ABSTRACT:We introduce the concept of saturated absorption competition (SAC) microscopy as a means of providing sub-diffraction spatial resolution in fluorescence imaging. Unlike the post-competition process between stimulated and spontaneous emission that is used in stimulated emission depletion (STED) microscopy, SAC microscopy breaks the diffraction limit by emphasizing a pre-competition process that occurs in the fluorescence absorption stage in a manner that shares similarities with ground-state depletion (GSD) microscopy. Moreover, unlike both STED and GSD microscopy, SAC microscopy offers a reduction in complexity and cost by utilizing only a single continuous-wave laser diode and an illumination intensity that is ~ 20x smaller than that used in STED. Our approach can be physically implemented in a confocal microscope by dividing the input laser source into a time-modulated primary excitation beam and a doughnut-shaped saturation beam, and subsequently employing a homodyne detection scheme to select the modulated fluorescence signal. Herein, we provide both a physico-chemical model of SAC and experimentally demonstrate by way of a proof-of-concept experiment a transverse spatial resolution of ~λ/6.Owing to the advantage of being non-invasive in observing living samples, far-field optical

Abstract:We present a quantitative second-harmonic generation (SHG) imaging technique that quantifies the 2D spatial organization of collagen fiber samples under dynamic conditions, as an image is acquired. The technique is demonstrated for both a well-aligned tendon sample and a randomly aligned, sparsely distributed collagen scaffold sample. For a fixed signal-to-noise ratio, we confirm the applicability of this method for various window sizes (pixel areas) as well as with using a gridded overlay map that allows for correlations of fiber orientations within a given image. This work has direct impact to in vivo biological studies by incorporating simultaneous SHG image acquisition and analysis. 245-257 (1989). 3. J. N. Gannaway and C. J. R. Sheppard, "Second-harmonic imaging in the scanning optical microscope," Opt.Quantum Electron. 10(5), 435-439 (1978). 4. R. Hellwarth and P. Christensen, "Nonlinear optical microscopic examination of structure in polycrystalline ZnSe," Opt. Commun. 12(3), 318-322 (1974). 5. I. Freund and M. Deutsch, "Second-harmonic microscopy of biological tissue," Opt. Lett. 11(2), 94-96 (1986). 6. P. J. Campagnola and L. M. Loew, "Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms," Nat. Biotechnol. 21 ( second-harmonic generation reveals shortened sarcomeres associated with myopathy induced by statin," PLoS ONE 6(9), e24764 (2011).

We demonstrate for the first time the imaging of unstained breast tissue biopsies using third-harmonic generation (THG) microscopy. As a label-free imaging technique, THG microscopy is compared to phase contrast and polarized light microscopy which are standard imaging methods for breast tissues. A simple feature detection algorithm is applied to detect tumour-associated lymphocyte rich regions in unstained breast biopsy tissue and compared with corresponding regions identified by a pathologist from bright-field images of hematoxylin and eosin stained breast tissue. Our results suggest that THG imaging holds potential as a complementary technique for analysing breast tissue biopsies.

We demonstrate nonlinear focal modulation microscopy (NFOMM) to achieve superresolution imaging. Traditional approaches to superresolution that utilize point scanning often rely on spatially reducing the size of the emission pattern by directly narrowing (e.g., through minimizing the detection pinhole in Airyscan, Zeiss) or indirectly peeling its outer profiles [e.g., through depleting the outer emission region in stimulated emission depletion (STED) microscopy]. We show that an alternative conceptualization that focuses on maximizing the optical system's frequency shifting ability offers advantages in further improving resolution while reducing system complexity. In NFOMM, a spatial light modulator and a suitably intense laser illumination are used to implement nonlinear focal-field modulation to achieve a transverse spatial resolution of ∼60  nm (∼λ/10). We show that NFOMM is comparable with STED microscopy and suitable for fundamental biology studies, as evidenced in imaging nuclear pore complexes, tubulin and vimentin in Vero cells. Since NFOMM is readily implemented as an add-on module to a laser-scanning microscope, we anticipate wide utility of this new imaging technique.

Second-harmonic generation (SHG) microscopy has gained popularity because of its ability to perform submicron, label-free imaging of noncentrosymmetric biological structures, such as fibrillar collagen in the extracellular matrix environment of various organs with high contrast and specificity. Because SHG is a two-photon coherent scattering process, it is difficult to define a point spread function (PSF) for this modality. Hence, compared to incoherent two-photon processes like two-photon fluorescence, it is challenging to apply the various PSF-engineering methods to improve the spatial resolution to be close to the diffraction limit. Using a synthetic PSF and application of an advanced maximum likelihood estimation (AdvMLE) deconvolution algorithm, we demonstrate restoration of the spatial resolution in SHG images to that closer to the theoretical diffraction limit. The AdvMLE algorithm adaptively and iteratively develops a PSF for the supplied image and succeeds in improving the signal to noise ratio (SNR) for images where the SHG signals are derived from various sources such as collagen in tendon and myosin in heart sarcomere. Approximately 3.5 times improvement in SNR is observed for tissue images at depths of up to ∼480 nm, which helps in revealing the underlying helical structures in collagen fibres with an ∼26% improvement in the amplitude contrast in a fibre pitch. Our approach could be adapted to noisy and low resolution modalities such as micro-nano CT and MRI, impacting precision of diagnosis and treatment of human diseases.