Seungrag Lee

Autofocusing and edge detection schemes in cell volume measurements with quantitative phase microscopy.

We have proposed and demonstrated a very sensitive volume measurement scheme for a live cell with a quantitative phase microscopy (QPM) utilizing auto-focusing and numerical edge detection schemes. An auto-focusing technique with two different focus measures is applied to find the focus dependent errors in our live cell volume measurement system. The volume of a polystyrene bead sample with 3 mum diameter has been measured for the validity test of our proposed method. We have shown that a small displacement of an object from its focusing position can cause a large volume error. A numerical edge detection technique is also used to accurately resolve the boundary between a cell and its suspension medium. We have applied this method to effectively suppress errors by the surrounding medium of a single red blood cell (RBC).

Bandwidth control in a hybrid fiber acousto-optic filter

We report a bandwidth variation technique in an acousto-optic filter. Utilizing the adiabatic conversion in both optical and acoustic modes, we obtain a novel hybrid waveguide composed of serial concatenation of single-mode fiber (SMF) and two-mode hollow optical fiber (HOF). On the basis of dissimilarity in the phase-matching conditions and beat-length dispersion in SMF and HOF, the FWHM of the resonant bands is varied from 3.8 to 190 nm near the 1.5-microm region in a single device. Furthermore, we theoretically analyze the acousto-optic coupling among the guided modes in HOF, which shows good agreement with experimental observations.

Dynamic analysis of pathogen-infected host cells using quantitative phase microscopy

We present the real-time quantitative analysis of Vibrio vulnificus-infected host cells using quantitative phase microscopy (QPM) based on interferometric techniques. This provides the ability to retrieve the phase or optical path-length distribution over the cell with nanometer path-length sensitivity from a single interferogram image. We have used QPM to study dynamic cell morphologic changes and to noninvasively quantify the cell volumes of rat basophilic leukemia RBL-2H3 cells infected with V. vulnificus strains: wild type (MO6-24∕O) and RtxA1 toxin mutant (CMM770). During the process of V. vulnificus infection in RBL-2H3 cells, the dynamic changes of quantitative phase images, cell volumes, and areas were observed in real time using QPM. In contrast, dramatic changes were not detected in RBL-2H3 cells infected with the noncytotoxic RtxA1 toxin mutant. The results showed good correlation between QPM analysis and biochemical assays, such as lactate dehydrogenase assay or β-hexosaminidase release assay. We suggest that QPM is a powerful quantitative method to study the dynamic process of host cells infected with pathogens in a noninvasive manner.

Detrended fluctuation analysis of membrane flickering in discocyte and spherocyte red blood cells using quantitative phase microscopy

Dynamic analyses of vibrational motion in cell membranes provide a lot of information on the complex dynamic motilities of a red blood cell (RBC). Here, we present the correlation properties of membrane fluctuation in discocyte and spherocyte RBCs by using quantitative phase microscopy (QPM). Since QPM can provide nanometer sensitivity in thickness measurement within a millisecond time scale, we were able to observe the membrane flicking of an RBC in nanometer resolution up to the bandwidth of 50 Hz. The correlation properties of the vibrational motion were analyzed with the detrended fluctuation analysis (DFA) method. Fractal scaling exponent α in the DFA method was calculated for the vibrational motion of a cell surface at various surface points for normal discocyte and abnormal spherocyte RBCs. Measured α values for normal RBCs are distributed between 0.7 and 1.0, whereas those for abnormal spherocyte RBCs are within a range from 0.85 to 1.2. We have also verified that the vibrational motion of background fluid outside of a cell has an α value close to 0.5, which is a typical property of an uncorrelated white noise.

Optimum phase shift for quantitative phase microscopy in volume measurement.

Volume measurement of a phase object is one of the most distinctive capabilities of quantitative phase microscopy (QPM). However, the accuracy of a measured volume is limited by the different noises of a measurement system and the finite bandpass filter used in the phase extraction algorithm. In this paper, we analyze the inherent errors in volume measurement with QPM and propose the optimum condition that can minimize these errors. We find that phase information of a sample in the frequency domain nonlinearly oscillates as a function of the phase shift corresponding to the sample and its medium, and that the phase information of a sample inside the bandpass filter can be maximized by a proper phase shift. Through numerical simulations and actual experiments, we demonstrate that the error in phase volume measurement can be effectively reduced by the enhancement of the phase signal inside the bandpass region using an optimum amount of phase, which can be controlled by changing either the medium index or the wavelength of illumination.

Three-dimensional Single Particle Tracking using Off-axis Digital Holographic Microscopy

Three dimensional particle tracking is useful technology to characterize live cell or surrounding environment by tracing small particles such as fluorescence beads or polystyrene beads which adhered to objective samples. In microscopy imaging system, the longitudinal(z axis) tracking of the particle is essential for implementation of three-dimensional particle tracking, however its been still a challenging topic to find the exact position of the particle in z axis with high precision. In this study, we present that a novel technique to find the longitudinal position of the particle, as well as the transverse position(x,y axis) by applying the numerical reconstruction and focusing with digital holographic microscope. Transmission type off-axis digital holographic microscope is implemented for this experiment, based on Mach-Zehnder interferometer and 632.8nm HeNe laser is used as a coherent light source of the microscope and high-speed CMOS camera is utilized for acquiring the hologram. Digital holographic microscope makes it possible to record and reconstruct the phase and amplitude image of the sample. In order to find the position of the particle in z axis, we apply the numerical focusing algorithm, which enables the translation of the imaging focus without actual longitudinal movement of the sample. To demonstrate the presented method, Brownian movement of 3μm polystyrene sphere suspended in water is investigated in this experiment.

Dynamic phase imaging of host cells attacked by Vibrio vulnificus using quantitative phase microscopy

We present the real time quantitative analysis of Vibrio vulnificus-infected host cells using high stability quantitative phase microscopy (HSQPM). It provides the ability to retrieve the phase or optical path length distribution over the cell from a single interferogram image, which has been measured with nanometer path length sensitivity for long periods of time. We have applied HSQPM to study dynamic cell morphologic changes and to quantify noninvasively cell volumes of rat basophilic leukemia RBL-2H3 cells infected with pathogenic bacteria V. vulnificus strains, wild type (MO6-24/O) and RTX toxin mutant (CMM770). During the process of V. vulnificus wild type infection to RBL-2H3 cells, the dynamic changes of quantitative phase images, cell volumes and areas were observed in real time using HSQPM. In contrast, the dramatic changes were not detected in RBL-2H3 cells infected with RTX toxin mutant. The results showed the good correlation between HSQPM analysis and biochemical assays such as lactate dehydrogenase (LDH) assay and β-hexosaminidase release assay. We suggest that HSQPM is useful real time quantitative method to study the dynamic process of host cells infected with pathogen in a noninvasive manner.

The measurement of red blood cell volume change induced by Ca2+ based on full field quantitative phase microscopy

We present the measurement of red blood cell (RBC) volume change induced by Ca2+ for a live cell imaging with full field quantitative phase microscopy (FFQPM). FFQPM is based on the Mach-Zehnder interferometer combined with an inverted microscopy system. We present the effective method to obtain a clear image and an accurate volume of the cells. An edge detection technique is used to accurately resolve the boundary between the cell line and the suspension medium. The measurement of the polystyrene bead diameter and volume has been demonstrated the validity of our proposed method. The measured phase profile can be easily converted into thickness profile. The measured polystyrene bead volume and the simulated result are about 14.74 μm3 and 14.14 μm3, respectively. The experimental results of our proposed method agree well with the simulated results within less than 4 %. We have also measured the volume variation of a single RBC on a millisecond time scale. Its mean volume is 54.02 μm3 and its standard deviation is 0.52 μm3. With the proposed system, the shape and volume changes of RBC induced by the increased intracellular Ca2+ are measured after adding ionophore A23187. A discocyte RBC is deformed to a spherocyte due to the increased intracellular Ca2+ in RBC. The volume of the spherocyte is 47.88 μm3 and its standard deviation is 0.19 μm3. We have demonstrated that the volume measurement technique is easy, accurate, and robust method with high volume sensitivity (<0.0000452 μm3) and this provides the ability to study a biological phenomenon in Hematology.

Optical property of red blood cell with a phase microscopy interferometer

We present a novel method to determine the effective elastic constant (EEC) and effective restoring force (ERF) by using volumetric analysis of Red blood cell (RBC)s with Full field quantitative phase microscopy (FFQPM). We use the simple harmonic oscillator model to determine EEC and ERF. We investigate the EECs and ERFs of different shape of RBCs (discocyte, acanocyte, stomatocyte, and spherocyte) and we investigate the effective temporal coherence of RBCs by analyzing temporal volumetric behavior of the RBCs.

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.