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. Last year, we
have reported how the SHG phase makes possible real-time nanometric 3D-tracking of SHG emitters, such as
nanoparticles (Shaffer et al., Opt. Express 18, p.17392-17403, 2010). Here, we investigate the phase of second
harmonic generated by a label-free biological specimen-more precisely collagen fibers forming the connective
tissue of a mouse dermis- and discuss of its interpretation. Notably, we show how the SHG phase, qualitatively
acting as an indicator of phase-matching conditions, tends to indicate that second harmonic generation, in
collagen, is dominated by coherent SHG scattering.
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.
Retrieval of the amplitude and phase of electromagnetic waves made digital holographic microscopy (DHM)
capable of revealing morphological details at ultrahigh resolution in the order of a few nanometers only and
precisely measuring the refractive index across a sample (e.g. cell or neuron). In short,DHM added a new
dimension to optical imaging,whic h explains why it is such an excellent instrument for metrological,but also
for biological applications. We believe that DHM is,b y nature,ideally suited for nonlinear microscopy. In this
work,w e review the advantages of DHM for nonlinear microscopy and present its application to determination
of the axial position of nonlinear nanoparticles capable of second harmonic generation.
Quantitative phase images make digital holographic microscopy (DHM) an excellent instrument for metrological, but
also for biological applications, where it can reveal deformations and morphological details at ultrahigh resolution in the
order of a few nanometers only, while also precisely determining the refractive index across a sample (e.g. cell or
neuron). On the other hand, non-linear light-matter interactions have also proved very useful in microscopy. For
instance, second harmonic generation (SHG) allows marker-free identification of cell structures, tubulin or membranes
and, because of its coherent nature, SHG is very sensitive to the local sample structure and to the direction of the laser
polarization. In addition, since SHG does not result from light absorption and subsequent re-emission, in opposition to
fluorescence, photo-bleaching of the studied material can be avoided by a judicious selection of the laser wavelength.
These characteristics make SHG very interesting for biomedical imaging. We have designed and built a microscope that
combines the fast and precise DHM imaging with tagging capabilities of non-linear light-matter interactions. Here, we
present the technique and look into its possible applications to biological and life sciences. Among promising
applications is the 3D tracking of colloidal gold nanoparticles.
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 recovering of the complex
diffraction wavefields from which it is possible to extract both amplitude-contrast and quantitative phase images.
We believe that application of digital holography to non-linear optical fields might ultimately be the key
to phase-related functional imaging. In a first approach, we report here on second harmonic generation digital
holographic microscopy, and present its application to (1) discrimination of nanoparticles of different nature
- here polystyrene microspheres and barium titanate (BaTiO3) nanoparticles - and to (2) 3D-mapping of a
distribution of BaTiO<sub>3</sub> nanoparticles.