Living specimen tomography by digital holographic microscopy: morphometry of testate amoeba

Florian Charrière,Nicolas Pavillon,Thierry J Heger,Pierre Marquet,Tristan Colomb,Christian Depeursinge,Edward A.D Mitchell,Benjamin Rappaz

doi:10.1364/oe.14.007005

Abstract

This paper presents an optical diffraction tomography technique based on digital holographic microscopy. Quantitative 2-dimensional phase images are acquired for regularly-spaced angular positions of the specimen covering a total angle of pi, allowing to built 3-dimensional quantitative refractive index distributions by an inverse Radon transform. A 20x magnification allows a resolution better than 3 microm in all three dimensions, with accuracy better than 0.01 for the refractive index measurements. This technique is for the first time to our knowledge applied to living specimen (testate amoeba, Protista). Morphometric measurements are extracted from the tomographic reconstructions, showing that the commonly used method for testate amoeba biovolume evaluation leads to systematic under evaluations by about 50%.

Highlights

Optical microscopy techniques nowadays offer non-contact, high-resolution and real-time cell imaging facilities
In a recent paper,[12] we have shown for the first time to our knowledge the quantitative 3D distribution of refractive index (RI) of a semi-transparent object, in our case a pollen grain, provided by backprojecting optical path length (OPL) values collected with digital holographic microscopy (DHM) on a series of projections of the preparation taken at various incidence angles
Tomographic reconstructions were performed on 5 different Hyalosphenia papilio during this study

Summary

Introduction

Optical microscopy techniques nowadays offer non-contact, high-resolution and real-time cell imaging facilities. Besides the techniques quoted in Ref. 1, different approaches developed to measure the refractive index (RI) of cells should be mentioned. Bereiter-Hahn et al have studied the refractive index variation at the surface of mammalian cells in culture with quantitative reflection contrast microscopy.[2] More recently, Curl et al have compared height measurement achieved with a confocal microscope and optical path length (OPL) measurements with a phase-sensitive technique to deduce the integrated RI through a muscle cell.[3] Rappaz et al have followed dynamically the integrated RI through neuronal cells during a hypotonic stress, comparing absolute phase measurements obtained with digital holographic microscopy for two different perfusion solutions.[4] these last two approaches allow to measure the 2D planar distribution of the RI integrated along the optical axis, making the visualization of individual intracellular organelles difficult

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Conclusion