Quantitative phase noise in a two-color low coherence Digital Holographic Microscope

In digital holographic microscopy (DHM), the long coherence length of laser light causes parasitic interferences due to multiple reflections in and by optical components in the optical path of the microscope and thus degrades the image quality. The parasitic effects are greatly reduced by using a short coherence length light. The main drawback of using a short coherence light source in an off-axis digital holographic microscope, is the reduction of the interference fringe contrast occurring in the field of view. Previously, we introduced a volume diffractive optical element (VDOE) placed in the reference arm of a DHM to correct the coherence plane tilt so as to obtain a high interference contrast throughout the field of view. Here, we experimentally quantify the spatial and temporal phase noise in the extracted phase image caused by non-homogeneities and scattering of the VDOE element itself. The results over five VDOEs show that the temporal phase noise is unchanged and a slight increase (up to 20%) is observed in the spatial phase noise. These results show that even with a low coherence source, a full field of view can be obtained with an off-axis DHM thanks to the VDOE without introducing significant additional phase noise.

Endoscopic low coherence interferometry applied to tri-dimensional contouring of upper airways

Yves Delacrétaz

This thesis presents a novel approach for optical tri-dimensional contouring, requiring only one acquisition to extract a contour depth. It is based on an interferometric setup, using a reduced coherence light source, and is fully adaptable to endoscopic procedure commonly in used in ear-nose-throat (ENT) medicine. It has been demonstrated that the method can be successfully applied to imaging of porcine upper airways. Although this method uses interferometry to obtain depth range measurement, the phase signal is not directly evaluated, rather the location of the coherent superposition of two waves is extracted, and thus provides a robust technique even in harsh environment. The key feature is to extract the coherent area on the image by locating intentionally induced fringes created by off-axis superposition of a smooth reference wave and an object wave coming from a rough or diffusing sample through an optical system with limited aperture. A model for the propagation of the light through the optical system has been developed. It takes into account the broadened emission spectrum of the light source, as well as the statistical speckle effect induced by the illumination of a rough surface with partially coherent light. Simulated results have been carefully compared with measurements, both in the spatial domain and in the Fourier domain (spatial frequencies response), which has permitted to better understand the processes involved in the creation of the interference fringes, and improve the design of the device. Different filters have been investigated in order to extract the depth information from acquired interferograms. By using Gabor filterbanks, which do not need any a priori knowledge of the signal, selectivity of the extracted signal has been enhanced by a factor of 1.3 compared to a simple local Fourier spectrum evaluation, and time of the extraction procedure divided by 6, even when compared to fast iterative calculation in the Fourier spectrum. Moreover, by imposing the frequency and/or orientation of the signal to detect, which can be known by a simple calibration procedure, it has been proved that the selectivity can be further enhanced by a factor of 2.5, keeping the calculation time as short or even shorter. A compact prototype has been designed and built, with a specific multiple fibers arrangement for illumination. The system is separated in two parts: the first one is directly attached to the rigid endoscope, and contains imaging as well as recombining object/reference wave optics, and the detector. It fits into a box with overall dimensions of 115 × 83 × 94 mm. The second part contains the laser source, the scanning arm and the injection optics for the fibered system. It is designed to be left on a small cart. This device can potentially be brought to the bedside, and used during routine endoscopic check-up. Associated with the device, a complete framework has been elaborated in order to simplify the acquisition procedure as well as the signal processing. This results in a fully automated environment taking in charge hardware control, data storage and signal processing, up to the tri-dimensional scene rendering.

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

Broadband Digital Holographic Camera for Microscopy

Zahra Monemhaghdoust

In this thesis, we explore a combination of analog and digital holographic techniques to demonstrate high speed quantitative phase imaging (QPI) using low coherence (broad bandwidth) light sources. Off-axis digital holographic microscopy is inherently a coherent method of phase quantification and thus suffers from coherent noise. However, only one measurement is required to retrieve field information which gives an advantage in term of image acquisition speed. While incoherent methods of phase quantification remove the problem of coherent noise, they require acquiring several measurements which is sometimes not acceptable in the study of dynamical systems. Therefore, this thesis is an attempt to extend off-axis digital holographic microscopy from the coherent to the incoherent regime to maintain the benefit of high speed measurement capability of off-axis holography while reducing the coherent noise. First, we demonstrate that by using a suitable combination of holographic gratings, off-axis digital holographic microscopy can work with spectrally broadband illumination sources. Holographic gratings introduce a tilt in the coherence plane of one of the reference or object arms of an off-axis digital holographic microscope to expand the interference area to the whole field of view of the camera. Although QPI provides useful information in addition to state-of-the-art intensity-based microscopic imaging, its use has not permeated yet far beyond research laboratories. One potential reason is the need for a stand-alone dedicated microscope system, which is expensive and not back-compatible to the existing wide-field microscopes. In this Regard, the second part of this thesis explores an add-on camera module which can be attached to the camera port of any standard intensity microscope. The techniques developed to extend the off-axis DHM in the first part of the thesis will be used to demonstrate a proof-of-concept camera module which operates with broadband light sources. Overall, through the combination of analog and digital holography, the goal of this work is to extend full-field off-axis holography to incoherent regime to benefit from real-time speckle-free measurements, optical sectioning and realization of a digital holographic camera which can be placed in the image plane of a standard optical microscope.

EPFL2015