Digital Holography Microscopy (DHM): Fast and robust systems for industrial inspection with interferometer resolution

With the recent technological advances, there is an increasing need for measurement systems providing interferometer resolution for inspection of large quantities of individual samples in manufacturing environments.. Such applications require high measurement rates, robustness, ease of use, and non-contact systems. We show here that Digital Holographic Microscopy (DIM), a new method that implements digitally the principle of holography, is particularly well suited for such industrial applications. With the present computers power and the developments of digital cameras, holograms can be numerically interpreted within a tenth of second to provide simultaneously: the phase information, which reveals object surface with vertical resolution at the nanometer scale along the optical axis, and intensity images, as obtained by conventional optical microscope. The strength of DHM lies in particular on the use of the so-called off-axis configuration, which enables to capture the whole information by a single image acquisition, i.e. typically during a few ten of microseconds. These extremely short acquisition times make DHM systems insensitive to vibrations. These instruments can operate without vibration insulation means, making them a cost effective solution not only for R&D, but also especially for an implementation on production lines. Numerous application examples are presented in this paper such as shape and surface characterization of high aspect ratio micro-optics, surface nanostructures, and surface roughness.

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

Digital holography applied to microscopy

Tristan Colomb, Etienne Cuche, Christian Depeursinge, Pierre Magistretti, Mihaela Marian, Pierre Marquet, Frédéric Montfort

We report on new developments of a technique, called Digital Holographic Microscopy (DHM), for the numerical reconstruction of digital holograms taken in microscopy, which allows simultaneous amplitude and quantitative phase contrast imaging. The reconstruction method computes the propagation of the complex optical wavefront diffracted by the object and is used to determine the refractive index and/or shape of the object with accuracies in the nanometer range along the optical axis. The method requires the acquisition of a single hologram. The technique comprises the recording of a digital hologram of the specimen by means of a standard CCD camera at the exit of a Mach-Zender or Michelson type interferometer. The quantitative nature of the reconstructed phase distribution has been demonstrated by an application to surface profilometry where step height differences of a few nanometers have been measured. Another application takes place in biology for transmission phase-contrast imaging of living cells in culture. The resolution for thickness measurements depends on the refractive index of the specimen and a resolution of approximately 30 nanometers in height, and about half of a micron in width, has been achieved for living neural cells in cultures by using a high numerical aperture.

SPIE2002

Advantages of digital holographic microscopy for real-time full field absolute phase imaging

Florian Charrière, Tristan Colomb, Christian Depeursinge, Jonas Kühn, Pierre Marquet

Different interferometric techniques were developed last decade to obtain full field, quantitative, and absolute phase imaging, such as phase-shifting, Fourier phase microscopy, Hilbert phase microscopy or digital holographic microscopy (DHM). Although, these techniques are very similar, DHM combines several advantages. In contrast, to phase shifting, DHM is indeed capable of single-shot hologram recording allowing a real-time absolute phase imaging. On the other hand, unlike to Fourier phase or Hilbert phase microscopy, DHM does not require to record in focus images of the specimen on the digital detector (CCD or CMOS camera), because a numerical focalization adjustment can be performed by a numerical wavefront propagation. Consequently, the depth of view of high NA microscope objectives is numerically extended. For example, two different biological cells, floating at different depths in a liquid, can be focalized numerically from the same digital hologram. Moreover, the numerical propagation associated to digital optics and automatic fitting procedures, permits vibrations insensitive full- field phase imaging and the complete compensation for a priori any image distortion or/and phase aberrations introduced for example by imperfections of holders or perfusion chamber. Examples of real-time full-field phase images of biological cells have been demonstrated.

SPIE2008

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

Florian Charrière

EPFL2007