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.

Label-free cytotoxicity screening assay by digital holographic microscopy.

We introduce a label-free technology based on digital holographic microscopy (DHM) with applicability for screening by imaging, and we demonstrate its capability for cytotoxicity assessment using mammalian living cells. For this first high content screening compatible application, we automatized a digital holographic microscope for image acquisition of cells using commercially available 96-well plates. Data generated through both label-free DHM imaging and fluorescence-based methods were in good agreement for cell viability identification and a Z'-factor close to 0.9 was determined, validating the robustness of DHM assay for phenotypic screening. Further, an excellent correlation was obtained between experimental cytotoxicity dose-response curves and known IC50 values for different toxic compounds. For comparable results, DHM has the major advantages of being label free and close to an order of magnitude faster than automated standard fluorescence microscopy.

Label-free second-harmonic phase imaging of biological specimen by digital holographic microscopy.

We have previously developed a new way for nonscanning second-harmonic generation (SHG) microscopy [Opt. Lett. 34, 2450 (2009)]. Based on digital holography, this technique captures, in single-shot hologram acquisition, both the amplitude and the phase of a coherent SHG radiation, which makes possible second harmonic phase microscopy. In this work, we present holographic SHG phase microscopy of a label-free biological tissue and discuss its added value to SHG microscopy.

Real time, nanometric 3D-tracking of nanoparticles made possible by second harmonic generation digital holographic microscopy.

In the past decade, quantitative phase imaging gave a new dimension to optical microscopy, and the recent extension of digital holography techniques to nonlinear microscopy appears very promising, for the phase of nonlinear signal provides additional information, inaccessible to incoherent imaging schemes. In this work, we show that the position of second harmonic generation (SHG) emitters can be determined from their respective phase, at the nanometer scale, with single-shot off-axis digital holography, making possible real-time nanometric 3D-tracking of SHG emitters such as nanoparticles.

Sub-Rayleigh resolution by phase imaging.

We report the experimental observation of systematically occurring phase singularities in coherent imaging of sub-Rayleigh distanced objects. A theory that relates the observation to the sub-Rayleigh distance is presented and compared with experimental measurements. As a consequence, the limit of resolution with coherent illumination is extended by a factor of 1.64x.

Endoscopic low-coherence topography measurement for upper airways and hollow samples.

To evaluate the severity of airway pathologies, quantitative dimensioning of airways is of utmost importance. Endoscopic vision gives a projective image and thus no true scaling information can be directly deduced from it. In this article, an approach based on an interferometric setup, a low-coherence laser source and a standard rigid endoscope is presented, and applied to hollow samples measurements. More generally, the use of the low-coherence interferometric setup detailed here could be extended to any other endoscopy-related field of interest, e.g., gastroscopy, arthroscopy and other medical or industrial applications where tri-dimensional topology is required. The setup design with a multiple fibers illumination system is presented. Demonstration of the method ability to operate on biological samples is assessed through measurements on ex vivo pig bronchi.

Digital holographic microscopy investigation of second harmonic generated at a glass/air interface.

Optical second-harmonic generation, thanks to its coherent nature, is a suitable signal for interferometric measurements such as digital holography, a well-established imaging technique that allows recovery of complex diffraction wave fields from which it is possible to extract both amplitude-contrast and quantitative phase images. Here, we report on a multifunctional form of microscopy, namely, second-harmonic generation digital holographic microscopy. As a proof of concept, we have investigated the second-harmonic signal generated at the glass/air interface of a microscope slide under focused femtosecond laser illumination, and we propose, for the first time to our knowledge, a representation and interpretation of the recovered phase. In this simple yet educative case study, we observe that the second harmonic is generated by the axial component of the incident field polarization.