Digital holographic microscopy: a quantitative label-free microscopy technique for phenotypic screening

Digital Holographic Microscopy (DHM) is a label-free imaging technique allowing visualization of transparent cells with classical imaging cell culture plates. The quantitative DHM phase contrast image provided is related both to the intracellular refractive index and to cell thickness. DHM is able to distinguish cellular morphological changes on two representative cell lines (HeLa and H9c2) when treated with doxorubicin and chloroquine, two cytotoxic compounds yielding distinct phenotypes. We analyzed parameters linked to cell morphology and to the intracellular content in endpoint measurements and further investigated them with timelapse recording. The results obtained by DHM were compared with other optical label-free microscopy techniques, namely Phase Contrast, Differential Interference Contrast and Transport of Intensity Equation (reconstructed from three bright-field images). For comparative purposes, images were acquired in a common 96-well plate format on the different motorized microscopes. In contrast to the other microscopies assayed, images generated with DHM can be easily quantified using a simple automatized on-the-fly analysis method for discriminating the different phenotypes generated in each cell line. The DHM technology is suitable for the development of robust and unbiased image-based assays.

Application of CMOS image sensors in optical sectioning and polarization microscopy

Jelena Mitic

In this thesis new imaging approaches for optical microscopy are proposed and studied. They are based on the use of dynamic structured illumination in combination with a demodulation detection concept employing CMOS image detectors. Two particular implementations are suggested: real-time optical sectioning microscopy and whole field quantitative polarization microscopy. Optical sectioning, i.e depth resolved imaging, allows observation of thin sections in volumetric samples. In its classical configuration the wide-field microscope does not allow optical sectioning of the investigated objects. In this thesis the optical sectioning in a wide-field microscope is achieved by illuminating the sample with a continuously moving single spatial frequency pattern, which temporarily modulates the signal obtained on the photodetector. Only the object parts that are in the focus have a good contrast and consequently generate stronger signal on the detector. The optically sectioned images are obtained employing a CMOS image detector, which demodulates the time-dependent component of the image. Two different systems for real-time acquisition of optically sectioned images are proposed. The first one is based on a specially designed smart-pixel-detector-array (SPDA), which allows performing the signal processing by an electronic circuit integrated to each pixel of the sensor. The second system utilizes a commercial CMOS image sensor. Here, the images are treated by a digital signal processor (DSP) integrated into the camera (iMVS-155). Both approaches provide real-time acquisition of optically sectioned images. The proof of principle for the new method is demonstrated by imaging artificial three-dimensional reflective samples. The optical sectioning imaging of biological samples in bright-field mode and in fluorescence mode is also demonstrated. The optical sectioning performances of the studied systems are similar to that of the confocal microscope. However the new method has some advantages over the classical confocal system: e.g. the difficulties with the alignment and the post-processing inherent to the confocal system are avoided. The theoretical framework presented in the thesis describes the image formation in structured illumination for both reflective and fluorescent samples. The influence of longitudinal chromatic aberration and spherical aberration caused by specimen induced index mismatch on depth discrimination property are studied. In extension to the work related to optically sectioned microscopy, the quantitative polarization microscopy imaging method is proposed as a new application for CMOS detectors. The polarization state of the illuminating light is dynamically changed and the demodulation detection approach is employed to extract the retardance and azimuth of the birefringent sample in real-time; for a whole field of view. Examples of polarization sensitive measurements for different samples are presented. The theoretical analysis is performed using the Jones formalism. Finally, the potentials and limitations of the CMOS detectors for applications in optical microscopy are discussed. Thanks to constant advancements in CMOS technology, the acquisition speed, resolution and sensitivity of new CMOS detectors generations have been improved. This will allow adapting the methods proposed in this thesis for investigations of fast dynamic process where high temporal and spatial resolution is required.

EPFL2004

Broadband Digital Holographic Camera for Microscopy

Zahra Monemhaghdoust

In this thesis, we explore a combination of analog and digital holographic techniques to demonstrate high speed quantitative phase imaging (QPI) using low coherence (broad bandwidth) light sources. Off-axis digital holographic microscopy is inherently a coherent method of phase quantification and thus suffers from coherent noise. However, only one measurement is required to retrieve field information which gives an advantage in term of image acquisition speed. While incoherent methods of phase quantification remove the problem of coherent noise, they require acquiring several measurements which is sometimes not acceptable in the study of dynamical systems. Therefore, this thesis is an attempt to extend off-axis digital holographic microscopy from the coherent to the incoherent regime to maintain the benefit of high speed measurement capability of off-axis holography while reducing the coherent noise. First, we demonstrate that by using a suitable combination of holographic gratings, off-axis digital holographic microscopy can work with spectrally broadband illumination sources. Holographic gratings introduce a tilt in the coherence plane of one of the reference or object arms of an off-axis digital holographic microscope to expand the interference area to the whole field of view of the camera. Although QPI provides useful information in addition to state-of-the-art intensity-based microscopic imaging, its use has not permeated yet far beyond research laboratories. One potential reason is the need for a stand-alone dedicated microscope system, which is expensive and not back-compatible to the existing wide-field microscopes. In this Regard, the second part of this thesis explores an add-on camera module which can be attached to the camera port of any standard intensity microscope. The techniques developed to extend the off-axis DHM in the first part of the thesis will be used to demonstrate a proof-of-concept camera module which operates with broadband light sources. Overall, through the combination of analog and digital holography, the goal of this work is to extend full-field off-axis holography to incoherent regime to benefit from real-time speckle-free measurements, optical sectioning and realization of a digital holographic camera which can be placed in the image plane of a standard optical microscope.

EPFL2015

Application of CMOS image sensors in optical sectioning and polarization microscopy

Jelena Mitic

EPFL2004

Broadband Digital Holographic Camera for Microscopy

Zahra Monemhaghdoust

EPFL2015