Multiple-wavelength digital holographic microscopy

Abstract

In this thesis work, we extend digital holographic microscopy (DHM) capabilities and applications range by using from two to twenty different wavelength laser sources, either simultaneously or sequentially. As an overview, we present methods to increase the measurement range to more than three decades, from demonstrated sub-nanometer precision to several micro-meters, as well as an approach to access difficult high-roughness surface measurement with DHM. Finally a technique achieving multiple-wavelength volume tomography of cells, is exposed and backed by promising preliminary results on red blood cells (RBC). Indeed, a classical limitation of single-wavelength DHM is the restricted measurement range due to the so-called phase ambiguity for sample optical height above one multiple of the wavelength. Although software unwrapping algorithms can solve this issue up to a certain extend, they are sensitive to noise or roughness, time-consuming and cannot cope with high aspect-ratio structures. The presented solution relies on the well-known dual-wavelength optical unwrapping thanks to a generated micron-range beat-wavelength, but with a major real-time advantage by being able to acquire the two-wavelength information simultaneously in one single hologram acquisition, thus maintaining the DHM high-speed and robustness unique features. It is demonstrated that not only this single-shot measurement range extension does not compromise or degrade the nanometer-range axial resolution under reasonable noise and beat-wavelength magnitudes, but that true experimentally-demonstrated sub-nanometer accuracy can be achieved with the proposed configuration at the same time, achieving a DHM investigation range of more than three decades. v In addition to this, the problematic of rough samples investigation by DHM is addressed in this work, as the strong scattering, also known as speckle phenomenon, remains an obstacle for DHM even when working in a two-wavelength mode. A new auto-correlation dual-wavelength hologram reconstruction process is introduced, enabling to successfully measure in two dimensions metallic roughness surfaces with arithmetic rugosity of up to 1.6 µm. Despite losing the digital propagation DHM feature, as well as sensibly reducing the data quantity, the method is able to discard scattering contributions and permits to recover a correct two-dimensional phase map of the surface where previous phase-adjustment reconstruction algorithms would fail. The technique is assessed on a roughness test-target with different types of manufacturing machining processes. Finally, the last part of this work concerns multiple-wavelength DHM tomography in order to achieve a slice-by-slice volume reconstruction of a fixed biological specimen. As will be shown, superposition of about twenty DHM-reconstructed complex wavefronts at different wavelengths in the 485-670 nm range enables true tomography with a sub-micrometer resolution in the axial direction, realizing a slicing effect similar to confocal microscopy but with instantaneous full-field acquisitions. Although the basic principles of this approach are already established, we are presenting major improvements not only enhancing the image reconstruction quality, but more importantly permitting to adapt the method to single-cell investigation. Actually, we propose, for the first time to the extend of our knowledge, a configuration enabling direct on-cell DHM reflection phase measurement, noticeably detecting the ultra-low signal reflected by a cellular membrane. By this means, we present results obtained on fixed erythrocyte cells with enough precision to retrieve the membrane position in the three-dimensional space.