Time-domain optical coherence tomography with digital holographic microscopy

We show that digital holography can be combined easily with optical coherence tomography approach. Varying the reference path length is the means used to acquire a series of holograms at different depths, providing after reconstruction images of slices at different depths in the specimen thanks to the short- coherence length of light source. A metallic object, covered by a 150-µm-thick onion cell, is imaged with high resolution. Applications in ophthalmology are shown: structures of the anterior eye, the cornea, and the iris, are studied on enucleated porcine eyes. Tomographic images of the iris border close to the pupil were obtained 165 µm underneath the eye surface.

Use of digital holographic microscopy in tomography

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

Digital Holographic Microscopy (DHM) provides three-dimensional (3D) images with a high vertical accuracy in the nanometer range and a diffracted limited transverse resolution. This paper focuses on 3 different tomographic applications based on DHM. First, we show that DHM can be combined with time gating: a series of holograms is acquired at different depths by varying the reference path length, providing after reconstruction images of slices at different depths in the specimen thanks to the short coherence length of the light source. Studies on enucleated porcine eyes will be presented. Secondly, we present a tomography based on the addition of several reconstructed wavefronts measured with DHM at different wavelengths. Each wavefront phase is individually adjusted to be equal in a given plane of interest, resulting in a constructive addition of complex waves in the selected plane and destructive addition in the others. Varying the plane of interest enables the scan of the object in depth. Thirdly, DHM is applied to perform optical diffraction tomography of a pollen grain: transmission phase images are acquired for different orientations of the rotating sample, then the 3D refractive index spatial distribution is computed by inverse radon transform. The presented works will exemplify the versatility of DHM, but above all its capability of providing quantitative tomographic data of biological specimen in a quick, reliable and non-invasive way.

SPIE2006

Low coherence digital holographic microscopy

Pia Massatsch

A new holographic technique combining digital holographic microscopy with the use of low coherence light has been developed and the potential of the resulting method for vision in turbid media and biomedical imaging has been evaluated. In digital holography the hologram is recorded on a CCD camera and reconstructed numerically on a PC, giving direct access to the phase and the amplitude of the reconstructed wave front. This technique is combined with low coherence light, creating the possibility to perform optical sectioning. Series of holograms corresponding to slices at different depths in the specimen are recorded and each of these holograms contain phase and amplitude information of the light originating from the corresponding tomographic slice in the object. Sub micrometer lateral resolution is obtained experimentally and the phase distribution of the reconstructed object corresponds to a depth resolution of approximately 10 nm for a depth range of a couple of micrometers. A second level depth resolution is achieved using low coherence illumination. The thickness of a tomographic layer in water is 11 µm. Vision through 18 mean free paths (mfp) of turbid media (micro sphere suspension) is demonstrated with depth resolved reconstruction of the phase. Both amplitude and phase is reconstructed through 17 mfp's of turbid media. Tomographic imaging of a USAF test target hidden behind a 100 µm thick onion cell membrane is performed with phase and amplitude reconstructions of the test target as well as the surface of the onion cell membrane. Tomography of a biological specimen is demonstrated by the images of an onion shell, with structures visible down to a depth of 1 mm and by the images of a St-Paulia leaf presenting two cellular layers separated by 200 µm. Two structures of the anterior eye, the iris and the cornea, are studied on enucleated porcine eyes. A tomography of the iris border close to the pupil was performed and the penetration into the iris tissue is good. The cornea is first imaged without contact medium giving images strongly dominated by the air-cornea reflection. These images contain valuable information on the superficial epithelial cells in spite the presence of the tear film on the cornea. A water immersion configuration is also applied to enable a tomography of the cornea. A z-scan profile of the cornea is obtained and phase and amplitude images are reconstructed for different locations in the cornea (epithelium, stroma and endothelium).

EPFL2003

Tomography using multiple wavelengths in digital holographic microscopy

Frédéric Montfort

This thesis presents a new tomographic imaging method using multiple wavelengths in digital holographic microscopy. It is based on the addition of several reconstructed wavefronts measured at different wavelengths. The resulting diffraction tomographic visibility is then enhanced and the position of the interfaces is determined with ultrahigh precision. Digital holographic microscopy is a method enabling the recording of complex wavefronts and its numerical reconstruction. In our case, it is based on the recording, in off-axis geometry, of the interference between a reference wave and an object wave reflected by a microscopic specimen and magnified by a microscope objective. A couple charged device (CCD) camera records consecutively the resulting holograms at several wavelengths equally separated in the frequency domain. An adapted reconstruction algorithm has been developed to perform an achromatic reconstruction of the different wavefronts. Tomography is then performed by adding the reconstructed wavefronts. Each wavefront phase is individually adjusted to be equal in a given plane of interest. The result of this operation is a constructive addition of complex waves in the selected plane and destructive addition in the others. The amplitude image is attenuated according to a function, called filter function. For perfectly constructive phases no attenuation is present. Varying the plane of interest enables the scan of the object in depth. The obtained diffraction tomographic resolution is better than the one obtained in OCT, with an equivalent spectral width, in low-coherence or short-pulse lasers in optical coherence tomography. Nevertheless, no axial scanning is needed and the shape of the spectrum can be post acquisition tailored. A method is then proposed to enhance the visibility and to precisely determine the position of the interfaces. An axial scan allows the extraction of depth profiles. The maxima detection and a least square fit algorithm are used to perform this enhancement. Moreover, weights are introduced in the addition of the wavefronts, in order to tailor the filter function. Different weights distributions are discussed in terms of separation limit and precision on the interface position. It is shown that position resolution increases for higher separation limit. Finally, the tomographic method has been simplified for cases of a total reflective surface to perform topography. This allows measurements on specimen of several microns high without phase ambiguities. The results are directly measured in optical pathlengths. Experimental measurements have been performed on a specifically designed and homemade target, as well as on a multilayer specimen. Twenty wavelengths in the range of 480-700 nm have been used, resulting in tomographic sections of 725 nm. It is shown that the experimental results perfectly correspond the simulations. Enhanced tomographic results show a separation limit of 725 nm with a position resolution under 130 nm. Measures performed using the Kaiser weight distribution allow a position resolution of a few nanometers, for an increasing separation limit to 3 µm.