Broadband Digital Holographic Camera for Microscopy

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.

Shot noise, refractive index tomography and aberrations estimation in digital holographic microscopy

Florian Charrière

The present thesis develops some specific aspects of digital holographic microscopy (DHM), namely the effect of shot noise on the phase image accuracy, the use of DHM in micro-tomography and in aberrations evaluation of a microscope objective (MO). DHM is an imaging technique, allowing to measure quantitatively the wavefront transmitted through or reflected by a specimen seen through a MO. A hologram, composed by the interference of the wave coming from the object with a reference wave, is recorded with a camera and then numerically processed to extract both amplitude and phase information. Thanks to its interferometric nature, DHM provides phase images, corresponding to a nanometric accuracy along the optical axis of the microscope, revealing extremely detailed information about the specimen surface in reflection configuration or its internal structure in transmission configuration. DHM has proven its efficiency on numerous applications fields going from cells biology to MEMS-MOEMS devices. In a first part, the use of DHM as metrological tools in the field of micro-optics testing is demonstrated. DHM measurement principle is compared with techniques employed in Twyman-Green, Mach-Zehnder, and white-light interferometers. Refractive microlenses are characterized with reflection DHM and the data are confronted with data obtained with standard interferometers. Specific features of DHM such as digital focussing, measurement of shape differences with respect to a perfect model, surface roughness measurements, and evaluation of a lens optical performance are discussed. The capability to image nonspherical lenses without modification of the optical setup, a key advantage of DHM against conventional interferometers, is demonstrated on a cylindrical mircrolens and a square lenses array. A second part treats the effect of shot noise in DHM. DHM is a single shot imaging technique, and its short hologram acquisition time (down to microseconds) offers a reduced sensitivity to vibrations. Real time observation is achievable, thanks to present performances of personal computers and digital camera. Fast dynamic imaging at low-light level involves few photons, requiring proper settings of the system (integration time and gain of the camera; power of the light source) to minimize the influence of shot noise on the hologram when the highest phase accuracy is aimed. With simulated and experimental data, a systematic analysis of the fundamental shot noise influence on phase accuracy in DHM is presented. Different configurations of the reference wave and the object wave intensities are also discussed, illustrating the detection limit and the coherent amplification of the object wave. In a third part, DHM has for the first time been applied to perform optical diffraction tomography of biological specimens: a pollen grain and living amoebas. Quantitative 2D phase images are acquired for regularly-spaced angular positions of the specimen covering a total angle of π, allowing to build 3D quantitative refractive index distributions by an inverse Radon transform. A precision of 0.01 for the refractive index estimation and a spatial resolution in the micron range are shown. For the amoebas, morphometric measurements are extracted from the tomographic reconstructions. The fourth part presents a DHM technique to determine the integral refractive index and morphology of cells. As the refractive index is a function of the cell dry mass, depending on the intra-cellular concentration and the organelles arrangement, the optical phase shift induced by the specimen on the transmitted wave can be regarded as a powerful endogenous contrast agent. The dual-wavelengths technique proposed in this thesis exploits the dispersion of the perfusion medium to obtain a set of equations, allowing decoupling the contributions of the refractive index and the cellular thickness to the total phase signal. The two wavelengths are chosen in the vicinity of the absorption peak of a dye added to the perfusion medium, where the absorption is accompanied by a strong variation of the refractive index as a function of the wavelength. The technique is demonstrated on yeasts. The last part exposes two methods capable of measuring the complex 3D amplitude point spread function (APSF) of an optical imaging system. The first approach consists in evaluating in amplitude and phase the image of a single emitting point, a 60 nm diameter tip of a Scanning Near Field Optical Microscopy (SNOM) fiber, with an original digital holographic setup. A single hologram giving access to the transverse APSF, the 3D APSF is obtained by performing an axial scan of the SNOM fiber. The method is demonstrated on an 20x 0.4 NA MO. For a 100x 1.3 NA MO, measurements performed with the new setup are compared with the prediction of an analytical aberrations model. The second method allows measuring the APSF of a MO with a single holographic acquisition of its pupil wavefront. The aberration function is extracted from this pupil measurement and then inserted in a scalar model of diffraction allowing to calculate the distribution of the complex wavefront propagated around the focal point. The results are compared with a direct measurement of the APSF achieved with the first proposed approach.

EPFL2007

Coherent imaging from bacteria to multicellular organisms

Séverine Coquoz

Structural and functional imaging of cells, tissues and organisms is crucial for understanding biomedical processes. Fluorescence microscopy is an established tool and has contributed to many discoveries in the life sciences. This technique provides molecular contrast but has limitations inherent to the fluorescent probes, namely phototoxicity and photobleaching. Phase microscopy provides a label-free alternative. Contrast is achieved by translating the phase variations induced by the sample into measurable intensity variations. While phase contrast and differential interference contrast microscopy offer a means to generate contrast without exogenous contrast agents, quantitative phase imaging (QPI) techniques have emerged, enabling the measurement of the phase delays. This measurement yields information on the thickness, refractive index and dry mass of the sample. QPI is thus a powerful tool and has proven its use in various biomedical applications. In the first part of this thesis, new concepts for extending the capabilities of QPI and applications exploiting them are presented. We implement piezo-based Fourier phase microscopy, exhibiting high spatial and temporal sensitivities and resolutions for label-free imaging of cell dynamics at various time scales. The phase is retrieved via phase-shifting intereferometry using a piezo-actuated module allowing very fast tunable phase modulation. Several examples of applications are described. Furthermore, we exploit the phase-to-mass equivalence for investigating the growth of uropathogenic E. coli and the effect of different antibiotics, thereby providing new insights into fundamental mechanisms of bacterial growth. Because QPI methods are limited to optically thin and weakly scattering objects, the second part of this thesis focuses on optical coherence microscopy (OCM). OCM is an interferometric technique providing 3D images of highly scattering biological samples with micrometric resolution and penetration depth of up to several hundreds of micrometers. Compared to optical coherence tomography, OCM uses high numerical aperture (NA) optics to achieve higher transverse resolution, leading to a decrease of the depth of field (DOF). Several methods have been developed to overcome this trade-off between resolution and DOF. Extended-focus OCM (xfOCM) provides a particularly interesting solution by illuminating the sample with a Bessel beam. Moreover, interferometric synthetic aperture microscopy (ISAM) achieves depth-independent resolution through an approximate solution to the inverse scattering problem. We develop extended ISAM (xISAM) to combine the benefits of both approaches, and demonstrate its potential on simulated and experimental data. We also propose a new application of visible OCM (visOCM), a xfOCM system using visible light and a high-NA objective to achieve 3D imaging with sub-micrometer spatial resolution. The capabilities of visOCM are well suited for 3D in vivo imaging of C. elegans, an extensively used model organism in biomedical research. We demonstrate that the anatomy of C. elegans can be visualized with high speed and high contrast down to the sub-cellular level. We further show that visOCM can be employed for imaging age-related morphological changes. Altogether, the methods developed in this thesis pave the way to many applications in the life sciences by featuring label-free imaging at various temporal and spatial scales. Several promising applications are pursued.

EPFL2017

Compact lensless digital holography

Manon Rostykus

Microscopy is of high interest for biology since it allows imaging features that are too small to be seen with naked eyes. However, cells are mostly transparent to visible and infrared light which makes it difficult to see with a traditional microscope. To do so, phase imaging was invented by F. Zernike, who received the Nobel prize in 1953 for this invention. It provides intensity contrast to visualize transparent samples such as those found in biology without any staining. Among the different implementations of phase imaging, digital holographic microscopy (DHM) is a well-known phase method which provides a quantitative value of the phase generated by the sample under test. Implementations of DHMs without the help of any lens element (so called lensless) offer the added advantage to provide large field of views (severalmm2 compared to several hundred ¹m2) and more compact setups than traditional DHMs which have high quality microscope objectives. The lateral resolution is limited by the pixel size of the camera. However, several techniques have been developed to obtain subpixel resolution with lensless setups, hence giving a resolution smaller than the pixel size. In this thesis, two lensless transmission DHMs are presented using a side illumination technique in order to further reduce the device size and keep a visual access to the sample. The first one operates in an in-line configuration: the most compact but using time-consuming image post-processing. The second one is also lensless and uses an off-axis configuration allowing quasi-real time imaging but less compact. Their practical use is described and images of transparent (phase only) samples are shown. Super-resolution (subpixel) using several subpixel shifted holograms was then investigated for the in-line configuration and demonstrated on absorptive samples. Since those microscopes are compact and made out of off-the-shelf low priced elements, they could be broadly spread in schools for educational purposes. They could also be implemented in cells incubators, allowing visualization inside processes at low cost and multiplication of the number of measurements made at the same time.

EPFL2018