Wenzhong Yang

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).

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.

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.

All-in-one multifunctional optical microscope with a single holographic measurement

An all-purpose multifunctional optical microscope based on digital holographic microscopy (DHM) is proposed, which enables DHM to be practically suitable for bioimaging applications. With our simple suggested digital signal-processing scheme, we have demonstrated that various optical images, including pure amplitude, pure phase, dark-field, phase-contrast, and differential interference contrast images, can be obtained from a single DHM measurement. Because these images obtained with our method are essentially the same as those obtainable with conventional optical imaging instruments, biologists can easily analyze them with their previous knowledge and experience.

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.

High-speed confocal fluorescence lifetime imaging microscopy by analog mean-delay method

We have demonstrated the high-speed confocal fluorescence lifetime imaging microscopy (FLIM) by analog mean-delay (AMD) method. The AMD method is a new signal processing technique for calculation of fluorescence lifetime and it is very suitable for the high-speed confocal FLIM with good accuracy and photon economy. We achieved the acquisition speed of 7.7 frames per second for confocal FLIM imaging. Here, the highest photon detection rate for one pixel was larger than 125 MHz and averaged photon detection rate was more than 62.5 MHz. Based on our system, we successfully obtained a sequence of confocal fluorescence lifetime images of RBL-2H3 cell labeled with Fluo-3/AM and excited by 4αPDD (TRPV channel agonist) within one second.

Silver nanoparticle-induced degranulation observed with quantitative phase microscopy

The use of AgNP is becoming more and more widespread in biomedical field. But compared with the promising bactericidal function, other physiological effects of AgNP on cells are relatively scant. In this research, we propose quantitative phase microscopy (QPM) as a new method to study the degranulation, and AgNP-induced RBL-2H3 cell degranulation is studied as well. Firstly, HeLa cells as the cell control and PBS as the solvent control, we measured the cell volume and cross section profile (x-z plane) with QPM. The results showed that the volume and cross section profile changed only the RBL-2H3 cells exposed to calcium ionophore A23187, which demonstrates the validity of QPM in degranulation research. Secondly, 50μg/mL of AgNP was used instead of A23187, and the measurement of cell volume and cross section profile was carried out again. RBL-2H3 cell volume increased immediately after AgNP was added, and cross section profile showed that the cell surface became granulated, but HeLa cell was lack of that effect. Phase images obviously indicated the RBL-2H3 cell deformation. Thirdly, stained with Fluo-3/AM, intracellular calcium Ca2+]i of single RBL-2H3 cell treated with AgNP was observed with fluorescent microscopy; incubated with AgNP for 20min, the supernatant of RBL-2H3 cells was collected and reacted with o-phthalaldehyde (OPA), then the fluorescent intensity of histamine-OPA complex was assayed with spectrofluorometer. The results of Ca2+]i and histamine increase showed that degranulation of AgNP-induced RBL-2H3 cell occurred. So, the cell volume was used as a parameter of degranulation in our study and AgNP-induced RBL-2H3 cells degranulation was confirmed by the cell volume increment, cross section profile change, and [Ca2+]i and histamine in supernatant increase.

Photobleaching Property of Confocal Laser Scanning Microscopy with Masked Illumination

Confocal laser scanning microscopy (CLSM) has become the tool of choice for high-contrast fluorescence imaging in the study of the three-dimensional and dynamic properties of biological system. However, the high cost and complexity of commercial CLSMs urges many researchers to individually develop low cost and flexible confocal microscopy systems. The high speed scanner is an influential factor in terms of cost and system complexity. Resonant galvo scanners at several kHz have been commonly used in custom-built CLSMs. However, during the repeated illumination for live cell imaging or 3D image formation, photobleaching and image distortion occurred at the edges of the scan field may be more serious than the center due to an inherent property (e.g. sinusoidal angular velocity) of the scan mirror. Usually, no data is acquired at the edges due to large image distortion but the excitation beam is still illuminated. Here, we present the photobleaching property of CLSM with masked illumination, a simple and low cost method, to exclude the unintended excitation illumination at the edges. The mask with a square hole in its center is disposed at the image plane between the scan lens and the tube lens in order to decrease photobleaching and image distortion at the edges. The excluded illumination section is used as the black level of the detected signals for a signal quantizing step. Finally, we demonstrated the reduced photobleaching at the edges on a single layer of fluorescent beads and real-time image acquisition without a standard composite video signal by using a frame grabber.