Imaging and measurement of the surface profile of an object with high resolution has become essential in both of
biological research and industry application. Many samples under investigation such as cultured cells are usually
immersed in liquid. Although the techniques such as scanning electron microscope and atomic force microscope can
provide imaging or measurement of the surface profile with nanometer resolution, it is difficult for them to image an
immersed object with their typical types. Recently, we have proposed and demonstrated a new technique based on
composite interferometer which can perform imaging and measurement of the surface profile of an object with accuracy
in the axial direction within 5 nm through a self-phase-compensation mechanism. In this research, an optical system
based on the concept of combination of optical coherence microscopy (OCM) and composite interferometer was built for
imaging of biological tissue immersed in water with axial accuracy at nanometer scale. In the system, a Ti:sapphire laser
with center wavelength at 800 nm and spectral width of 140 nm was used as the light source. The composite
interferometer comprises two Michelson interferometers sharing common light source, reference arm and photodetector.
One of the two interferometers served as a typical OCM system and the other was used to measure the phase shift in the
reference arm in each axial scan with the sample being a fixed reflection mirror. The system was used to image the
surface profiles of various immersed biological samples with accuracy at nanometer scale through the self-phasecompensation
mechanism.
The measurement of surface morphology of a material with high resolution is important in both the industrial and
biomedical applications. Furthermore, a precise measurement of the morphology of the internal interface is usually
needed for materials with multilayered structures. Although some optical techniques can provide subsurface imaging of
materials, their resolutions are difficult to achieve nanometer scale. In our research, an optical system based on a
composite interferometer which can image the internal interface of a material with nanometer resolution is proposed and
demonstrated. The system consists of a Michelson interferometer and a Mach-Zehnder interferometer. The Michelson
interferometer with a broadband light source is used for three-dimensional imaging of the sample. In the Mach-Zehnder
interferometer, a prism and a retro-reflector are arranged for an optical delay line with adjustable length. The two
interferometers share common light source and a rapid scanning optical delay system used for axial scanning. In the
experiment, the adjustable optical delay line in the second interferometer is adjusted for the optical path lengths to match
that relative to the interface under investigation. With a phase compensation mechanism, the interface can be imaged
with an axial accuracy at nanometer scale.
Cytokinesis is a consecutive process during cell division. For systems biological studies, it is important to precisely
monitor and quantify proteins in different cell stages and mitosis processes. However, the absolute quantities in living
cells are usually difficult to quantify. Fluorescent protein tagged protein is one of the techniques that are usually applied
to monitor biological behaviors and phenomena. In this study, an insect cell line, DPnE, which can stably express both
green fluorescent protein (EGFP) and red fluorescent protein (DsRed) was established. This dual fluorescent cell line
was chosen as a model system to monitor the protein partition during cytokinesis. A spectrum analysis system was established and integrated in an inverted microscope. The two-dimensional distribution of the full fluorescent spectra of the two fluorescent proteins was obtained in a time-lapse series. Furthermore, we also developed an algorithm to analyze the quantities of both fluorescent proteins in the daughter cells and parent cells during the process of cytokinesis, respectively. With this innovative optical system and algorithm, the proteins partition during cytokinesis can be monitored and quantified precisely.
We proposed and demonstrated an optical system for high-speed and high-resolution imaging of surface profiles. The
system is a fiber-based composite interferometer in which a Michelson interferometer is used for the measurement of the
surface profile of the sample and a Mach-Zehnder interferometer is used for compensation of phase deviation due to the
systematic errors and environmental perturbations. In the system, a laser diode with wavelength of 531 nm was used as
the light source. A two-axis translation stage was used for lateral scanning of the sample. In the reference arm, a
reflection mirror was arranged on a translation stage driven by a piezoelectric transducer as the axial scanning
component. The phase difference between the interferograms of both interferometers was acquired to obtain the surface
height of the sample. The axial accuracy of ±5 nm was achieved where the imaging was acquired within one minute for a
frame. The lateral resolution was at the diffraction limit of light. The system possesses the advantages of low cost,
portability and flexibility. Furthermore, it can perform
high-resolution imaging in large area without special shielding of
the system as well as any special preparation of the sample.
The cloning and transcription techniques on gene cloned fluorescent proteins have been widely used in many
applications. They have been used as reporters of some conditions in a series of reactions. However, it is usually difficult
to monitor the specific target with the exactly number of proteins during the process in turbid media, especially at
micrometer scales. We successfully revealed an alternative way to monitor the cell cycle behavior and quantitatively
analyzed the target cells with green and red fluorescent proteins (GFP and RFP) during different phases of the cell cycle
by quantitatively analyzing its behavior and also monitoring its spatial distribution.
We proposed and developed an optical system for imaging of the surface profiles of biological cells and tissues with
nanometer resolution. The system is a low-cost system which can perform the imaging of the surface morphology in a
large area and large depth range without any special preparation of the sample. The system consists of two
interferometers in which one is in the configuration of a Michelson interferometer and the other is in the configuration of
a Mach-Zehnder interferometer. The former is used for scanning of the surface profile of the sample, and the latter is
used to compensate the phase shift due to the different traveling ranges of the reference mirror in successive scannings.
The phase difference between the interferograms detected in both interferometers is proportional to the surface height of
the sample at that point. The system was demonstrated to possess the axial resolution within ±5 nm and its lateral
resolution is at the diffraction limit. We used the system for the imaging of various samples including biological cells
and tissues. The system was also used for dynamic imaging to observe the morphological change of the surface of biological cells.
Exogenous fluorescent agents such as green fluorescent protein (GFP) have been widely used as biological indicators in
bioimaging techniques. Although GFP and its mutants have been used in many applications, their optical properties have
not been completely investigated, especially when they are under various environmental conditions. In this research, we
developed a spectrum-analyzing system to investigate the fluorescent properties of GFP in the environments of different
temperatures. We found that the fluorescent spectrum of GFP consisted of two components that might come from the
transitions between different electronic energy states where the quantum efficiencies of the two components varied with
different temperature. This effect was expected to come from the thermal effect on the electron populations in the
molecular energy states of GFP. Furthermore, GFP was used as fluorescent marker to monitor the infection process of
cells by viruses with a dynamic spectral imaging system. The recombinant baculoviruses containing the red and green
fluorescent protein gene that can simultaneously produce dual fluorescence were used as vectors in insect cells. The
system was used to monitor the spatial distribution of fluorescent spectra of cells infected by virus during the process of
infection.
The theory of optical coherence tomography (OCT) was conventionally considered as the light beams propagating in the
system to be in the forms of planar waves. However, the actual behaviors of the light beams in an OCT system are more
likely to be Gaussian beams. With the consideration of the light beam passing through the focal lens in the sample arm to
be a Gaussian beam, we deduced the theory of OCT in an analytic form. We also simulated and analyzed the interference
signals with different positions of the photodetector and the interface in the sample as well as their transverse patterns
spectrally. The results were demonstrated by experiments with a Fourier-domain OCT system.
We constructed a dynamic spectroscopy system that can simultaneously measure the intensity and spectral distributions
of samples with multi-fluorophores in a single scan. The system was used to monitor the fluorescence distribution of
cells infected by the virus, which is constructed by a recombinant baculoviruses, vAcD-Rhir-E, containing the red and
green fluorescent protein gene that can simultaneously produce dual fluorescence in recombinant virus-infected
Spodoptera frugiperda 21 cells (Sf21) under the control of a polyhedrin promoter. The system was composed of an
excitation light source, a scanning system and a spectrometer. We also developed an algorithm and fitting process to
analyze the pattern of fluorescence distribution of the dual fluorescence produced in the recombinant virus-infected cells.
All the algorithm and calculation are automatically processed in a visualized scanning program and can monitor the
specific region of sample by calculating its intensity distribution. The spectral measurement of each pixel was performed
at millisecond range and the two dimensional distribution of full spectrum was recorded within several seconds. We have
constructed a dynamic spectroscopy system to monitor the process of virus-infection of cells. The distributions of the
dual fluorescence were simultaneously measured at micrometer resolution.
One of the most interested aspects in the development of optical coherence tomography (OCT) is to construct a stable
and easy fabricated optical delay line that can perform real-time imaging for clinical applications. There are many
methods that have been used for fast scanning in OCT. However, most of the configurations are difficult to construct or
variational intensity loss exists during the scanning because of the walk-off of different wavelength components and
different tilted angle of the scanner. We proposed and constructed an optical delay line with all reflective components
that is compact, easy to fabricate and can avoid the intensity loss during the scanning. The optical delay line was
constructed of a retro-reflector fabricated with two right-angled prisms assembled on an aluminum jig, a reflection
mirror and a galvoscanner. The size of the optical delay line is within 2 cm x 2 cm and the system can provide a scanning
rate in the kilohertz range. The achieved imaging depth is 3.3 mm when the vibration angle of the scanning mirror is ±9°.
This system can perform a real-time scanning and thus is supposed suitable for clinical applications.
We proposed and developed a low-cost system for profilometry and thin film measurement at nanometer resolution. The
system is based on the concept of a dual interferometer. The sample was measured while a simultaneous compensation
of the phase deviation due to the instability of piezoelectric transducer served as an optical delay component was
performed. In the application of profilometry, the information of the surface profile of a material was obtained from the
phase shift of the interference signal. By using the proposed compensation mechanism, an axial resolution of 1.09 nm
was achieved. For the measurement of a thin film or membrane, the probe beam was prepared to polarize at 45° and was
oblique incident on the sample. The system can perform a simultaneous measurement of the refractive index and the
geometrical thickness as well as the position when the thin film is suspended. In order to calculate the refractive index
and thickness of the thin film, the phase shifts and intensities of the interferograms of TE and TM waves were measured
independently. By comparing the ratio of intensities and the phase shifts of the interferograms, the refractive index and
the thickness of the thin film can then be obtained simultaneously.
A polarization-sensitive optical coherence tomography (PSOCT) system using a femtosecond-laser as the broadband light source is implemented with the axial resolution of 5 µm in free space. Through the design of path-length difference between the two polarization inputs and the modulation of one of the polarization inputs, the PSOCT images of various input and output polarization combinations can be distinguished and simultaneously collected. The PSOCT system is then used for in vitro scanning of the myocardium tissues of normal and infarcted rat hearts. The destruction of the birefringence nature of the fiber muscle in the infarcted heart can be clearly observed.
We demonstrate a new method for simultaneous compensation of time-dependent and time-independent second- and third-order dispersion mismatches in an optical coherence tomography (OCT) system. There have been several methods for dispersion compensation in scanning interferometry by translating or tilting the diffraction grating in a rapid-scanning optical delay line (RSODL). Although these methods can provide a time-independent or time-dependent compensation of the second- or third-order dispersion, they cannot compensate the dispersion mismatch at both orders at the same time. However, the effects of both orders of dispersion mismatch caused by different lengths of fibers in the sample and reference arms in a fiber-based OCT system are significant and cannot be neglected. In this paper, we propose a new method for simultaneous compensation of time-dependent and time-independent second- and third-order dispersions by placing and adjusting a prism between the grating and the lens of the RSODL. The resulting dispersion can be calculated from the phase response of the RSODL with ray tracing analysis. The dependence of each order of dispersion on the configuration of the system is discussed with numerical analysis. The effects of the dispersion compensation are demonstrated with experiments.
The longitudinal resolution of an optical coherence tomography (OCT) system is conventionally defined as the full-width at half maximum (FWHM) of the interference fringe envelope, which depends on the center wavelength as well as the spectral width of the light source. One can obtain an FWHM of an interference fringe envelope larger or smaller than that resulted from a Gaussian spectrum of the same spectral FWHM when the light source spectrum is non-Gaussian distributed. In this paper, we first study the dependencies of OCT resolution on the spectral shape and dispersion mismatch with numerical simulations. We will demonstrate the capability of enhancing the longitudinal resolution of an OCT system with a proper control of spectral distribution and dispersion mismatch. Then, in experiments we built an OCT system with its light source generated from nonlinear optics effects of 12-fsec Ti:sapphire laser pulses in an optical fiber. With proper control of dispersion mismatch between the sample and reference arms, the FWHM of the interference fringe envelope was smaller than that of a Gaussian spectrum with the same spectral FWHM by a factor of two. Furthermore, the side lobes were suppressed with a process algorithm to significantly improve the longitudinal resolution.
We proposed and demonstrated a novel technique of improving the spatial resolution of an optical coherence tomography (OCT) system given a certain light source spectrum. By using dispersive materials in the reference arm of the OCT system, the resultant dispersion compensation led to a FWHM of interference fringe envelope smaller than the Fourier transform limited value, at the expense of significant tails. The effects of the tails, which would blur the OCT images, were tremendously reduced with retrieval algorithms. Simulation results and processed OCT scanning images have shown the capability of the proposed technique. Two retrieval algorithms were proposed and compared.
A polarization controllable optical coherence tomography (OCT) system was built with the broadband source generated with femtosecond Ti:sapphire laser pulses in fiber. Spectral broadening in such fiber originated from self-phase modulation, four-wave mixing, Raman scattering, and other nonlinear-optics effects. Two different mode-locked Ti:sapphire lasers with 100 fsec and 12 fsec pulses were used. The generated spectral shape and width were compared in terms of the application to the OCT system. The relationship between the OCT resolution and the source spectrum shape was studied. Also, an algorithm was built for increasing the effective longitudinal resolution in data processing. The scheme of this algorithm meant to separate the contribution of the central portion from those of the tails in the interference fringe envelope. By removing the tail contribution to the scanning results, the effective longitudinal resolution was improved. Such a procedure is particularly important when the light source spectrum is not a well-defined shape. This procedure involved in the computation of a matrix inversion. The OCT system and the process algorithm were used for oral cancer study. Features of oral cancer were well identified. A probe was also fabricated for in vivo scan of oral tissues.
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