KEYWORDS: Microscopy, Sensors, Signal to noise ratio, Image enhancement, Point spread functions, Confocal microscopy, Diffraction, Image resolution, Super resolution, Single photon
Technological advancements in the creation, manipulation and detection of quantum states of light have motivated the application of such states to overcome classical limits in sensing and imaging. In particular, there has been a surge of recent interest in super-resolution imaging based on principles of quantum optics. However, the application of such schemes for practical imaging of biological samples is demanding in terms of signal-to-noise ratio, speed of acquisition and robustness with respect to sample labeling. Here, we re-introduce the concept of quantum image scanning microscopy (Q-ISM), a super-resolution method that enhances the classical image scanning microscopy (ISM) method by measuring photon correlations. Q-ISM was already utilized to achieve super-resolved images of a biological sample labeled with fluorescent nanoscrystals whose contrast is based entirely on a quantum optical phenomenon, photon antibunching. We present here an experimental demonstration of the method and discuss with further details its prospects for application in life science microscopy.
Far-field optical microscopy beyond the Abbe diffraction limit, making use of nonlinear excitation (e.g. STED), or temporal fluctuations in fluorescence (PALM, STORM, SOFI) is already a reality. In contrast, overcoming the diffraction limit using non-classical properties of light is very difficult to achieve due to the fragility of quantum states of light. Here, we experimentally demonstrate superresolution microscopy based on quantum properties of light naturally emitted by fluorophores used as markers in fluorescence microscopy. Our approach is based on photon antibunching, the tendency of fluorophores to emit photons one by one rather than in bursts. Although a distinctively quantum phenomenon, antibunching is readily observed in most common fluorophores even at room temperature.
This nonclassical resource can be utilized directly to enhance the imaging resolution, since the non-classical far-field intensity correlations induced by antibunching carry high spatial frequency information on the spatial distribution of emitters. Detecting photon statistics simultaneously in the entire field of view, we were able to detect non-classical correlations of the second and third order, and reconstructed images with resolution significantly beyond the diffraction limit.
Alternatively, we demonstrate the utilization of antibunching for augmenting the capabilities of localization-based superresolution imaging in the presence of multiple emitters, using a novel detector comprised of an array of single photon detectors connected to a densely packed fiber bundle. These features allow us to enhance the spatial and temporal resolution with which multiple emitters can be imaged compared with other techniques that rely on CCD cameras.
Discrete nonlinear optical systems exhibit unique properties unknown from wave propagation in bulk materials. Among them are the possibilities to form highly localized discrete solitons and the ability of a wide beam to propagate without diffraction and modulational instability across the array. The interaction between a highly localized discrete soliton and a non-diffracting beam has potential applications for all optical routing and switching. We present our results on the experimental investigation of this kind of beam interactions in a one-dimensional AlGaAs array at a wavelength of 1550 nm. A discrete soliton, almost completely confined to a single waveguide, was excited and the interaction with a wide beam of the same or orthogonal polarization was studied. We confirmed that the wide beam is able to drag the soliton over multiple waveguides towards itself while the soliton is able to maintain its original, highly confined shape. The outcome of the coherent interaction depends on the power of the wide beam and the relative phase between the two beams. This phase-dependence is due to linear interference in the case of co-polarized beams and due to four-wave mixing for orthogonally polarized beams.
The ability to perform optical sectioning is one of the great
advantages of laser-scanning microscopy. This introduces, however,
a number of difficulties due to the scanning process, such as
lower frame rates due to the serial acquisition process. Here we
show that by introducing spatiotemporal pulse shaping techniques
to multiphoton microscopy it is possible to obtain full-frame
depth resolved imaging completely without scanning. Our method
relies on temporal focusing of the illumination pulse. The pulsed
excitation field is compressed as it propagates through the
sample, reaching its shortest duration at the focal plane, before
stretching again beyond it. Combining temporal focusing with
line-scanning microscopy results in an enhanced depth resolution,
equivalent to that achieved by point scanning. Both the
scanningless and the line-scanning techniques are applied to
obtain depth-resolved two-photon excitation fluorescence (TPEF)
images of drosophila egg-chambers.
The field of multiphoton microscopy has undergone significant advances since its beginning just over a decade ago. One of these was the development of coherent multiphoton techniques, which typically image intrinsic properties of the sample, without resorting to staining or labeling. Here we describe some recent technological developments in the two leading coherent multiphoton techniques: third-harmonic generation and coherent anti-Stokes Raman spectroscopy. These techniques are applied for visualization of a variety of biological and other samples.
Intensity and interferometric autocorrelation measurements of femtosecond pulses by third harmonic generation are demonstrated. The third harmonic signal is generated by tight focusing of the femtosecond pulses on the surface of an ordinary glass slide, which serves as the nonlinear medium. The method is shown to be applicable to real-time measurements.
1D and 2D linear spectral interference measurements of ultrashort pulses are shown. The methods involve measurements of the spectral interference pattern between ultrashort reference and signal pulses, from which the spectral phase difference can be extracted. 1D measurements require post-processing to extract the phase. 2D measurements require no post-processing, and are therefore most suitable for visual interpretation. Dispersion of optical glasses, as well as crafted spectral phase discontinuities are measured. The methods are applicable to single shot measurements of extremely weak pulses.
Third harmonic generation near the focal point of a tightly focused beam is used to probe microscopical structures of transparent samples. The axial resolution at which this method can resolve interfaces and inhomogeneities is shown to be comparable with the confocal length of the beam. Using 125 femtosecond pulses at 1.5 micrometers , we were able to resolve interfaces with a resolution of 1.2 micrometers and to produce 2D cross-sectional images of the samples used.
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