Multimodal Highlighting of Structural Abnormalities in Diabetic Rat and Human Corneas.

PURPOSE: This study aimed to highlight structural corneal changes in a model of type 2 diabetes, using in vivo corneal confocal microscopy (CCM). The abnormalities were also characterized by transmission electron microscopy (TEM) and second harmonic generation (SHG) microscopy in rat and human corneas. METHODS: Goto-Kakizaki (GK) rats were observed at age 12 weeks (n = 3) and 1 year (n = 6), and compared to age-matched controls. After in vivo CCM examination, TEM and SHG microscopy were used to characterize the ultrastructure and the three-dimensional organization of the abnormalities. Human corneas from diabetic (n = 3) and nondiabetic (n = 3) patients were also included in the study. RESULTS: In the basal epithelium of GK rats, CCM revealed focal hyper-reflective areas, and histology showed proliferative cells with irregular basement membrane. In the anterior stroma, extracellular matrix modifications were detected by CCM and confirmed in histology. In the Descemet's membrane periphery of all the diabetic corneas, hyper-reflective deposits were highlighted using CCM and characterized as long-spacing collagen fibrils by TEM. SHG microscopy revealed these deposits with high contrast, allowing specific detection in diabetic human and rat corneas without preparation and characterization of their three-dimensional organization. CONCLUSION: Pathologic findings were observed early in the development of diabetes in GK rats. Similar abnormalities have been found in corneas from diabetic patients. TRANSLATIONAL RELEVANCE: This multidisciplinary study highlights diabetes-induced corneal abnormalities in an animal model, but also in diabetic donors. This could constitute a potential early marker for diagnosis of hyperglycemia-induced tissue changes

Characterization of the Temperature-Sensitive Mutations un-7 and png-1 in Neurospora crassa

The model filamentous fungus Neurospora crassa has been studied for over fifty years and many temperature-sensitive mutants have been generated. While most of these have been mapped genetically, many remain anonymous. The mutation in the N. crassa temperature-sensitive lethal mutant un-7 was identified by a complementation based approach as being in the open reading frame designated NCU00651 on linkage group I. Other mutations in this gene have been identified that lead to a temperature-sensitive morphological phenotype called png-1. The mutations underlying un-7 result in a serine to phenylalanine change at position 273 and an isoleucine to valine change at position 390, while the mutation in png-1 was found to result in a serine to leucine change at position 279 although there were other conservative changes in this allele. The overall morphology of the strain carrying the un-7 mutation is compared to strains carrying the png-1 mutation and these mutations are evaluated in the context of other temperature-sensitive mutants in Neurospora

Ptychographic electron microscopy using high-angle dark-field scattering for sub-nanometre resolution imaging

Diffractive imaging, in which image-forming optics are replaced by an inverse computation using scattered intensity data, could, in principle, realize wavelength-scale resolution in a transmission electron microscope. However, to date all implementations of this approach have suffered from various experimental restrictions. Here we demonstrate a form of diffractive imaging that unshackles the image formation process from the constraints of electron optics, improving resolution over that of the lens used by a factor of five and showing for the first time that it is possible to recover the complex exit wave (in modulus and phase) at atomic resolution, over an unlimited field of view, using low-energy (30 keV) electrons. Our method, called electron ptychography, has no fundamental experimental boundaries: further development of this proof-of-principle could revolutionize sub-atomic scale transmission imaging

Ptychography

Ptychography is a computational imaging technique. A detector records an extensive data set consisting of many inference patterns obtained as an object is displaced to various positions relative to an illumination field. A computer algorithm of some type is then used to invert these data into an image. It has three key advantages: it does not depend upon a good-quality lens, or indeed on using any lens at all; it can obtain the image wave in phase as well as in intensity; and it can self-calibrate in the sense that errors that arise in the experimental set up can be accounted for and their effects removed. Its transfer function is in theory perfect, with resolution being wavelength limited. Although the main concepts of ptychography were developed many years ago, it has only recently (over the last 10 years) become widely adopted. This chapter surveys visible light, x-ray, electron, and EUV ptychography as applied to microscopic imaging. It describes the principal experimental arrangements used at these various wavelengths. It reviews the most common inversion algorithms that are nowadays employed, giving examples of meta code to implement these. It describes, for those new to the field, how to avoid the most common pitfalls in obtaining good quality reconstructions. It also discusses more advanced techniques such as modal decomposition and strategies to cope with three-dimensional () multiple scattering

Importance of wavelength for ultrashort laser pulses in healthy and pathological corneas

International audienceA study on the role of laser wavelength in keratoplasty assisted by ultrashort pulse lasers is presented.This article gives a summary of the principal physical mechanisms contributing to the transparency of healthy corneas and presents transparency measurements as well as laboratory experiments on tissue with lasers at different wavelengths.The transparency of a healthy cornea is strongly related to its regular structure at micrometer and nanometer length scales. Many indications for keratoplasty are associated with a perturbation of this structure and therefore with a sometimes strongly reduced tissue transparency. This explains the often unsatisfactory results obtained when using ultrashort pulse lasers for the procedure. Theoretical considerations and laboratory experiments show that the light scattering processes responsible for the loss in laser beam quality depend strongly on wavelength and the use of wavelengths longer than those presently used allows these processes to be almost completely eliminated. The use of a spectral transparency window close to 1.65 A mu m is suggested.The use of laser wavelengths close to 1.65 A mu m represents an interesting alternative for the improvement of keratoplasty assisted by ultrashort pulse lasers

Local measurements of thermal diffusivity in homogeneous and heterogeneous samples by photoreflectance microscopy

We present a variant of the traditional photoreflectance microscopy. It allows thermal diffusivity measurements on samples as small as a few tens of microns, possibly of mediocre surface quality

Light efficiency versus image acquisition time: considerations for parallel confocal microscopy applied to biological tissue observation

The concept of parallel confocal microscopy with aperture de-correlation has been developed in order to reduce acquisition time and to increase light efficiency over conventional scanning confocal microscopy. It consists in simultaneously observing numerous confocal volumes of a specimen, in lieu of scanning one confocal spot over the region of interest. The cross-talk between neighbouring parallel confocal systems is eliminated by capturing images using a sequence of linearly independent illumination and detection patterns which permit to de-correlate the confocal from the non-confocal image. In this paper, we compare the relative efficiency of parallel and conventional confocal microscopy, using a simple analytical approach, and show that this depends strongly upon the illumination configuration. In particular it is shown that despite its drawbacks in other applications, conventional confocal microscopy still is more efficient than parallel confocal microscopy when the dose, i.e. the total energy onto the sample, must be limited. This may represent a limitation to the advent of parallel confocal microscopy for the imaging of photo-sensitive tissues

Quantitative Measures of Corneal Transparency, Derived from Objective Analysis of Stromal Light Backscattering with Full-Field Optical Coherence Tomography

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