In-situ measurement and characterization of cloud particles using digital in-line holography

Satellite measurement validations, climate models, atmospheric radiative transfer models and cloud models, all depend on accurate measurements of cloud particle size distributions, number densities, spatial distributions, and other parameters relevant to cloud microphysical processes. And many airborne instruments designed to measure size distributions and concentrations of cloud particles have large uncertainties in measuring number densities and size distributions of small ice crystals. HOLODEC (Holographic Detector for Clouds) is a new instrument that does not have many of these uncertainties and makes possible measurements that other probes have never made. The advantages of HOLODEC are inherent to the holographic method. In this dissertation, I describe HOLODEC, its in-situ measurements of cloud particles, and the results of its test flights. I present a hologram reconstruction algorithm that has a sample spacing that does not vary with reconstruction distance. This reconstruction algorithm accurately reconstructs the field to all distances inside a typical holographic measurement volume as proven by comparison with analytical solutions to the Huygens-Fresnel diffraction integral. It is fast to compute, and has diffraction limited resolution. Further, described herein is an algorithm that can find the position along the optical axis of small particles as well as large complex-shaped particles. I explain an implementation of these algorithms that is an efficient, robust, automated program that allows us to process holograms on a computer cluster in a reasonable time. I show size distributions and number densities of cloud particles, and show that they are within the uncertainty of independent measurements made with another measurement method. The feasibility of another cloud particle instrument that has advantages over new standard instruments is proven. These advantages include a unique ability to detect shattered particles using three-dimensional positions, and a sample volume size that does not vary with particle size or airspeed. It also is able to yield two-dimensional particle profiles using the same measurements.

Writing and characterisation methods for UV-photosensitivity based devices and their applications

The underlying work to this thesis focused on the exploitation and investigation of photosensitivity mechanisms in optical fibres and planar waveguides for the fabrication of advanced integrated optical devices for telecoms and sensing applications. One major scope is the improvement of grating fabrication specifications by introducing new writing techniques and the use of advanced characterisation methods for grating testing. For the first time the polarisation control method for advanced grating fabrication has successfully been converted to apodised planar waveguide fabrication and the development of a holographic method for the inscription of chirped gratings at arbitrary wavelength is presented. The latter resulted in the fabrication of gratings for pulse-width suppression and wavelength selection in diode lasers. In co-operation with research partners a number of samples were tested using optical frequency domain and optical low coherence reflectometry for a better insight into the limitations of grating writing techniques. Using a variety of different fabrication methods, custom apodised and chirped fibre Bragg gratings were written for the use as filter elements for multiplexer-demultiplexer devices, as well as for short pulse generation and wavelength selection in telecommunication transmission systems. Long period grating based devices in standard, speciality and tapered fibres are presented, showing great potential for multi-parameter sensing. One particular scope is the development of vectorial curvature and refractive index sensors with potential for medical, chemical and biological sensing. In addition the design of an optically tunable Mach-Zehnder based multiwavelength filter is introduced. The discovery of a Type IA grating type through overexposure of hydrogen loaded standard and Boron-Germanium co-doped fibres strengthened the assumption of UV-photosensitivity being a highly non-linear process. Gratings of this type show a significantly lower thermal sensitivity compared to standard gratings, which makes them useful for sensing applications. An Oxford Lasers copper-vapour laser operating at 255 nm in pulsed mode was used for their inscription, in contrast to previous work using CW-Argon-Ion lasers and contributing to differences in the processes of the photorefractive index change

Methods of Fourier optics in digital holographic microscopy

In this paper, processing methods of Fourier optics implemented in a digital holographic microscopy system are presented. The proposed methodology is based on the possibility of the digital holography in carrying out the whole reconstruction of the recorded wave front and consequently, the determination of the phase and intensity distribution in any arbitrary plane located between the object and the recording plane. In this way, in digital holographic microscopy the field produced by the objective lens can be reconstructed along its propagation, allowing the reconstruction of the back focal plane of the lens, so that the complex amplitudes of the Fraunhofer diffraction, or equivalently the Fourier transform, of the light distribution across the object can be known. The manipulation of Fourier transform plane makes possible the design of digital methods of optical processing and image analysis. The proposed method has a great practical utility and represents a powerful tool in image analysis and data processing. The theoretical aspects of the method are presented, and its validity has been demonstrated using computer generated holograms and images simulations of microscopic objects. (c) 2007 Elsevier B.V. All rights reserved.

Comparative study of human erythrocytes by digital holographic microscopy, confocal microscopy, and impedance volume analyzer.

Red blood cell (RBC) parameters such as morphology, volume, refractive index, and hemoglobin content are of great importance for diagnostic purposes. Existing approaches require complicated calibration procedures and robust cell perturbation. As a result, reference values for normal RBC differ depending on the method used. We present a way for measuring parameters of intact individual RBCs by using digital holographic microscopy (DHM), a new interferometric and label-free technique with nanometric axial sensitivity. The results are compared with values achieved by conventional techniques for RBC of the same donor and previously published figures. A DHM equipped with a laser diode (lambda = 663 nm) was used to record holograms in an off-axis geometry. Measurements of both RBC refractive indices and volumes were achieved via monitoring the quantitative phase map of RBC by means of a sequential perfusion of two isotonic solutions with different refractive indices obtained by the use of Nycodenz (decoupling procedure). Volume of RBCs labeled by membrane dye Dil was analyzed by confocal microscopy. The mean cell volume (MCV), red blood cell distribution width (RDW), and mean cell hemoglobin concentration (MCHC) were also measured with an impedance volume analyzer. DHM yielded RBC refractive index n = 1.418 +/- 0.012, volume 83 +/- 14 fl, MCH = 29.9 pg, and MCHC 362 +/- 40 g/l. Erythrocyte MCV, MCH, and MCHC achieved by an impedance volume analyzer were 82 fl, 28.6 pg, and 349 g/l, respectively. Confocal microscopy yielded 91 +/- 17 fl for RBC volume. In conclusion, DHM in combination with a decoupling procedure allows measuring noninvasively volume, refractive index, and hemoglobin content of single-living RBCs with a high accuracy.

Submicrometer tomography of cells by multiple-wavelength digital holographic microscopy in reflection

We present first results on a method enabling mechanical scanning-free tomography with submicrometer axial resolution by multiple-wavelength digital holographic microscopy. By sequentially acquiring reflection holograms and summing 20 wavefronts equally spaced in spatial frequency in the 485-670 nm range, we are able to achieve a slice-by-slice tomographic reconstruction with a 0.6-1 mum axial resolution in a biological medium. The method is applied to erythrocytes investigation to retrieve the cellular membrane profile in three dimensions.

Determination of Transmembrane Water Fluxes in Neurons Elicited by Glutamate Ionotropic Receptors and by the Cotransporters KCC2 and NKCC1: A Digital Holographic Microscopy Study.

Digital holographic microscopy (DHM) is a noninvasive optical imaging technique that provides quantitative phase images of living cells. In a recent study, we showed that the quantitative monitoring of the phase signal by DHM was a simple label-free method to study the effects of glutamate on neuronal optical responses (Pavillon et al., 2010). Here, we refine these observations and show that glutamate produces the following three distinct optical responses in mouse primary cortical neurons in culture, predominantly mediated by NMDA receptors: biphasic, reversible decrease (RD) and irreversible decrease (ID) responses. The shape and amplitude of the optical signal were not associated with a particular cellular phenotype but reflected the physiopathological status of neurons linked to the degree of NMDA activity. Thus, the biphasic, RD, and ID responses indicated, respectively, a low-level, a high-level, and an "excitotoxic" level of NMDA activation. Moreover, furosemide and bumetanide, two inhibitors of sodium-coupled and/or potassium-coupled chloride movement strongly modified the phase shift, suggesting an involvement of two neuronal cotransporters, NKCC1 (Na-K-Cl) and KCC2 (K-Cl) in the genesis of the optical signal. This observation is of particular interest since it shows that DHM is the first imaging technique able to monitor dynamically and in situ the activity of these cotransporters during physiological and/or pathological neuronal conditions.

Simultaneous Optical Recording in Multiple Cells by Digital Holographic Microscopy of Chloride Current Associated to Activation of the Ligand-Gated Chloride Channel GABA(A) Receptor.

Chloride channels represent a group of targets for major clinical indications. However, molecular screening for chloride channel modulators has proven to be difficult and time-consuming as approaches essentially rely on the use of fluorescent dyes or invasive patch-clamp techniques which do not lend themselves to the screening of large sets of compounds. To address this problem, we have developed a non-invasive optical method, based on digital holographic microcopy (DHM), allowing monitoring of ion channel activity without using any electrode or fluorescent dye. To illustrate this approach, GABA(A) mediated chloride currents have been monitored with DHM. Practically, we show that DHM can non-invasively provide the quantitative determination of transmembrane chloride fluxes mediated by the activation of chloride channels associated with GABA(A) receptors. Indeed through an original algorithm, chloride currents elicited by application of appropriate agonists of the GABA(A) receptor can be derived from the quantitative phase signal recorded with DHM. Finally, chloride currents can be determined and pharmacologically characterized non-invasively simultaneously on a large cellular sampling by DHM.

Measurement of absolute cell volume, osmotic membrane water permeability, and refractive index of transmembrane water and solute flux by digital holographic microscopy.

ABSTRACT. A dual-wavelength digital holographic microscope to measure absolute volume of living cells is proposed. The optical setup allows us to reconstruct two quantitative phase contrast images at two different wavelengths from a single hologram acquisition. When adding the absorbing dye fast green FCF as a dispersive agent to the extracellular medium, cellular thickness can be univocally determined in the full field of view. In addition to the absolute cell volume, the method can be applied to derive important biophysical parameters of living cells including osmotic membrane water permeability coefficient and the integral intracellular refractive index (RI). Further, the RI of transmembrane flux can be determined giving an indication about the nature of transported solutes. The proposed method is applied to cultured human embryonic kidney cells, Chinese hamster ovary cells, human red blood cells, mouse cortical astrocytes, and neurons.

Adding functionalities to precomputed holograms with random mask multiplexing in holographic optical tweezers

In this study, we present a method designed to generate dynamic holograms in holographic optical tweezers. The approach combines our random mask encoding method with iterative high-efficiency algorithms. This hybrid method can be used to dynamically modify precalculated holograms, giving them new functionalities¿temporarily or permanently¿with a low computational cost. This allows the easy addition or removal of a single trap or the independent control of groups of traps for manipulating a variety of rigid structures in real time.

Digital holographic microscopy: a noninvasive contrast imaging technique allowing quantitative visualization of living cells with subwavelength axial accuracy.

We have developed a digital holographic microscope (DHM), in a transmission mode, especially dedicated to the quantitative visualization of phase objects such as living cells. The method is based on an original numerical algorithm presented in detail elsewhere [Cuche et al., Appl. Opt. 38, 6994 (1999)]. DHM images of living cells in culture are shown for what is to our knowledge the first time. They represent the distribution of the optical path length over the cell, which has been measured with subwavelength accuracy. These DHM images are compared with those obtained by use of the widely used phase contrast and Nomarski differential interference contrast techniques.

Axial sub-nanometer accuracy in digital holographic microscopy

We present state-of-the-art dual-wavelength digital holographic microscopy (DHM) measurement on a calibrated 8.9 nm high chromium thin step sample and demonstrate sub-nanometer axial accuracy. By using a modified DHM reference calibrated hologram (RCH) reconstruction method, a temporal averaging procedure and a specific dual-wavelength DHM arrangement, it is shown that specimen topography can be measured with an accuracy, defined as the axial standard deviation, reduced to at least 0.9 nm. Indeed for the first time to the best of our knowledge, it is reported that averaging each of the two wavefronts recorded with real-time dual-wavelength DHM can provide up to 30% spatial noise reduction for the given configuration. Moreover, the presented experimental configuration achieves a temporal stability below 0.8 nm, thus paving the way to Angström range for dual-wavelength DHM.

Automated segmentation of multiple red blood cells with digital holographic microscopy.

We present a method to automatically segment red blood cells (RBCs) visualized by digital holographic microscopy (DHM), which is based on the marker-controlled watershed algorithm. Quantitative phase images of RBCs can be obtained by using off-axis DHM along to provide some important information about each RBC, including size, shape, volume, hemoglobin content, etc. The most important process of segmentation based on marker-controlled watershed is to perform an accurate localization of internal and external markers. Here, we first obtain the binary image via Otsu algorithm. Then, we apply morphological operations to the binary image to get the internal markers. We then apply the distance transform algorithm combined with the watershed algorithm to generate external markers based on internal markers. Finally, combining the internal and external markers, we modify the original gradient image and apply the watershed algorithm. By appropriately identifying the internal and external markers, the problems of oversegmentation and undersegmentation are avoided. Furthermore, the internal and external parts of the RBCs phase image can also be segmented by using the marker-controlled watershed combined with our method, which can identify the internal and external markers appropriately. Our experimental results show that the proposed method achieves good performance in terms of segmenting RBCs and could thus be helpful when combined with an automated classification of RBCs.

Review of quantitative phase-digital holographic microscopy: promising novel imaging technique to resolve neuronal network activity and identify cellular biomarkers of psychiatric disorders.

Quantitative phase microscopy (QPM) has recently emerged as a new powerful quantitative imaging technique well suited to noninvasively explore a transparent specimen with a nanometric axial sensitivity. In this review, we expose the recent developments of quantitative phase-digital holographic microscopy (QP-DHM). Quantitative phase-digital holographic microscopy (QP-DHM) represents an important and efficient quantitative phase method to explore cell structure and dynamics. In a second part, the most relevant QPM applications in the field of cell biology are summarized. A particular emphasis is placed on the original biological information, which can be derived from the quantitative phase signal. In a third part, recent applications obtained, with QP-DHM in the field of cellular neuroscience, namely the possibility to optically resolve neuronal network activity and spine dynamics, are presented. Furthermore, potential applications of QPM related to psychiatry through the identification of new and original cell biomarkers that, when combined with a range of other biomarkers, could significantly contribute to the determination of high risk developmental trajectories for psychiatric disorders, are discussed.

Fabrication and Characterization of Dye Sensitized Polymer Films for Holographic Applications

Holographic technology is at the dawn of quick evolution in various new areas including holographic data storage, holographic optical elements, artificial intelligence, optical interconnects, optical correlators, commerce, medical practice, holographic weapon sight, night vision goggles and games etc. One of the major obstacles for the success of holographic technology to a large extent is the lack of suitable recording medium. Compared with other holographic materials such as dichromated gelatin and silver halide emulsions, photopolymers have the great advantage of recording and reading holograms in real time and the spectral sensitivity could be easily shifted to the type of recording laser used by simply changing the sensitizing dye. Also these materials possess characteristics such as good light sensitivity, real time image development, large dynamic range, good optical properties, format flexibility, and low cost. This thesis describes the attempts made to fabricate highly economic photopolymer films for various holographic applications. In the present work, Poly (vinyl alcohol) (PVA) and poly (vinyl chloride) (PVC) are selected as the host polymer matrices and methylene blue (MB) is used as the photosensitizing dye. The films were fabricated using gravity settling method. No chemical treatment or pre/post exposures were applied to the films. As the outcome of the work, photopolymer films with more than 70% efficiency, a permanent recording material which required no fixing process, a reusable recording material etc. were fabricated.