Subtraction Microscopy Research Articles

Resolution and contrast enhancement in weighted subtraction microscopy by deep learning

In subtraction microscopy, the negative sidelobes are inevitably generated by the difference between the envelopes of Gaussian and doughnut point spread functions (PSFs), resulting in undesired information loss. Therefore, the trade-off between high resolution and information loss hinders further improvement in the performance of subtraction microscopy. Moreover, the postprocessing subtraction algorithms derived from PSF algebra tend to cause artifacts in dense samples. Herein, we propose an adaptive algorithm for assignment of the subtractive coefficient based on deep learning, termed Deep-IWS, to enhance the performance of subtraction microscopy. Both simulation and experiment reveal that Deep-IWS increases the resolution 1.8 times better than confocal microscopy, and significantly outperforms the previous subtraction microscopy. Furthermore, the reconstructed images also have fewer artifacts with a higher signal-to-noise ratio (SNR), demonstrating the validity and superiority of our method.

Resolution and contrast enhancement in optical subtraction microscopy with annular aperture

A method for improving lateral resolution and reducing imaging distortion of optical subtraction microscopy is proposed. First, an azimuthally polarized (AP) light is modulated by an annular aperture. Then, an image with higher lateral resolution is obtained by subtracting an image obtained by an annular AP beam from an image obtained by a radially polarized beam. The simulation results demonstrate that compared with the case without the annular aperture, negative side-lobes in the effective point spread function are effectively suppressed, and the imaging quality of subtraction microscopy is obviously enhanced.

Orthogonal linear polarization subtraction confocal method for lateral resolution enhancement

Confocal microscopy is a valuable tool for modern life science research. Subtraction microscopy can improve the resolution of the confocal system by the simple addition of components, but the image distortion that is produced can be problematic. In this paper, an orthogonal linear polarization subtraction confocal method (OLSCM) is proposed. The OLSCM obtains images using orthogonal linearly polarized illumination separately and obtains the calculated image by subtracting the absolute difference between the images from the arithmetic mean of the images. A resolution improvement of 14.6% without image distortion can be achieved with a pinhole of one airy unit. In addition, we experimented with 60nm gold particles and achieved a resolution improvement of 10.3%. In the OLSCM, solid-shaped and hollow-shaped point spread functions (PSFs) are both calculated from the PSFs of pairs of orthogonal linearly polarized illuminations. A resolution improvement can be achieved by using the subtraction method without image distortion.

Spatial-domain filter enhanced subtraction microscopy and application to mid-IR imaging.

We have experimentally investigated the enhancement in spatial resolution by image subtraction in mid-infrared central solid-immersion lens (c-SIL) microscopy. The subtraction exploits a first image measured with the c-SIL point-spread function (PSF) realized with a Gaussian beam and a second image measured with the beam optically patterned by a silicon π-step phase plate, to realize a centrally hollow PSF. The intense sides lobes in both PSFs that are intrinsic to the SIL make the conventional weighted subtraction methods inadequate. A spatial-domain filter with a kernel optimized to match both experimental PSFs in their periphery was thus developed to modify the first image prior to subtraction, and this resulted in greatly improved performance, with polystyrene beads 1.4 ± 0.1 µm apart optically resolved with a mid-IR wavelength of 3.4 µm in water. Spatial-domain filtering is applicable to other PSF pairs, and simulations show that it also outperforms conventional subtraction methods for the Gaussian and doughnut beams widely used in visible and near-IR microscopy.

Intensity Weighted Subtraction Microscopy Approach for Image Contrast and Resolution Enhancement.

We propose and demonstrate a novel subtraction microscopy algorithm, exploiting fluorescence emission difference or switching laser mode and their derivatives for image enhancement. The key novelty of the proposed approach lies in the weighted subtraction coefficient, adjusted pixel-by-pixel with respect to the intensity distributions of initial images. This method produces significant resolution enhancement and minimizes image distortions. Our theoretical and experimental studies demonstrate that this approach can be applied to any optical microscopy techniques, including label free and non-linear methods, where common super-resolution techniques cannot be used.

Label Free Linear and Non-Linear Excitation Nanoscopy

Despite tremendous progress in super-resolution microscopy techniques, the imaging of non-fluorescent species with sub-diffraction resolution is a challenging task. To overcome this limit the subtraction microscopy approach has been proposed recently [1,2]. The concept is based on the acquisition and the successive mathematical subtraction of two images acquired with Gaussian and doughnut shaped focal spots, respectively.Here, we will show a novel rescaling method that avoids artifacts, maintaining the contrast and resolution enhancement. Nevertheless, Infrared absorption/saturation microscopy is also designed to be a label free super-resolution approach[3]. The main idea of InfraRed Nanoscopy (IRN) is an absorption/saturation effect similar to the conventional pump-probe technique[4], where the pump beam modifies the carrier density inside the sample, followed by the intensity changes in the transmitted probe beam. The introduction of an additional doughnut shaped pump beam can transiently saturate the induced transition in the periphery of the focal spot and enabling super-resolution. Finally we will show a novel implementation based on single wavelength 2PE-STED nanoscopy[5] by means of random access scanning, and a phasor approach for the analysis of polarization SHG images, that, providing quantitative information, can be used to extract a fingerprint of tissues.The LANIR research leading to these results has received funding from FP7/20012-2015 under grant agreement n°280804.1.Harold, D., Piche, M., De Koninck, Y.(2013) Optics express 21,13:15912-15925.2.Wang, N., Miyazaki, J., He, J., Seto, K., Kobayashi, T.(2015). Optics Communications 343,28-33.3.Silien, C., Liu, N., Hendaoui, N., Tofail, SAM., Peremans, A.(2012). Optics Express 20,29694-29704.4.Dong CY, So PT, French T, Gratton E.(1995) Biophysical Journal. 69(6):2234-2242.5. Bianchini, P., Peres, C., Oneto, M., Galiani, S., Vicidomini, G., Diaspro, A. (2015). Cell and Tissue Research 360,143-150.

Subtraction threshold for an isotropic fluorescence emission difference microscope

Isotropic fluorescence emission difference microscopy proposed recently provides a simple method to enhance the spatial resolution in three-dimensions (3D) for fluorescence imaging. However, the subtraction threshold to achieve the condition for appropriately resolving the sample in 3D have not been studied. Then the subtraction factors used in this type of microscopes are still experientially chosen. Based on vector diffraction theory and a 3D numerical model developed here, the subtraction threshold is numerically investigated for the isotropic fluorescence subtraction microscopy. The subtraction factors and peak intensities at the threshold are obtained and comparied both in lateral and axial planes for achieving most appropriate subtraction and inspecting the isotropic characteristic. The effects of radius ratios of implemented 0-π annular phase plate for generating three dimensional donut spot on the subtracted resolution, peak intensity and negative sidebands are also discussed.

Numerical calibration of the spatial overlap for subtraction microscopy

Recently proposed subtraction microscopy provides a simple and compact approach to enhance the spatial resolution. However, inevitable imperfect overlap of the Gaussian and donut spots could deteriorate the quality of the subtracted images, while this effect is rarely investigated quantitatively. Based on vector diffraction theory, the effects of spatial displacements on the image resolution, signal intensity, and subtraction threshold are studied with several microscope parameters here. The peak-position shift is found to be a useful and convenient parameter to inspect and calibrate the spatial overlap of excitation spots in such microscopes.

Numerical study of the subtraction threshold for fluorescence difference microscopy.

Subtraction microscopy has recently been promoted for its compactness and simplicity of enhancing spatial resolution. However, until now, the subtraction factors used in such microscopes to process raw images have been chosen experientially, and it has been impossible to determine whether the resolved structures after subtraction are appropriate or over-processed. Based on vector diffraction theory and two-dimensional convolution, this paper presents numerical investigations of the parameters that may offer the selection criterion of subtraction factors used in subtraction microscopy. It proposes two essential parameters for appropriately evaluating the subtraction factor: the fluorescence peak intensity after subtraction and resolution derivative.

Saturated structured confocal microscopy with theoretically unlimited resolution

The resolution of fluorescence microscopes is limited by diffraction, which determines the extension of their point spread functions. We propose and study numerically a simple method, based on a combination of subtraction microscopy with regular and annular excitation beams, which permits to double the resolution compared to wide field microscopy. When combined with the fluorescence saturation phenomenon, this approach would be able to deliver a resolution of a few tens of nanometers.

Development of an x-ray microscopy and microtomography station at the VEPP-3 storage ring

The current status of the experimental station for x-ray scanning transmission subtraction microscopy and microtomography using synchrotron radiation produced by a wiggler-magnet installed at the storage ring VEPP-3 (2 GeV, 100–200 mA, 2 T) is described. The working x-radiation energies range from 5 to 35 keV. The results are presented concerning the achievement of 1-μm spatial resolution in this spectral range.

Application of synchrotron radiation to medical diagnostics at Novosibirsk: Status and prospects

The work on medical diagnostics with synchrotron radiation at the Institute of Nuclear Physics (Novosibirsk) is discussed. The plans for work and the results obtained at the experimental stations on digital subtraction angiography, digital subtraction microscopy and microtomography and X-ray fluorescence elemental analysis are presented.