Phase-contrast and fluorescence X-ray imaging of soft and bio matter

The high brightness and coherence of X-ray synchrotron sources and steady development in X-ray optics are critical ingredients in novel imaging techniques for a variety of domains. We employed several different approaches and a wide energy spectrum of radiation to demonstrate the applicability of synchrotron X-ray imaging in soft granular matter and life science. We applied refraction-enhanced contrast tomography to the Distinct Element Method (DEM) to predict instant granular jamming in an hourglass. The validation of numerical simulations as a DEM is mostly confined to global properties. We present a stringent microscopic test in the case of instant flow jamming in hourglasses: synchrotron X-ray microtomography coupled to advanced image analysis. The results show that DEM is amazingly successful in predicting the actual microscopic position of individual particles from given initial conditions through to the end of the instant jamming process. We further report refraction-enhanced radiology employed in the study of Drosophila Melanogaster wing development. The objective of the study was to reveal the morphological difference between two different phenotypes of fruit flies. Refraction-enhanced radiology is highly sensitive to morphological information, whereas fluorescent X-ray microanalysis techniques can reveal the distribution of heavy elements containing molecules. Here we report the design of a fast fluorescence detection system coupled to an existing radiology setup at the B01A beamline, NSRRC, Taiwan and the successful test on cartilage samples. Since its invention in 1930, Zernike phase contrast has been a pillar in visible microscopy – and more recently in X-ray microscopy – of low-absorption-contrast objects such as biological specimens. We experimentally demonstrated hard-X-ray Zernike microscopy with lateral resolution beyond 30 nm, opening up many new research opportunities in biomedicine and materials science. In the soft X-ray regime we show that synchrotron X-ray fluorescence microscopy can map molecules with fluorine-containing labels and quantitatively measure the fluorine concentration with micrometer spatial resolution. This is an important result for life science since fluorine is a very widely used molecule label for a variety of investigations such as metabolic processes or therapeutic agents. Our results specifically concern Fluorodeoxyglucose and are therefore directly relevant to the crucial neurobiology issue of glucose metabolism.

Hard x-ray Zernike microscopy reaches 30 nm resolution

Vincent Gajdosik, Giorgio Margaritondo

Since its invention in 1930, Zernike phase contrast has been a pillar in optical microscopy and more recently in x-ray microscopy, in particular for low-absorption-contrast biological specimens. We experimentally demonstrate that hard-x-ray Zernike microscopy now reaches a lateral resolution below 30nm while strongly enhancing the contrast, thus opening many new research opportunities in biomedicine and materials science. (C) 2011 Optical Society of America

Optical Society of America2011

Application of CMOS image sensors in optical sectioning and polarization microscopy

Jelena Mitic

In this thesis new imaging approaches for optical microscopy are proposed and studied. They are based on the use of dynamic structured illumination in combination with a demodulation detection concept employing CMOS image detectors. Two particular implementations are suggested: real-time optical sectioning microscopy and whole field quantitative polarization microscopy. Optical sectioning, i.e depth resolved imaging, allows observation of thin sections in volumetric samples. In its classical configuration the wide-field microscope does not allow optical sectioning of the investigated objects. In this thesis the optical sectioning in a wide-field microscope is achieved by illuminating the sample with a continuously moving single spatial frequency pattern, which temporarily modulates the signal obtained on the photodetector. Only the object parts that are in the focus have a good contrast and consequently generate stronger signal on the detector. The optically sectioned images are obtained employing a CMOS image detector, which demodulates the time-dependent component of the image. Two different systems for real-time acquisition of optically sectioned images are proposed. The first one is based on a specially designed smart-pixel-detector-array (SPDA), which allows performing the signal processing by an electronic circuit integrated to each pixel of the sensor. The second system utilizes a commercial CMOS image sensor. Here, the images are treated by a digital signal processor (DSP) integrated into the camera (iMVS-155). Both approaches provide real-time acquisition of optically sectioned images. The proof of principle for the new method is demonstrated by imaging artificial three-dimensional reflective samples. The optical sectioning imaging of biological samples in bright-field mode and in fluorescence mode is also demonstrated. The optical sectioning performances of the studied systems are similar to that of the confocal microscope. However the new method has some advantages over the classical confocal system: e.g. the difficulties with the alignment and the post-processing inherent to the confocal system are avoided. The theoretical framework presented in the thesis describes the image formation in structured illumination for both reflective and fluorescent samples. The influence of longitudinal chromatic aberration and spherical aberration caused by specimen induced index mismatch on depth discrimination property are studied. In extension to the work related to optically sectioned microscopy, the quantitative polarization microscopy imaging method is proposed as a new application for CMOS detectors. The polarization state of the illuminating light is dynamically changed and the demodulation detection approach is employed to extract the retardance and azimuth of the birefringent sample in real-time; for a whole field of view. Examples of polarization sensitive measurements for different samples are presented. The theoretical analysis is performed using the Jones formalism. Finally, the potentials and limitations of the CMOS detectors for applications in optical microscopy are discussed. Thanks to constant advancements in CMOS technology, the acquisition speed, resolution and sensitivity of new CMOS detectors generations have been improved. This will allow adapting the methods proposed in this thesis for investigations of fast dynamic process where high temporal and spatial resolution is required.

EPFL2004

Biomedical optical techniques for intracochlear cellular imaging

Marilisa Romito

Imaging the inner ear microanatomy is of great importance for the assessment of the state of the cells responsible for sound detection. Hearing disorders are mainly due to a malfunction of the cochlea, the bone containing the cells inside the inner ear. Under this situation, using suitable imaging techniques would be extremely helpful in detecting disease and diagnosing pathologies of the inner ear. However, cochlear small size and encasement in bone provide challenging obstacles, preventing visualization of intracochlear microanatomy using standard clinical imaging modalities. In this thesis, we explore optical techniques to image inner ear cells and we report an innovative method to observe intracochlear structures through the scattering cochlear bone. Firstly, we report different imaging techniques we used for intracochlear cellular investigation, starting with conventional optical microscopy. Two-photon excitation fluorescence (TPEF) microscopy is the most suitable technique for high quality images of the hair cells within the organ of Corti. Our results from extracted mouse organ of Corti show that we are able to distinguish between healthy and damaged sensory cells and nerve fibers, analyzing TPEF images from different mouse samples: young, old, exposed to noise and not exposed to noise. We then describe different noninvasive methods that have been used to image intact cochleae, such as Optical Coherence Tomography (OCT), X-ray Computed Tomography (X-Ray CT), micro-Computed Tomography (X-Ray µCT) and Optical Diffraction Tomography (ODT). These techniques allow the observation of the internal ear anatomy, despite the bone. We report tomographic volumetric reconstructions of mouse cochlea and we compare them in terms of achievable image resolution. µ-CT is the most appropriate techniques providing both penetration through bone and high-level resolution images down to 2µm. However, the high dose of radiation used in µCT studies prevent translation of these techniques to living humans. Due to the discussed challenges for imaging the hair cells through the highly scattering bone, the aim of my thesis is to develop a new method for intracochlear cellular imaging with minimum trauma. The last chapter represents the core of this PhD project. In this chapter, after considering the advantages and disadvantages of all the possible imaging techniques, we report our innovative technique for visualization of cochlear cells through the overlying scattering bone by combining femtosecond laser bone ablation and TPEF microscopy. The controlled ultrafast laser ablation reduces the optical scattering of the cochlear bone while the TPEF provides a contrast mechanism to resolve individual cells behind the bone. We implemented a simultaneous OCT with the laser ablation to enhance the precision of ablation and prevent inadvertent violation of the delicate cells hidden behind bone. An additional bright-field camera shows real-time images of the sample. We demonstrate that our approach improves the light focusing capability through the cochlear bone, allowing imaging of intracochlear structures in intact cochleae with high resolution. This extremely highly promising approach can be further developed for clinical ablation instruments and endoscopes that can reach the cochlea and produce images of cells within the inner ear to establish precise diagnosis, guide treatment and assess response to therapies, which is not yet possible in the clinic.