Correction of spherical surface measurements by confocal microscopy

Refractive microlenses are nowadays widely used in optical systems. Characterizing their surface is essential to ensure their quality and to optimize their fabrication process. This is realized by optical surface profilers thanks to their vertical resolution, short measurement time and areal information. However, when measuring non-flat surfaces, errors appear caused by aberrations of the microscope objective used in such systems, which significantly limit the achievable quality of the manufactured spherical surfaces. Approaches have been proposed to tackle these errors, but none of them demonstrated its validity for measurements of high quality microlenses. In this work, we demonstrate that the surface error depends on the surface position within the field of view of the microscope objective and on the surface slope. We then explain how to record the value of this error experimentally: this can be done by measuring a reference ball placed at different positions in the field of view. We finally use a machine learning algorithm to fit the experimental data in order to correct subsequent measurements. We apply this approach to measurements performed by a 20x numerical aperture 0.6 microscope objective of a confocal microscope. The effectiveness of the proposed method is demonstrated by showing that the surface error corresponds to a RMS wavefront error of lambda/7 before correction and of lambda/50 after correction for glass microlenses used in the visible range. This method thus allows the use of high numerical aperture microscope objectives for an accurate characterization of microlenses. Likewise, the fabrication capability of microlenses in terms of slope and quality is greatly extended, which is especially important for aspheres or freeforms.

On the complex three-dimensional amplitude point spread function of lenses and microscope objectives: theoretical aspects, simulations and measurements by digital holography

Florian Charrière, Tristan Colomb, Christian Depeursinge, Jonas Kühn, Mihaela Marian, Pierre Marquet, Frédéric Montfort

The point spread function is widely used to characterize the three-dimensional imaging capabilities of an optical system. Usually, attention is paid only to the intensity point spread function, whereas the phase point spread function is most often neglected because the phase information is not retrieved in noninterferometric imaging systems. However, phase point spread functions are needed to evaluate phase-sensitive imaging systems and we believe that phase data can play an essential role in the full aberrations' characterization. In this paper, standard diffraction models have been used for the computation of the complex amplitude point spread function. In particular, the Debye vectorial model has been used to compute the amplitude point spread function of ×63/0.85 and ×100/1.3 microscope objectives, exemplifying the phase point spread function specific for each polarization component of the electromagnetic field. The effect of aberrations on the phase point spread function is then analyzed for a microscope objective used under nondesigned conditions, by developing the Gibson model (Gibson & Lanni, 1991), modified to compute the three-dimensional amplitude point spread function in amplitude and phase. The results have revealed a novel anomalous phase behaviour in the presence of spherical aberration, providing access to the quantification of the aberrations. This work mainly proposes a method to measure the complex three-dimensional amplitude point spread function of an optical imaging system. The approach consists in measuring and interpreting the amplitude point spread function by evaluating in amplitude and phase the image of a single emitting point, a 60-nm-diameter tip of a Near Field Scanning Optical Microscopy fibre, with an original digital holographic experimental setup. A single hologram gives access to the transverse amplitude point spread function. The three-dimensional amplitude point spread function is obtained by performing an axial scan of the Near Field Scanning Optical Microscopy fibre. The phase measurements accuracy is equivalent to λ/60 when the measurement is performed in air. The method capability is demonstrated on an Achroplan ×20 microscope objective with 0.4 numerical aperture. A more complete study on a ×100 microscope objective with 1.3 numerical aperture is also presented, in which measurements performed with our setup are compared with the prediction of an analytical aberrations model.

2007

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

Correction of surface error occurring in microlenses characterization performed by optical profilers

Jeremy Béguelin, Wilfried Noell, Toralf Scharf

Characterizing the surface of microlenses by optical profilers has the important advantages of measurement speed, flexibility and automation. Nevertheless, the accuracy of such characterization is limited by error occurring in non-flat measurements. Here, we propose a method that uses multiple measurements of a single reference ball combined with a machine learning algorithm that fits the experimental data to correct the measurements. The success of the method is demonstrated by showing that the residual error after correction reaches 20nm RMS. Such results extend greatly the quality of microlens characterization by optical profilers.