Tomographic Diffractive Microscopy: Principles, Implementations, and Applications in Biology (original) (raw)

Diffraction tomography for biological cells imaging using digital holographic microscopy

Laser Applications in Life Sciences, 2010

Many biological objects are mainly transparent and weakly scattering, thus a promising (and already widely used) way of imaging them consists in considering optical refractive index variations. The method proposed here permits 3D imaging of the refractive index distribution with a tomographic approach. Usually, the classical Radon transform does not sufficiently take into account the physical interaction between light and biological cells, therefore diffraction has to be considered.

Refractive Index Tomography by Digital Holographic Microscopy

Digital Holography and Three-Dimensional Imaging, 2008

Full 3D Refractive Index (RI) tomography appears as a challenging perspective in the observation of microscopic 3D objects, biological cells in particular. Recent developments in Digital Holographic Microscopy (DHM) have permitted to achieve accurate RI 3D images.

Sub-cellular quantitative optical diffraction tomography with digital holographic microscopy - art. no. 64410K

Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues V, 2007

Digital holographic microscopy (DHM) is an interferometric technique, providing quantitative mapping of the phase shift induced by semi-transparent microscopic specimens, such as cells, with sub-wavelenght resolution along the optical axis. Thanks to actual PCs and CCDs, DHM provides nowadays cost-effective instruments for real-time measurements at very high acquisition rates, with sub-micron transverse resolution. However, DHM phase images do not reveal the threedimensional (3D) internal distribution of refractive index, but a phase shift resulting from a mean refractive index (RI) integrated over the cellular thickness. Standard optical diffraction tomography (ODT) techniques can be efficiently applied to reveal internal structures and to measure 3D RI spatial distributions, by recording 2D DHM phase data for different sample orientations or illumination beam direction, in order to fill up entirely the Ewald sphere in the Fourier space. The 3D refractive index can then be reconstructed, even in the direct space with backpropagation algorithms or from the Fourier space with inverse Fourier transform. The presented technique opens wide perspectives in 3D cell imaging: the DHM-based micro-tomography furnishes invaluable data on the cell components optical properties, potentially leading to information about organelles intracellular distribution. Results obtained on biological specimens will be presented. Morphometric measurements can be extracted from the tomographic data, by detection based on the refractive index contrast within the 3D reconstructions. Results and perspectives about sub-cellular organelles identification inside the cell will also be exposed.

Cell refractive index tomography by digital holographic microscopy

Optics Letters, 2006

For what we believe to be the first time, digital holographic microscopy is applied to perform optical diffraction tomography of a pollen grain. Transmission phase images with nanometric axial accuracy are numerically reconstructed from holograms acquired for different orientations of the rotating sample; then the threedimensional refractive index spatial distribution is computed by inverse radon transform. A precision of 0.01 for the refractive index estimation and a spatial resolution in the micrometer range are demonstrated.

Tomographic diffractive microscopy: basics, techniques and perspectives

Journal of Modern Optics, 2010

Tomographic diffractive microscopy (TDM) is an advanced digital imaging technique, which combines the recording of multiple holograms with the use of inversion procedures to retrieve quantitative information on the sample. In this review, we discuss the basic theory of TDM in the framework of electromagnetism and draw a comparison with conventional widefield microscopes. We describe various implementations of TDM, highlighting

Use of digital holographic microscopy in tomography

Biophotonics and New Therapy Frontiers, 2006

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.

Use of digital holographic microscopy in tomography

Biophotonics and New Therapy Frontiers, 2006

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.

Tomographic diffractive microscopy of transparent samples

We report a tomographic diffractive microscope, which permits imaging non-labelled transparent or semi-transparent samples. Based on a combination of microholography with a tomographic illumination, our set-up creates 3-D images of the index of refraction distribution within the sample. One acquires successively interferograms, rotating the illumination (the specimen being static) and using phase-shifting holography. Within the first Born approximation, each interferogram is interpreted as a subset of the Fourier transform of the specimen index of refraction distribution. The reconstruction is therefore similar to synthetic aperture imaging: one recombines the information in the Fourier space, and a final Fourier transform gives a 3-D image of the specimen. First recalling the theoretical foundations, we then describe our experiment, and show initial results obtained on biological samples.