Imaging nanometre-scale structure in cells using in situ aberration correction

Accurate distance measurements of cellular structures on a length scale relevant to single macromolecules or macromolecular complexes present a major challenge for biological microscopy. In addition to the inherent challenges of overcoming the limits imposed by the diffraction of light, cells themselves are a complex and poorly understood optical environment. We present an extension of the high-resolution colocalization method to measure three dimensional distances between diffraction-limited objects using standard widefield fluorescence microscopy. We use this method to demonstrate that in three dimensions, cells intrinsically introduce a large and variable amount of chromatic aberration into optical measurements. We present a means of correcting this aberration in situ [termed 'Colocalization and In-situ Correction of Aberration for Distance Analysis' (CICADA)] by exploiting the fact that there is a linear relationship between the degree of aberration between different wavelengths. By labelling a cellular structure with redundantly multi-colour labelled antibodies, we can create an intracellular fiducial marker for correcting the individual aberrations between two different wavelengths in the same cells. Our observations demonstrate that with suitable corrections, nanometre scale three-dimensional distance measurements can be used to probe the substructure of macromolecular complexes within cells.     

Simulation of specimen-induced aberrations for objects with spherical and cylindrical symmetry

Wavefront aberrations caused by the refractive index structure of the specimen are known to compromise signal intensity and three‐dimensional resolution in confocal and multiphoton microscopy. However, adaptive optics can measure and correct specimen‐induced aberrations. For the design of an adaptive optics system, information on the type and amount of the aberration is required. We have previously described an interferometric set‐up capable of measuring specimen‐induced aberrations and a method for the extraction of the Zernike mode content. In this paper we have modelled specimen‐induced aberrations caused by spherical and cylindrical objects using a ray tracing method. The Zernike mode content of the wavefronts was then extracted from the simulated wavefronts and compared with experimental results. Aberrations for a simple model of an oocyte cell consisting of two spherical regions and for a model of a well‐characterized optical fibre are calculated. This simple model gave Zernike mode data that are in good agreement with experimental results.     

Three‐dimensional optical transfer functions in the aberration‐corrected scanning transmission electron microscope

In the scanning transmission electron microscope, hardware aberration correctors can now correct for the positive spherical aberration of round electron lenses. These correctors make use of nonround optics such as hexapoles or octupoles, leading to the limiting aberrations often being of a nonround type. Here we explore the effect of a number of potential limiting aberrations on the imaging performance of the scanning transmission electron microscope through their resulting optical transfer functions. In particular, the response of the optical transfer function to changes in defocus are examined, given that this is the final aberration to be tuned just before image acquisition. The resulting three‐dimensional optical transfer functions also allow an assessment of the performance of a system for focal‐series experiments or optical sectioning applications.     

低剂量~(60)Coγ线诱发人血淋巴细胞早熟凝聚染色体畸变的剂量效应曲线研究

本文报道应用人外周血淋巴细胞在离体条件下,用低剂量~(60)Coγ线照射后与M期CHO细胞融合产生G_1-PCC,计数其畸变产额,得到线性剂量效应刻度曲线,Y=0.26+0.028.Dr=0.99,并与常规细胞遗传学方法作了比较,结果表明G_1-PCC方法比常规遗传学方法快速、灵敏。对其作为生物剂量计尚存在的问题也进行了讨论。     

Linear versus non-linear structural information limit in high-resolution transmission electron microscopy

A widely used performance criterion in high-resolution transmission electron microscopy (HRTEM) is the information limit. It corresponds to the inverse of the maximum spatial object frequency that is linearly transmitted with sufficient intensity from the exit plane of the object to the image plane and is limited due to partial temporal coherence. In practice, the information limit is often measured from a diffractogram or from Young's fringes assuming a weak phase object scattering beyond the inverse of the information limit. However, for an aberration corrected electron microscope, with an information limit in the sub-angstrom range, weak phase objects are no longer applicable since they do not scatter sufficiently in this range. Therefore, one relies on more strongly scattering objects such as crystals of heavy atoms observed along a low index zone axis. In that case, dynamical scattering becomes important such that the non-linear and linear interaction may be equally important. The non-linear interaction may then set the experimental cut-off frequency observed in a diffractogram. The goal of this paper is to quantify both the linear and the non-linear information transfer in terms of closed form analytical expressions. Whereas the cut-off frequency set by the linear transfer can be directly related with the attainable resolution, information from the non-linear transfer can only be extracted using quantitative, model-based methods. In contrast to the historic definition of the information limit depending on microscope parameters only, the expressions derived in this paper explicitly incorporate their dependence on the structure parameters as well. In order to emphasize this dependence and to distinguish from the usual information limit, the expressions derived for the inverse cut-off frequencies will be referred to as the linear and non-linear structural information limit. The present findings confirm the well-known result that partial temporal coherence has different effects on the transfer of the linear and non-linear terms, such that the non-linear imaging contributions are damped less than the linear imaging contributions at high spatial frequencies. This will be important when coherent aberrations such as spherical aberration and defocus are reduced.     

5.4 nm spatial resolution in biological photoemission electron microscopy

We report a spatial resolution of 5.4 nm in images of sarcoplasmic reticulum from rabbit muscle. The images were obtained in an aberration-corrected photoemission electron microscope with a hyperbolic mirror as the correcting element for spherical and chromatic aberration. In-situ measurements and numerical simulations confirm the low residual aberration in the instrument and indicate the ultimate resolution in this type of microscopy to be below 2 nm.     

Aberration correction for confocal imaging in refractive-index-mismatched media

A major limitation to the use of confocal microscopes to image thick biological tissue lies in the dramatic reduction in both signal level and resolution when focusing deep into a refractive-index-mismatched specimen. This limitation may be overcome by measuring the wavefront aberration and pre-shaping the input beam so as to cancel the effects of aberration. We consider the images of planar and point objects in brightfield, single-photon fluorescence and two-photon fluorescence imaging. In all cases, the specimens are imaged using an oil-immersion objective through various thicknesses of water. The question of finite-sized pinhole is addressed and it is found, in general, that it is sufficient to correct only the first two or three orders of spherical aberration to restore adequate image signal level and optical resolution, at imaging depths of up to 50-100 wavelengths.     

215名放射工作人员染色体畸变率及微核率的测定

摘要采用常规方法检测了215名放射工作人员及86名普通行业人员（对照）的外周血淋巴细胞染色体畸变率和微核率。结果表明，放射工作人员染色体畸变率和微核率分别为0．53%和2．37‰，均显著高于对照组，并随工龄的增加而增加。     

26例广岛原子弹爆炸受照者的染色体畸变分析

本研究工作是日本放射线影响研究所细胞遗传学研究方面有关工作的继续。对26名广岛原爆受照者(0—786rad)的染色体畸变的分析结果,发现畸变率与每一剂量组的平均剂量之间呈现良好的线性关系。大体看来,剂量每增加100rad,畸变细胞约增加3%,总畸变数约增加4%。对畸变类型的分析表明,大多数畸变为异常的单着丝点染色体,包括相互易位、倒位及缺失。它们约占总畸变数的86.3%。     

职业性受照人员的稳定性染色体畸变观察

对日本原爆受照生存者的大量染色体畸变分析结果表明,残存于受照者外周血淋巴细胞中的染色体畸变主要是稳定性畸变(相互易位、倒位及缺失),且畸变率与剂量之间有一定的关系~([1,2])。原爆受照人群所接受的是一次性照射;而对于那些在较长时间中连续或多次受小剂量照射的职业性工作人员,其染色体漪变是否也以稳定性畸变为主?畸变率与累积剂量之间是否有一定关系?这在国内外文献中尚未见报道。最近我们对一核工厂部分长期从事放射性工作的职工进行了稳定性畸变的分析,并对畸变率与累积剂量关系作了初步探讨。