Thanh Nguyen

Fully automated, high speed, tomographic phase object reconstruction using the transport of intensity equation in transmission and reflection configurations

While traditional transport of intensity equation (TIE) based phase retrieval of a phase object is performed through axial translation of the CCD, in this work a tunable lens TIE is employed in both transmission and reflection configurations. These configurations are extended to a 360° tomographic 3D reconstruction through multiple illuminations from different angles by a custom fabricated rotating assembly of the phase object. Synchronization circuitry is developed to control the CCD camera and the Arduino board, which in its turn controls the tunable lens and the stepper motor to automate the tomographic reconstruction process. Finally, a MATLAB based user friendly graphical user interface is developed to control the whole system and perform tomographic reconstruction using both multiplicative and inverse radon based techniques.

Accurate quantitative phase digital holographic microscopy with single- and multiple-wavelength telecentric and nontelecentric configurations.

In this work, we investigate, both theoretically and experimentally, single-wavelength and multiwavelength digital holographic microscopy (DHM) using telecentric and nontelecentric configurations in transmission and reflection modes. A single-wavelength telecentric imaging system in DHM was originally proposed to circumvent the residual parabolic phase distortion due to the microscope objective (MO) in standard nontelecentric DHM configurations. However, telecentric configurations cannot compensate for higher order phase aberrations. As an extension to the telecentric and nontelecentric arrangements in single-wavelength DHM (SW-DHM), we propose multiple-wavelength telecentric DHM (MW-TDHM) in reflection and transmission modes. The advantages of MW-TDHM configurations are to extend the vertical measurement range without phase ambiguity and optically remove the parabolic phase distortion caused by the MO in traditional MW-DHM. These configurations eliminate the need for a second reference hologram to subtract the two-phase maps and make digital automatic aberration compensation easier to apply compared to nontelecentric configurations. We also discuss a reconstruction algorithm that eliminates the zero-order and virtual images using spatial filtering and another algorithm that minimizes the intensity of fluctuations using apodization. In addition, we employ two polynomial models using 2D surface fitting to compensate digitally for chromatic aberration (in the multiwavelength case) and for higher order phase aberrations. A custom-developed user-friendly graphical user interface is employed to automate the reconstruction processes for all configurations. Finally, TDHM is used to visualize cells from the highly invasive MDA-MB-231 cultured breast cancer cells.

3D High Speed Characterization Of Phase Objects Using The Transport Of Intensity Equation

In this work we will extend the traditional TIE setup of phase retrieval of a phase object through axial translation of the CCD by employing a tunable lens (TL-TIE). This setup is also extended to a 360° tomographic 3D reconstruction through multiple illuminations from different angles by rotating the phase object. Finally, synchronization between the CCD, and the tunable lens is employed using a reconfigurable hardware to automate the 3D 360° tomographic reconstruction process.

Non-Interferometric Tomography of Phase Objects Using Spatial Light Modulators

Quantitative 3D phase retrieval techniques are based on either interferometric techniques such as holography or noninterferometric intensity-based techniques such as the transport of intensity equation (TIE). Interferometric techniques are vibration-sensitive and often use a reference beam requiring complicated optical alignment. In this work we develop a simple, fast, and noninterferometric tomographic 3D phase retrieval technique based on the TIE which does not suffer from such drawbacks. The optical setup is a modified 4f TIE system which uses an SLM to replace the slow translation of the CCD required to record several diffraction patterns in a traditional TIE system. This novel TIE setup is suitable for dynamical events such as imaging biological processes. A rotating mechanical stage is constructed to obtain tomographic phase images of the object. The tomographic reconstruction algorithm is based on the Fourier slice theorem (backprojection algorithm) which applies to objects with a small refractive index span. Simulation and experimental results are shown as part of this work. A graphical user interface is developed to perform the TIE tomographic reconstruction algorithm and to synchronize the captured intensities by the CCD, the phase patterns displayed on the SLM, and the Arduino controlled rotating stage assembly.

Quantitative assessment of cancer cell morphology and movement using telecentric digital holographic microscopy

Digital holographic microscopy (DHM) provides label-free and real-time quantitative phase information relevant to the analysis of dynamic biological systems. A DHM based on telecentric configuration optically mitigates phase aberrations due to the microscope objective and linear high frequency fringes due to the reference beam thus minimizing digital aberration correction needed for distortion free 3D reconstruction. The purpose of this work is to quantitatively assess growth and migratory behavior of invasive cancer cells using a telecentric DHM system. Together, the height and lateral shape features of individual cells, determined from time-lapse series of phase reconstructions, should reveal aspects of cell migration, cell-matrix adhesion, and cell cycle phase transitions. To test this, MDA-MB-231 breast cancer cells were cultured on collagen-coated or un-coated glass, and 3D holograms were reconstructed over 2 hours. Cells on collagencoated glass had an average 14% larger spread area than cells on uncoated glass (n=18-22 cells/group). The spread area of cells on uncoated glass were 15-21% larger than cells seeded on collagen hydrogels (n=18-22 cells/group). Premitotic cell rounding was observed with average phase height increasing 57% over 10 minutes. Following cell division phase height decreased linearly (R2=0.94) to 58% of the original height pre-division. Phase objects consistent with lamellipodia were apparent from the reconstructions at the leading edge of migrating cells. These data demonstrate the ability to track quantitative phase parameters and relate them to cell morphology during cell migration and division on adherent substrates, using telecentric DHM. The technique enables future studies of cell-matrix interactions relevant to cancer.

Detailed analysis of an optimized FPP-based 3D imaging system

In this paper, we present detail analysis and a step-by-step implementation of an optimized fringe projection profilometry (FPP) based 3D shape measurement system. First, we propose a multi-frequency and multi-phase shifting sinusoidal fringe pattern reconstruction approach to increase accuracy and sensitivity of the system. Second, phase error compensation caused by the nonlinear transfer function of the projector and camera is performed through polynomial approximation. Third, phase unwrapping is performed using spatial and temporal techniques and the tradeoff between processing speed and high accuracy is discussed in details. Fourth, generalized camera and system calibration are developed for phase to real world coordinate transformation. The calibration coefficients are estimated accurately using a reference plane and several gauge blocks with precisely known heights and by employing a nonlinear least square fitting method. Fifth, a texture will be attached to the height profile by registering a 2D real photo to the 3D height map. The last step is to perform 3D image fusion and registration using an iterative closest point (ICP) algorithm for a full field of view reconstruction. The system is experimentally constructed using compact, portable, and low cost off-the-shelf components. A MATLAB® based GUI is developed to control and synchronize the whole system.

Portable, Low-cost, Lensless DHM System Using RGB LEDs

We built a portable, low-cost, and lensless DHM with RGB LEDs to obtain holograms. We also developed a GUI to digitally reconstruct the holograms and to control the webcam and the microcontroller that drives the LEDs.

Optical and Digital Aberration Compensation in DHM

We propose optical and digital automatic phase aberration compensation for single and multi-wavelength DHM. The optical technique employs telecentric configuration to compensate for parabolic phase distortion due to the MO and the digital technique uses Zernike polynomials model to compensate for higher order and chromatic aberrations.