White-light diffraction tomography of unlabelled live cells

Abstract

We present a technique called white-light diffraction tomography (WDT) for imaging microscopic transparent objects such as live unlabelled cells. The approach extends diffraction tomography to white-light illumination and imaging rather than scattering plane measurements. Our experiments were performed using a conventional phase contrast microscope upgraded with a module to measure quantitative phase images. The axial dimension of the object was reconstructed by scanning the focus through the object and acquiring a stack of phase-resolved images. We reconstructed the threedimensional structures of live, unlabelled, red blood cells and compared the results with confocal and scanning electron microscopy images. The 350 nm transverse and 900 nm axial resolution achieved reveals subcellular structures at high resolution in Escherichia coli cells. The results establish WDT as a means for measuring three-dimensional subcellular structures in a non-invasive and label-free manner.A transparent object illuminated by an electromagnetic field generates a scattering pattern that carries specific information about its internal structure. Inferring this information from measurements of the scattered field, that is, solving the inverse scattering problem, is the fundamental principle that has allowed X-ray diffraction measurements to reveal the molecular-scale organization of crystals 1 and more recently, image cells with nanoscale resolution 2,3 . The scattered field is related to the spatially varying dielectric susceptibility of the scattering object by a transformation that simplifies considerably and, more importantly, becomes invertible, when the incident field is only weakly perturbed by the presence of the object. In this regime, the first-order Born approximation 4 and the Rytov approximation 5 have been used to unambiguously retrieve the three-dimensional spatial distribution of the dielectric constant. Implementation of inverse scattering requires knowledge of both the amplitude and phase of the scattered field. This obstacle, known as the phase problem, has been associated with X-ray diffraction measurement throughout its century-old history (for a review, see ref. 6).In 1969, Wolf proposed diffraction tomography as a reconstruction method combining the X-ray diffraction principle with optical holography 7 . Unlike X-rays, light at lower frequencies can be used in phase imaging measurements, as demonstrated by Gabor 8 . In recent years, as a result of new advances in light sources, detector arrays and computing power, quantitative phase imaging (QPI), in which optical path-length delays are measured at each point in the field of view, has become a very active field of study 9 . Whether involving holographic or non-holographic methods 10-16 , QPI presents new opportunities for studying cells and tissues non-invasively, quantitatively and without the need for staining or tagging [17][18][19][20][21][22][23] . Projection tomography using laser QPI has made use of ideas from X-ray imaging and enabled three-dimensional ...

3D intensity and phase imaging from light field measurements in an LED array microscope

“…2D phase retrieval methods are easily combined with tomography [4][5][6][7][8][9][10][11][12], but when wave-optical effects become more prominent (as in microscopy), diffraction tomography [13][14][15][16][17][18][19] becomes necessary. All these methods require both angle scanning and multiple measurements at each angle, or a reference beam.…”

Rapid quantitative phase imaging for partially coherent light microscopy

“…In addition, the partial coherence mitigates speckle noise. Recently, low coherent light sources such as halogen lamps or light emission diodes (LEDs) have also been applied in digital holographic microscopy, [15][16][17][18]. For instance, in [15,16] the noise reduction in the reconstructed phase has been experimentally demonstrated for LED illumination.…”

“…We underline that a pinhole of few microns (e.g. 25 μm) is often used as a spatial filter for LED and halogen sources in order to obtain high spatial coherence but low temporal coherence [15][16][17][18]. In contrast, we consider quantitative phase imaging with an illumination light exhibiting significantly reduced spatial and temporal coherence.…”

Realistic optical cell modeling and diffraction imaging simulation for study of optical and morphological parameters of nucleus

“…Optical detection through coherent light scattering offers a much valued platform for its label-free nature and capacities to extract both morphology and molecular information. Characterization of nucleus by scattered light signals thus attracts active research efforts [2][3][4][5][6][7]. Determination of cellular and nuclear morphology is fundamentally a challenging inverse problem for their complex 3D structures.…”