Optical projection tomography for whole organ imaging

In the past twenty years, far-reaching studies of molecular and cellular processes have reached a milestone in their maturation, and the knowledge from these studies was ready to apply at higher organizational levels. At that time, rodent models were long established. However, methods were inappropriate to image a whole rodent organ, such as the mouse brain, which drove the emergence of a new range of imaging techniques, later gathered under the name mesoscopy. Mesoscopic techniques filled a gap between classical microscopy and medical imaging techniques, such as magnetic resonance imaging, and X-ray computed tomography. They allow the acquisition of centimeter-sized samples. In this thesis, we focus on one of these mesoscopic imaging techniques called optical projection tomography, or OPT, and its potential application to Alzheimer's disease (AD) research. We review the fundamentals of OPT and describe the filtered back-projection algorithm, which is the primary tomographic reconstruction method of this technique. We also go through the implementation of OPT for whole mouse brain imaging, including sample preparation. We show that OPT is suitable to image the whole brain anatomy based on endogenous fluorescence, and the whole neural vasculature as well as amyloid plaques (a hallmark of AD) with adequate fluorescent markers. Then, we dwell on the characterization of OPT instruments. We give some insights on the instrument point spread function and discuss the influence of the number of projections on the quality of the reconstructed image. Afterward, we illustrate the application of OPT to study amyloidosis progression in a preliminary cross-sectional study, where we have used supervised learning to quantify the amyloid plaque load. In this study, we show that OPT can be used to quantify amyloidosis in whole mouse brains and that comparison between individuals of different age can be performed. Imaging of a whole mouse brain is unquestionably necessary. At this scale though, it has some constraints. We present the limitations of OPT, and we share how we think they can be circumvented by combining this modality with another microscopy technique, namely structured illumination microscopy. We see that this other microscopy technique has the potential to produce high-resolution zooms in selected regions of interest based on a prior OPT acquisition. The results presented in this work have led to the duplication of our OPT instrument in Lund University, and we hope they will help to foster advances in OPT and broaden its range of application. We also hope that this work will contribute to making OPT more accessible and user-friendly.

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.

EPFL2019

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

Vincent Gajdosik

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.

EPFL2009

Extended Focus Optical Coherence Microscopy

Martin Villiger

Optical coherence tomography (OCT) is an interferometric imaging technique that can provide depth resolved cross-sectional views of biological tissue. OCT employs light with low temporal and yet high spatial coherence. The sample is illuminated point by point and raster scanned in the lateral directions. Part of the incident light is back-scattered by differences in the tissue refractive index and combined with a strong reference signal. The detection in the Fourier, or frequency domain (FDOCT), separates the interference signal spectrally and records the resulting pattern with a line camera. Processing of this pattern extracts the sample structure along the axial direction but without axial scanning and in parallel with high resolution of 2-3 µm. The interferometric detection conveys a very high sensitivity that allows imaging several hundred micrometers deep into the tissue. A single two dimensional scan thus can extract the three dimensional structure of the object, resulting in very high volume acquisition rates and offering an exceptional speed advantage over other optical imaging methods. Combined with its ability to measure an intrinsic sample property without the need of adding extrinsic labels or contrasting agents, FDOCT has become a confirmed tool for minimally invasive in vivo measurements and comprehensive volumetric imaging. While the axial resolution in OCT is defined by the coherence length of the employed light source, it is the optical focusing that specifies the resolution in the lateral direction, which reaches in general only approximately 10 µm. To improve the resolution, classical optical coherence microscopy (OCM) uses higher numerical apertures. However, this implies a reduction of the lateral dimension of the focal spot which leads to a dramatic decrease in the depth of field (DOF). This imposes a severe limitation on FDOCT's parallel depth extraction. Therefore, the motivation behind this thesis work was to circumvent the compromise between the lateral resolution and the depth of field by engineering an extended focal volume (xf). Combining FDOCT's assets of speed and sensitivity with the high lateral resolution of microscopy provided a very promising tool for rapid in vivo imaging at close to cellular resolution. To study the mechanisms limiting the DOF and to investigate what impact they have on the tomogram reconstruction process, we developed a model for image formation in FDOCT. Generally, the tomogram is reconstructed from the two dimensional interference patterns in the individual spectral channels of the detection. By making use of the coherent transfer functions (CTF), the spatial frequency content made accessible by each channel, was analyzed. This provided a novel perspective on the entire tomogram formation. We were able to show that the out-of-focus structures suffer from two signal degrading mechanisms. First, a de-focusing effect, induced by additional phase arguments in the spatial frequency domain provokes a lateral blurring. Second, the setup's reduced transmission efficiency for out-of-focus structures results in an amplitude scaling. The application of the CTF enabled us to illustrate the intrinsic link between these two effects and to develop optical design strategies and novel configurations to circumvent their detrimental impact on the quality of the tomogram. It is known that with the use of a conical lens, it is possible to produce a so-called diffraction-less beam. Close to the optical axis, the radial envelope of its field amplitude follows a Bessel function of order zero and is maintained upon propagation over a long axial range. We could show that a combination of such a Bessel-like beam with a Gaussian detection indeed limits both the de-focusing and amplitude scaling phenomenon, all the while improving the lateral resolution. The price to pay is a reduced signal amplitude also at the in-focus position, as compared to a Gaussian focal volume of a classical confocal setting. Another possibility is to combine the Bessel-like illumination with a Bessel-like detection, although with differently spaced side lobes in its envelope. These different combinations were evaluated both analytically and with computer simulations. Several of the proposed solutions were implemented in practice to confirm the theoretical predictions. Considering that the theoretical analysis was performed in the spatial frequency domain and in terms of the CTF, the corresponding system parameters had to be measured. Lacking an adequate technique, we developed and validated a method which derived characteristic parameters of the optical system's CTF from the measurement of a simple rubber surface. The tomogram of this sample consists of a fully developed speckle field. Although generally disfavored for degrading the tomogram quality, in this case the speckle shape provided information on the system characteristics because it was imposed by the illumination and detection apertures of the system in question. Since the principal interest behind the development of xfOCM was its amenability to live imaging, we used this novel concept to visualize individual islets of Langerhans in a mouse pancreas. These endocrine islets are assemblies of insulin-secreting beta cells contained within the exocrine tissue of the organ. They are of primary importance in the research on diabetes, but their large diversity in size, down to the micrometer scale, and their localization in the pancreas have so far hindered their observation in vivo. To meet the conditions for successful live imaging, we implemented the xfOCM into a specially designed setup that could provide enough mechanical robustness to allow for measurements over long periods. In addition, the set up was equipped with a fast beam scanning device to achieve high volume acquisition rates. The signal processing and the user interface were conceived in such a way that they offered a rapid visualization of the tomograms, providing an instantaneous feedback and facilitating sample handling. With the developed architecture we achieved label-free in vivo visualization of pancreatic lobules, ducts, blood vessels and individual islets of Langerhans and could demonstrate the potential of the xfOCM in diabetes research for high resolution studies on live animals. Besides the aim of an extended focal volume, the control over the design of the CTF also enabled the realization of a dark field configuration. Signals with low spatial frequencies are suppressed in this configuration, which enabled the measurement of the weak scattering signature of near transparent live cell samples. In conclusion, this thesis provides a better understanding of the image formation involved in the FDOCT. Several strategies to increase the trade-off between lateral resolution and DOF were proposed and validated experimentally. The suitability and potential of this approach for small animal in vivo imaging was demonstrated in the context of a relevant biological question.

EPFL2010