Multi-kernel deconvolution applied to confocal fluorescence microscopy with engineered point spread function

Fluorescence microscopy is a powerful technique in biology, because of the immense variety of markers now available. Compared to other methods, its resolution is however limited. In wide-field microscopy, the technique of structured illumination permits to improve the lateral resolution by a factor of two, even surpassing confocal microscopy, which permits a theoretical gain of about 40%. We propose an alternate technique, combining laterally interfering focused beams, which should permit the same gain of resolution in a confocal microscope. Furthermore, this technique, combined with multiple acquisition and multikernel deconvolution, permits a better object reconstruction than classical monokernel deconvolution using a regular excitation point spread function

Maximal Spontaneous Photon Emission and Energy Loss from Free Electrons

Free electron radiation such as Cerenkov, Smith--Purcell, and transition radiation can be greatly affected by structured optical environments, as has been demonstrated in a variety of polaritonic, photonic-crystal, and metamaterial systems. However, the amount of radiation that can ultimately be extracted from free electrons near an arbitrary material structure has remained elusive. Here we derive a fundamental upper limit to the spontaneous photon emission and energy loss of free electrons, regardless of geometry, which illuminates the effects of material properties and electron velocities. We obtain experimental evidence for our theory with quantitative measurements of Smith--Purcell radiation. Our framework allows us to make two predictions. One is a new regime of radiation operation---at subwavelength separations, slower (nonrelativistic) electrons can achieve stronger radiation than fast (relativistic) electrons. The second is a divergence of the emission probability in the limit of lossless materials. We further reveal that such divergences can be approached by coupling free electrons to photonic bound states in the continuum (BICs). Our findings suggest that compact and efficient free-electron radiation sources from microwaves to the soft X-ray regime may be achievable without requiring ultrahigh accelerating voltages.Comment: 7 pages, 4 figure

Exponential decay of Laplacian eigenfunctions in domains with branches

The behavior of Laplacian eigenfunctions in domains with branches is investigated. If an eigenvalue is below a threshold which is determined by the shape of the branch, the associated eigenfunction is proved to exponentially decay inside the branch. The decay rate is twice the square root of the difference between the threshold and the eigenvalue. The derived exponential estimate is applicable for arbitrary domains in any spatial dimension. Numerical simulations illustrate and further extend the theoretical estimate

Stain-Free Quantification of Chromosomes in Live Cells Using Regularized Tomographic Phase Microscopy

Refractive index imaging is a label-free technique that enables long-term monitoring of the internal structures and molecular composition in living cells with minimal perturbation. Existing tomographic methods for the refractive index imaging lack 3-D resolution and result in artifacts that prevent accurate refractive index quantification. To overcome these limitations without compromising the capability to observe a sample in its most native condition, we have developed a regularized tomographic phase microscope (RTPM) enabling accurate refractive index imaging of organelles inside intact cells. With the enhanced accuracy, we quantify the mass of chromosomes in intact living cells, and differentiate two human colon cancer lines, HT-29 and T84 cells, solely based on the non-aqueous (dry) mass of chromosomes. In addition, we demonstrate chromosomal imaging using a dual-wavelength RTPM, which shows its potential to determine the molecular composition of cellular organelles in live cells.National Institute of Biomedical Imaging and Bioengineering (U.S.) (9P41EB015871-26A1

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

Tomographic diffractive microscopy of transparent samples

We report a tomographic diffractive microscope, which permits imaging non-labelled transparent or semi-transparent samples. Based on a combination of microholography with a tomographic illumination, our set-up creates 3-D images of the index of refraction distribution within the sample. One acquires successively interferograms, rotating the illumination (the specimen being static) and using phase-shifting holography. Within the first Born approximation, each interferogram is interpreted as a subset of the Fourier transform of the specimen index of refraction distribution. The reconstruction is therefore similar to synthetic aperture imaging: one recombines the information in the Fourier space, and a final Fourier transform gives a 3-D image of the specimen. First recalling the theoretical foundations, we then describe our experiment, and show initial results obtained on biological samples