Here we achieved record-high >500 volumes/second two-photon imaging by improving lateral and axial scanning speed via 32-channel multifocal excitation/detection, and a tunable acoustic gradient-index lens, respectively. We developed a deconvolution process to reduce scattering-induced crosstalk in multifocal detection scheme, thus enabling whole brain imaging of Drosophila with millisecond and micrometer spatiotemporal resolution. Potential applications toward brain science include studying millisecond dynamics in a neuronal network, and resolving 3D microfluidics in blood vessels.
Confocal reflectance quantitative phase microscopy system is developed in our lab to quantify nuclear membrane fluctuations. This system able to provide the µm level depth resolved phase information of the back scattered signal from nucleic membrane at ms temporal resolution. The phase information quantify the height fluctuations of nucleic membranes, which are subject to thermal fluctuations around the stable equilibrium in viscoelastic mediums. We further combined this system with Brillouin spectroscopic system, which measures longitudinal modulus of the nuclear material in the gigahertz (GHz) frequency range. Combining the information from confocal phase and Brillouin spectroscopy provides the nuclear membrane and material mechanical properties. Studies of anti-cancer drug effect on nuclear stiffness is performed on human lung cancer cells. Chemotherapeutic agent Doxorubicin (Dox) were used to treat these cancer cells and mechanical properties of nucleus were studied using the combined confocal and Brillouin spectroscopic system, as discussed above. The combined study of membrane fluctuations and stiffness measurements represent the positive correlation and indicate the softening the nuclei of tumor cells after treating with chemotherapeutic drug.
Quantitative phase microscopy (QPM) techniques developed so far primarily belongs to high speed transmitted light based systems that has enough sensitivity to resolve membrane fluctuations and dynamics, but has no depth resolution. Therefore, most biomechanics studies using QPM today is confined to simple cells, such as RBCs, without internal organelles. An important instrument that will greatly extend the biomedical applications of QPM is to develop next generation microscope with 3D capability and sufficient temporal resolution to study biomechanics of complex eukaryotic cells including the mechanics of their internal compartments. For eukaryotic cells, the depth sectioning capability is critical and should be sufficient to distinguish nucleic membrane fluctuations from plasma membrane fluctuations. Further, this microscope must provide high temporal resolution since typical eukaryotes membranes are substantially stiffer than RBCs. A confocal reflectance quantitative phase microscope is presented based on multi-pinhole scanning, with the capabilities of higher temporal resolution and sensitivity for nucleic and plasma membranes of eukaryotic cells. System hardware is developed based on an array of confocal pinhole generated by using the ‘ON’ state of subset of micro-mirrors of digital micro-mirror device (DMD, from Texas Instruments) and high-speed raster scanning provides 14ms imaging speed in wide-field mode. A common path interferometer is integrated at the imaging arm for detection of specimens’ quantitative phase information. Theoretical investigation of quantitative phase reconstructed from system is investigated and application of system is presented for dimensional fluctuations measurements of both cellular plasma and nucleic membranes of embryonic stem cells.
Most of the quantitative phase microscopy systems are unable to provide depth-resolved information for measuring complex biological structures. Optical diffraction tomography provides a non-trivial solution to it by 3D reconstructing the object with multiple measurements through different ways of realization. Previously, our lab developed a reflection-mode dynamic speckle-field phase microscopy (DSPM) technique, which can be used to perform depth resolved measurements in a single shot. Thus, this system is suitable for measuring dynamics in a layer of interest in the sample. DSPM can be also used for tomographic imaging, which promises to solve the long-existing “missing cone” problem in 3D imaging. However, the 3D imaging theory for this type of system has not been developed in the literature. Recently, we have developed an inverse scattering model to rigorously describe the imaging physics in DSPM. Our model is based on the diffraction tomography theory and the speckle statistics. Using our model, we first precisely calculated the defocus response and the depth resolution in our system. Then, we further calculated the 3D coherence transfer function to link the 3D object structural information with the axially scanned imaging data. From this transfer function, we found that in the reflection mode excellent sectioning effect exists in the low lateral spatial frequency region, thus allowing us to solve the “missing cone” problem. Currently, we are working on using this coherence transfer function to reconstruct layered structures and complex cells.
Quantitative phase microscopy (QPM), used to measure the refractive index, provides the optical path delay measurement at each point of the specimen under study and becomes an active field in biological science. In this work we present development of confocal reflection phase microscopy system to provide depth resolved quantitative phase information for investigation of intracellular structures and other biological specimen. The system hardware development is mainly divided into two major parts. First, creates a pinhole array for parallel confocal imaging of specimen at multiple locations simultaneously. Here a digital micro mirror device (DMD) is used to generate pinhole array by turning on a subset micro-mirrors arranged on a grid. Second is the detection of phase information of confocal imaging foci by using a common path interferometer. With this novel approach, it is possible to measure the nuclei membrane fluctuations and distinguish them from the plasma membrane fluctuations. Further, depth resolved quantitative phase can be correlated to the intracellular contents and 3D map of refractive index measurements.
Light induced fluorescent microscopy has long been developed to observe and understand the object at microscale, such as cellular sample. However, the transfer function of lense-based imaging system limits the resolution so that the fine and detailed structure of sample cannot be identified clearly. The techniques of resolution enhancement are fascinated to break the limit of resolution for objective given. In the past decades, the resolution enhancement imaging has been investigated through variety of strategies, including photoactivated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), stimulated emission depletion (STED), and structure illuminated microscopy (SIM). In those methods, only SIM can intrinsically improve the resolution limit for a system without taking the structure properties of object into account. In this paper, we develop a SIM associated with Bayesian estimation, furthermore, with optical sectioning capability rendered from HiLo processing, resulting the high resolution through 3D volume. This 3D SIM can provide the optical sectioning and resolution enhancement performance, and be robust to noise owing to the Data driven Bayesian estimation reconstruction proposed. For validating the 3D SIM, we show our simulation result of algorithm, and the experimental result demonstrating the 3D resolution enhancement.
This paper presents an intensity normalization method for analysis of liver tissue images, acquired using the two-photon
microscopy system at different stages of fibrosis. Image informatics methods require precise intensity segmentation for
analysis of collagen, vessel and cellular structures. Intensities of the images recorded at different time intervals
corresponding to the progression of fibrosis could vary spatially and temporally depending on the experimental
conditions. These variations significantly affect the image segmentation process and thus the final image analysis,
especially when automatic computer-based methods are used for diagnostic parameters quantification. We propose an
adaptive intensity normalization method that facilitates spatial and temporal intensity variations of the images before the
segmentation process. The images are first portioned into a tessellation of regions with relatively uniform background
pixels intensities and then the normalization is performed to make sure the intensity range is unified throughout the
whole set of image data. This approach is further extended for montage of images acquired from multianode
photomultiplier tube based multifocal multiphoton microscope (MMM) system. The proposed approach significantly
improves the automated analysis of images with varying intensities without any user intervention.
A simple, full-field method to characterize phase modulation property of translucent twisted nematic spatial light
modulator (TN-LCSLM) is presented using Digital Holography (DH). Generally, amplitude and phase modulation is
coupled due to simultaneous distortion in twist alignment and tilt order with applied field. The change in birefringence of
liquid crystal material with respect to input grey scale produces phase modulation of wavefront transmitted through TNLCSLM.
Most existing techniques for phase modulation characterization of LCSLM are complex requiring lengthy
calculations and measure a very small region of total active region of SLM. In this paper, a transmission Digital
Holographic system is used to extract phase information from the digitally recorded and numerically reconstructed
wavefront. The advantage of phase modulation characterization using DH is that the phase value from larger area inside
the active region of TN-LCSLM can be visualized and quantified in real time.
MEMS are tiny mechanical devices that are built onto semiconductor chips and are measured in micrometers and
nanometers. Testing of MEMS device is an important part in carrying out their functional assessment and reliability
analysis. Development of systems based on digital holography (DH) for MEMS inspection and characterization is
presented in this paper. Two DH reflection systems, table-top and handheld types, are developed depending on the
MEMS measurement requirements and their capabilities are presented. The methodologies for the systems are
developed for 3D profile inspection and static & dynamic measurements, which is further integrated with in-house
developed software that provides the measurement results in near real time. The applications of the developed systems
are demonstrated for different MEMS devices for 3D profile inspection, static deformation/deflection measurements and
vibration analysis. The developed systems are well suitable for the testing of MEMS and Microsystems samples, with
full-field, static & dynamic inspection as well as to monitor micro-fabrication process.
Development of a commercial prototype of reflection handheld digital holographic microscope system is presented in
this paper. The concept is based on lensless magnification using diverging wave geometry and the miniaturized optical
design which provides a compact packaged system. The optical geometry design provides the same curvature of object
and reference waves and thus phase aberration is automatically compensated. The basic methodology of the system is
developed and it further explored for 3D imaging, static deflection and vibration measurements applications. Based on
the developed methodology an user-friendly software is developed suitable for industrial shop floor environment. The
applications of the system are presented for 3D imaging, static deflection measurement and vibration analysis of MEMS
samples. The developed system is well suitable for the testing of MEMS and Microsystems samples, with full-field and
real-time features, for static and dynamic inspection and characterization and to monitor micro-fabrication process.
In this paper, dynamic imaging of micro-particles in 3D using lensless in-line digital holographic microscopy (LIDHM)
is explored. The methodology presented retains the reconstructed pixels size used for the particles analysis at different
depth locations under converging wave reconstruction geometry. Experiments studying the 3D visualization of copolymer
microsphere suspensions in distilled water are presented. The dynamic behavior of microspheres in 1mm cube
volume is captured and the numerical reconstruction provides their volumetric flow behavior. From the plot the 3D
visualization and the location of each particle, its depth and dynamic behavior can be observed clearly. The proposed
work is useful for tracking the 3D dynamic behavior of particles and can be used to predict the motion of the moving
particles in volume.
The inspection and characterisation of laser marks using digital holographic microscopy (DHM) is presented in this
paper. A DHM system in transmission mode was designed and the reconstruction algorithm for this configuration was
investigated. The software was developed to provide live reconstruction of holograms for real time numerical evaluation
of amplitude and phase contrast images. A CO2 laser-based marking system was employed to create marks on glass
substrates. By analysing the quality, 3D profile measurement, and material distribution of the marked area, the
parameters of the laser system could be optimised to achieve the desired mark. The phase contrast images provide
quantitative refractive index analysis and 3D profile studies. The results were compared with those obtained using white
light confocal microscopy. The capabilities and advantages of the DHM system for the analysis of laser marks are also
presented.
In this paper vibration characterization of MEMS cantilevers are presented using lens-less in-line digital holographic
microscope (LDHM). In-line digital holography provides larger information capability with higher phase sensitivity,
and full CCD sensor area is utilized for real image reconstruction. In lensless in-line digital holographic microscope, a
highly diverging beam replaces the conventional microscope objectives to provide the required magnification. The
diverging wave geometry also reduces the effect of twin-image wave caused by the in-line holographic geometry. For
vibration analysis, the time averaged holograms were recorded corresponding to different vibration states of the
cantilevers. Direct numerical evaluation of the amplitude and phase information from single time averaged hologram
provides the full-field real time quantitative analysis. The experimental study of vibration measurements of Aluminum
nitride (AlN) driven cantilevers is performed. The full field study shows the simultaneous vibration behavior of many
cantilevers corresponding to same input conditions. Our study shows the shift in the resonant condition of cantilevers
both for first and second resonant frequencies. This kind of analysis is most suitable to optimize and monitoring the
fabrication process of cantilevers.
KEYWORDS: Digital holography, Holograms, Holography, Microscopes, 3D metrology, 3D image processing, Reflection, 3D image reconstruction, Glasses, Silicon
We report the development of a simple commercial digital holographic microscope. The hologram is
recorded using a CCD sensor and numerically reconstructed to provide quantitative analysis of the
object. The laser source is coupled via fibre optics and the opto-mechanical setup is flexible and
customizable for either the reflection or transmission mode. The user-friendly software allows live
reconstruction, simultaneously providing both the amplitude and phase images. System performance is
improved with phase unwrapping and interferometric comparison. Additional features include various
image enhancements, cross-sectional and line profiling, measurement and data analysis tools for
quantitative 3D imaging and surface topography measurement. The performance of the product is
tested on different micro devices, glass and silicon surfaces.
Theory, analysis and applications of digital in-line holography are presented for metrological applications.
Particularly time averaged in-line digital holography is explored for dynamic characterization of membranes and MEMS
diaphragms. The analysis and capability of numerically reconstructed amplitude and phase information from time
averaged holograms is presented. Reconstructed amplitude provides the vibration mode shapes by showing the time
average fringes that are modulated by zero-order Bessel function, same as in conventional time-averaged holography.
However the numerical phase information divided in two parts, the first part represents the surface roughness
information of object and is a source of noise for single exposure, and the second part called the time average phase. By
using a novel double exposure method, the reconstructed phase information from time averaged holograms can be used
for mean static deformation as well for better visualization of time averaged fringes. In case of the vibrating objects with
simultaneous mean static deformation, the phase information mixes together and used for precise analysis of vibration
behaviors. The use of double exposure method also suppress the noise from the real image wave, caused by overlapping
of zero-order term and twin image wave because of in-line geometry. The experimental results are presented for
vibrations of aluminum membrane with 10mm in size, and also for a MEMS diaphragm with 6mm in size.
Digital holographic interferometry allows accurate measurements on a microscopic level. As the number and size
of the recorded digital holograms increase so does their data volume. As a result the volume of holographic data can
substantially constrain applications where storage or transmittance of such data is required. Compression of holographic
data in order to reduce their storage requirements has been studied. The speckled nature of the interferograms makes
their compression nontrivial; however image compression algorithms such as JPEG, JPEG2000 and Set Partitioning In
Hierarchical Trees (SPIHT) have been shown to perform adequately. So far the compression effects of the holographic
interferograms using such coding methods have mainly been studied in terms of errors at the reconstruction intensity.
On the other hand, metrology applications usually rely on the holograms' reconstructed phase. In this paper we
investigate hologram compression and how it affects the reconstructed phase. Holographic interferometry experiments
are carried out to investigate measurement error due to interferograms compression using image compression methods.
The results indicate that compression can be achieved while the measurement error due to compression is retained low.
In this paper a lensless in-line digital holographic microscope is presented as interferometric applications for dynamic measurements. A diverging beam is used to illuminate the object to get the required magnification. In particular, time average in-line digital holographic interferometry is studied for vibration measurements of a smaller membrane. The sensitivities of the numerically reconstructed amplitude and phase information are studied with the distance from object to the CCD, during hologram recording. It is shown that, the increase in object recording distance results the increase in the sensitivity of the Bessel type of fringes representing the vibration amplitude information, while it shows the opposite behaviour for phase information which represent the mean deformation fringes. To explain this phenomenon, the samplings of the interference of object and reference beams, and of the diffracted speckled wavefront from the object are individually studied. A double exposure approach is used for the suppression of noise from real image wave caused by zero-order term and twin image waves because of in-line geometry. The experiment is performed for the study of vibration behaviour of harmonically excited aluminium membranes of 5 mm in size and results are presented.
In this paper in-line digital holography has been explored for dynamic micro metrological applications. In in-line digital holography, full CCD sensor area is utilized for real image reconstruction of the objects with less speckle noise. Numerical evaluation of the amplitude and phase information during reconstruction process finds promising applications in optical micro-metrology. Vibration analysis of the smaller object has been performed by combining the time average principle with in-line digital holographic methods. A double exposure method has been explored for measurements, which is simultaneously used to suppress the overlapping of zero-order and twin image wave with real image wave. The vibration amplitude and mean static state deformation of the harmonically excited object are analysed separately from time average in-line digital holograms. The experimental results are presented for a thin aluminium membrane of 5mm diameter.
In-line digital holography is presented for the particles analysis in 3-D. The presence of zero order wave, conjugate image wave, and defocused images degrade the image quality in in-line digital holography. In this paper we utilise the numerical reconstruction process to minimise these effects. We present a new subtraction method of reconstructed wave fields for particles analysis. The effects of zero order term, conjugate image wave and more importantly the particles outside the plane of interest (defocused images) are simultaneously minimized, and thus improve the contrast of reconstructed amplitude images. This method is useful to slice in-line digital holograms to display only in-focus particles at any particular plane. Furthermore all this is accomplished from a single hologram with no additional pre or post processing required.
Nanomaterials have gained considerable importance in various fields such as chemistry, physics, materials science, biology, and bio-engineering due to the great demand in designing nanomaterial with controlled size, shape and physico-chemical properties. The conventional methods for the preparing metal chalcogenide suffer from several limitations, such as high processing temperature, relatively high cost, non-stoichiometric compositions and poor crystallinity. Metal chalcogenide particles with fine size and spherical morphology and non-aggregation have important applications for the on going technological advancement. It is important to develop a process that can produce particles having controlled characteristics such as morphology, composition and size. In recent years, sonochemistry offers an advantageous alternate in design of nanomaterial with desired properties. This review is mainly focused on the preparation of nanosized metal chalcognide using sonochemistry and their practical applications.
The response of fine wire resistance temperature transducer was analyzed including the effects of (i) conductive heat transfer between wire and the supporting prong and (ii) a thermal boundary layer which can form around the prongs especially at low stream velocities. The transfer function H(w) varied substantially with frequency of turbulence and density and velocity of the air stream. The a.c. gain of the sensor was evaluated as a function of altitude (0-30 km) and air stream velocity. Variation in a.c. gain with altitude can lead to a significant error in the measurement of turbulence with (delta)T probes. The error becomes even larger if the velocity of air stream (relative to sensor) becomes very small.
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