Extended Focus Optical Coherence Microscopy

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.

Optical microscopy for imaging through scattering media

Xin Yang

Optical microscopy has been widely used in the life sciences and biological research for several decades. It possesses many advantages over x-ray radiography, computed tomography, ultrasound imaging and magnetic resonance imaging, in terms of being low cost, high resolution, harmless and capable of multiple contrast mechanism, etc. However, the weakness of optical microscopy is the inability to perform deep tissue imaging. Owing to the short wavelength of optical waves, it experiences much more scattering than longer waves in inhomogeneous materials such as biological tissues. Scattering scrambles the wavefront and limits the penetration depth of optical microscopy to only hundreds of microns. We explore in this thesis, novel optical approaches to image through scattering media. There are two main categories of the state of art techniques: imaging with ballistic photons, which requires separation of ballistic photons from the sneak or multiple scattered ones; and imaging with scattered photons, which relies on manipulation of the scattered photons according to the specific scattering properties. We primarily focus on the second category in this thesis. Taking advantage of the reversibility of light, phase conjugation has a long history in aberration compensation. A deterministic wave is incident on a scattering medium and results in a random scattered field. If we record the full information of the scattered field (amplitude and phase) and generate a phase conjugated replica of it, the phase conjugated wave can follow the same path and reconstruct the original deterministic wave after passing back through the scattering medium. In this work, phase conjugation is achieved using digital method, called digital phase conjugation. The scattered field is recorded through digital holography, and a spatial light modulator is applied to construct the phase conjugated wave. Second harmonic radiation imaging probes are used as the original light source, rendering the generation of a focus behind the scattering medium. The memory effect, which indicates the correlation between speckles with different incident angles, is exploited to move the focus around and obtain three-dimensional scanning microscopy behind scattering medium. Speckle scanning microscopy is the second method demonstrated in this thesis for imaging through turbid media. In contrast to digital phase conjugation, which requires access from both sides of the scatterers to measure the scattered field, in speckle scanning microscopy, excitation and collection can be achieved from the same side. The specific scattering events are not taken into account, instead, the statistic property of the scatterers plays a major role. This technique makes full use of two facts: 1. The statistically averaged autocorrelation of speckle fields approaches a delta function; 2. When a speckle pattern scans across a fluorescence object, the collected fluorescence intensity is proportional to the convolution of the speckle field and the object. Linking these two facts with delicate calculation, we can eventually minimize the influence of the speckle, and reconstruct two-dimensional images of objects behind turbid medium. Encouraged by the success of two-dimensional speckle scanning microscopy, we investigate more advanced possibilities by simulation, including three-dimensional speckle scanning and reference based speckle scanning techniques. [...]

EPFL2014

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

Towards high resolution optical imaging of beta cells in vivo

Joan Goulley, Anne Grapin-Botton, Theo Lasser, Erica Martin-Williams, Martin Villiger

Endocrine beta cells produce and release insulin in order to tightly regulate glucose homeostasis and prevent metabolic pathologies such as Diabetes Mellitus. Optical imaging has contributed greatly to our current understanding of beta cell structure and function. In vitro microscopy of beta cell lines has revealed the localization of molecular components in the cell and more recently their dynamic behavior. In cultured islets, interactions of beta cells with other islet cells and the matrix as well as paracrine and autocrine signaling or reaction to nutrients have been studied. Lastly, microscopy has been performed on tissue sections, visualizing the islets in an environment closer to their natural surroundings. In most efforts to date, the samples have been isolated for investigation and hence have by definition been divorced from their natural environments and deprived of vascularization and innervations. In such a setting the beta cells lack the metabolic information that is primordial to their basic function of maintaining glucose homeostasis. We review optical microscopy; its general principles, its impact in decoding beta cell function and its recent developments towards the more physiologically relevant assessment of beta cell function within the environment of the whole organism. This requires both large imaging depth and fast acquisition times. Only few methods can achieve an adequate compromise. We present extended focus Optical Coherence Microscopy (xfOCM) as a valuable alternative to both confocal microscopy and two photon microscopy (2PM), and discuss its potential in interpreting the mechanisms underlying glucose homeostasis and monitoring impaired islet function.