. When a cell is attached to a metal thin film, the lifetime of a fluorescence probe attached to the membrane of the cell is reduced by the metal-induced energy transfer (MIET). Fluorescence lifetime imaging microscopy (FLIM) is used to monitor the attachment condition of a cell to a metal surface using FRET. However, this requires high numerical aperture (NA) objective lens because axial confocal resolution must be smaller than the cell thickness. This requirement limits the field of view of the measurement specimen. In this study we provides a new method which can measure adhesion properties of endothelial cells even with a low NA objective lens by resolving two lifetime components in FLIM.

The study on the parallel processing based time series correlation analysis of RBC membrane flickering in quantitative phase imaging

Not only static characteristics but also dynamic characteristics of the red blood cell (RBC) contains useful information for the blood diagnosis. Quantitative phase imaging (QPI) can capture sample images with subnanometer scale depth resolution and millisecond scale temporal resolution. Various researches have been used QPI for the RBC diagnosis, and recently many researches has been developed to decrease the process time of RBC information extraction using QPI by the parallel computing algorithm, however previous studies are interested in the static parameters such as morphology of the cells or simple dynamic parameters such as root mean square (RMS) of the membrane fluctuations. Previously, we presented a practical blood test method using the time series correlation analysis of RBC membrane flickering with QPI. However, this method has shown that there is a limit to the clinical application because of the long computation time. In this study, we present an accelerated time series correlation analysis of RBC membrane flickering using the parallel computing algorithm. This method showed consistent fractal scaling exponent results of the surrounding medium and the normal RBC with our previous research.

Referencing techniques for high-speed confocal fluorescence lifetime imaging microscopy (FLIM) based on analog mean-delay (AMD) method

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 (FLIM). Even though the photon economy and the lifetime precision of the AMD method are proven to be as good as the state-of-the-art time-correlated single photon counting (TC-SPC) method, there have been some speculations and concerns about the accuracy of this method. 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 lifetime to be measured. We 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. As a referencing technique, we already proposed dual-channel waveform measurement scheme that may suppress these errors. In this study, we have demonstrated real-time confocal AMD-FLIM system with dual-channel waveform measurement technique.

A study on the characteristics of the Analog Mean Delay (AMD) method for high-speed Fluorescence Lifetime Imaging Microscopy (FLIM)

We present a study on the characteristics of the AMD method. We have demonstrated that the photon economy of the AMD method is not degraded for longer lifetimes even when the applied integration window size is increased. By an extension of MCS, the photon economy with respect to different designs of the Gaussian low-pass filter (GLPF) used in the AMD setup was also studied. When a GLPF with the highest cutoff frequency of 100 MHz is applied, the most effective photon economy performance is achieved for lifetimes of 1, 3.2, 5, and 8 ns.

The study on RBC characteristic in paroxysmal nocturnal hemoglobinuria (PNH) patients using common path interferometric quantitative phase microscopy

We have studied the RBC membrane properties between a normal RBC and a RBC in Paroxysrnal nocturnal hemoglobinuria (PNH) patient using common path interferometric quantitative phase microscopy (CPIQPM). CPIQPM system has provided the subnanometer optical path length sensitivity on a millisecond. We have measured the dynamic thickness fluctuations of a normal RBC membrane and a RBC membrane in PNH patient over the whole cell surface with CPIQPM. PNH is a rare and serious disease of blood featured by destruction of red blood cells (RBCs). This destruction happens since RBCs show the defect of protein which protects RBCs from the immune system. We have applied CPIQPM to study the characteristic of RBC membrane in PNH patient. We have shown the morphological shape, volume, and projected surface for both different RBC types. The results have showed both RBCs had the similar shape with donut, but membrane fluctuations in PNH patient was shown to reveal the difference of temporal properties compared with a normal RBC. In order to demonstrate the practical tool of the CPIQPM technique, we have also obtained the time series thickness fluctuation outside a cell.

Fluorescence lifetime measurement with confocal endomicroscopy for direct analysis of tissue biochemistry in vivo

Confocal endomicroscopy is a powerful tool for in vivo real-time imaging at cellular resolution inside a living body without tissue resection. Microscopic fluorescence lifetime measurement can provide information about localized biochemical conditions such as pH and the concentrations of oxygen and calcium. We hypothesized that combining these techniques could assist accurate cancer discrimination by providing both biochemical and morphological information. We designed a dual-mode experimental setup for confocal endomicroscopic imaging and fluorescence lifetime measurement and applied it to a mouse xenograft model of activated human pancreatic cancer generated by subcutaneous injection of AsPC-1 tumor cells. Using this method with pH-sensitive sodium fluorescein injection, we demonstrated discrimination between normal and cancerous tissues in a living mouse. With further development, this method may be useful for clinical cancer detection.