Second-harmonic generation (SHG) microscopy is a label-free imaging technique to study collagenous materials in extracellular matrix environment with high resolution and contrast. However, like many other microscopy techniques, the actual spatial resolution achievable by SHG microscopy is reduced by out-of-focus blur and optical aberrations that degrade particularly the amplitude of the detectable higher spatial frequencies. Being a two-photon scattering process, it is challenging to define a point spread function (PSF) for the SHG imaging modality. As a result, in comparison with other two-photon imaging systems like two-photon fluorescence, it is difficult to apply any PSF-engineering techniques to enhance the experimental spatial resolution closer to the diffraction limit. Here, we present a method to improve the spatial resolution in SHG microscopy using an advanced maximum likelihood estimation (AdvMLE) algorithm to recover the otherwise degraded higher spatial frequencies in an SHG image. Through adaptation and iteration, the AdvMLE algorithm calculates an improved PSF for an SHG image and enhances the spatial resolution by decreasing the full-width-at-halfmaximum (FWHM) by ~20%. Similar results are consistently observed for biological tissues with varying SHG sources, such as gold nanoparticles and collagen in porcine feet tendons. By obtaining an experimental transverse spatial resolution of ~400 nm, we show that the AdvMLE algorithm brings the practical spatial resolution closer to the theoretical diffraction limit. Our approach is suitable for adaptation in micro-nano CT and MRI imaging, which has the potential to impact diagnosis and treatment of human diseases.
We describe the principles of using orientation-independent differential interference contrast (OI-DIC) microscopy for mapping optical path length (OPL). Computation of the scalar two-dimensional OPL map is based on an experimentally received map of the OPL gradient vector field. Two methods of contrast enhancement for the OPL image, which reveal hardly visible structures and organelles, are presented. The results obtained can be used for reconstruction of a volume image. We have confirmed that a standard research grade light microscope equipped with the OI-DIC and 100×/1.3 NA objective lens, which was not specially selected for minimum wavefront and polarization aberrations, provides OPL noise level of ∼0.5 nm and lateral resolution if ∼300 nm at a wavelength of 546 nm. The new technology is the next step in the development of the DIC microscopy. It can replace standard DIC prisms on existing commercial microscope systems without modification. This will allow biological researchers that already have microscopy setups to expand the performance of their systems.
We describe a combined orientation-independent differential interference contrast OI-DIC and polarization microscope and its biological applications. Several conventional DIC images were recorded with the specimen oriented in different directions followed by digital alignment and processing of the images. Then the obtained images are used for computation of the phase gradient magnitude and azimuth distribution and, further, the phase image. The OI-DIC images were obtained using optics having numerical aperture (NA) 1.4, thus achieving a level of resolution not previously achieved with phase contrast or interference microscope. The combined system yields two complementary phase images of thin optical sections of the specimen: distribution of refractive index and distribution of birefringence due to anisotropy of the cell structure. For instance, in a live dividing cell, the OI-DIC image clearly shows the detailed shape of the chromosomes, while the polarization image quantitatively depicts the distribution of birefringent microtubules in the spindle, both without any need for staining or other modifications of the cell. We present pseudo-color combined images of a crane fly spermatocyte at diakinesis and metaphase of meiosis I. Those images provide clear evidence that the proposed technique can reveal fine architecture and molecular organization in live cells without perturbation associated with staining or fluorescent labeling.
We describe a new DIC technique, which records phase gradients within microscopic specimens independently of their
orientation. The proposed system allows the generation of images representing the distribution of dry mass (optical path
difference) in the specimen. Unlike in other forms of interference microscopes, this approach does not require a narrow
illuminating cone. The orientation-independent differential interference contrast (OI-DIC) system can also be combined
with orientation-independent polarization (OI-Pol) measurements to yield two complementary images: one showing dry
mass distribution (which is proportional to refractive index) and the other showing distribution of birefringence (due to
structural or internal anisotropy). With a model specimen used for this work -- living spermatocytes from the crane fly,
Nephrotoma suturalis --- the OI-DIC image clearly reveals the detailed shape of the chromosomes while the polarization
image quantitatively depicts the distribution of the birefringent microtubules in the spindle, both without any need for
staining or other modifications of the cell. We present examples of a pseudo-color combined image incorporating both
orientation-independent DIC and polarization images of a spermatocyte at diakinesis and metaphase of meiosis I. Those
images provide clear evidence that the proposed technique can reveal fine architecture and molecular organization in live
cells without perturbation associated with staining or fluorescent labeling. The phase image was obtained using optics
having a numerical aperture 1.4, thus achieving a level of resolution never before achieved with any interference
microscope.
KEYWORDS: Deconvolution, Super resolution, Point spread functions, Microscopes, 3D image processing, Microscopy, Signal to noise ratio, Cameras, Diffraction, Image processing
Optical light microscopy is a predominant modality for imaging living cells, with the maximum resolution typically diffraction limited to approximately 200nm. The objective of this project is to enhance the resolution capabilities of optical light microscopes using image-processing algorithms, to produce super-resolved imagery at a sub-pixel level. The sub-pixel algorithm is based on maximum-likelihood iterative deconvolution of photon-limited data, and reconstructs the image at a finer scale than the pixel limitation of the camera. The software enhances the versatility of light microscopes, and enables the observation of sub-cellular components at a resolution two to three times finer than previously. Adaptive blind deconvolution is used to automatically determine the point spread function from the observed data. The technology also allows camera-binned or sub-sampled (aliased) data to be correctly processed. Initial investigations used computer simulations and 3D imagery from widefield epi-fluorescence light microscopy.
Imaging through a stochastically varying distorting medium, such as a turbulent atmosphere, requires multiple short-exposure frames to ensure maximum resolution of object features. Restoration methods are used to extract the common underlying object from the speckle images, and blind deconvolution techniques are required as typically there is little prior information available about either the image or individual PSFs. A method is presented for multiframe restoration based on iterative blind deconvolution, which alternates between restoring the image and PSF estimates. A maximum-likelihood approach is employed via the Richardson-Lucy (RL) method which automatically ensures positively and conservation of the total number of photons. The restoration is accelerated by applying a vector sequence is treated as a 3D volume of data and processed to produce a 3D stack of PSFs and a single 2D image of the object. The problem of convergence to an undesirable solution, such as a delta function, is addressed by weighting the number of image or PSF iterations according to how quickly each is converging, this leads to the asymmetrical nature of the algorithm. Noise artifacts are suppressed by using a dampened RL algorithm to prevent over fitting of the corrupted data. Results are presented for real single frame and simulated multiframe speckle imaging.
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