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Spectral confocal microscopy using white light supercontinuum sources

使用白光超连续谱源的光谱共焦显微镜

基本信息

批准号：
BB/E01240X/1
负责人：
Martin Booth
金额：
$ 12.41万
依托单位：
University of Oxford
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2006
资助国家：
英国
起止时间：
2006 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FE01240X%2F1
关键词：
Spectral confocal microscopy using white

项目摘要

The confocal microscope is a powerful imaging tool that is widely used across the biological sciences. Its strength lies in its ability to image specimens at high resolution in three-dimensions, rather than the two-dimensions provided by a conventional optical microscope. Taking advantage of this three-dimensional resolution, one can produce images of cellular structures and processes that can shed light upon many biological processes. The confocal microscope is particularly useful when used to image fluorescence that can be either naturally occurring or can be deliberately introduced in order to label a particular part of the specimen. Fluorescence involves the absorption of light of one wavelength and the subsequent emission of light at a different, longer wavelength. Different fluorescent markers generally absorb and emit light at different wavelengths. A fluorescence microscope therefore normally requires more than one light source, in order to excite the various fluorophores, and a detection system that is wavelength specific, to separate the emission wavelengths. Confocal microscopes have relied upon the use of lasers to produce a bright, point-like source of light. However, lasers that might be practically used for microscopy have only been available in a limited number of discrete wavelengths. For this reason, commercial confocal microscopes have only typically incorporated two or three laser wavelengths. As a consequence, only certain fluorescent markers could be used and then, in some cases, only in an inefficient manner. Whilst the illumination light has been constrained in this way, the detection of different wavelengths has also been limited to a small number of channels. In order to alleviate these restrictions, we propose to build a new microscope incorporating a 'white light laser' and a spectrally resolved detector. The white light source, based upon on a photonic crystal optical fibre, will produce a wide continuous spectrum of illumination wavelengths including those produced by standard lasers. The spectral detector will provide wavelength resolution greater than that previously used. Together they will permit the acquisition of detailed, high resolution, spectrally resolved, three dimensional images that will provide more information about biological specimens than is possible using present microscopes. The new microscope will also permit 'real colour' confocal reflection microscopy, where the colour of an image corresponds to the light reflected from the specimen. In traditional reflection confocal microscopes, where the illumination was only at one wavelength, only bright or dark regions of the specimen could be seen. The bright regions occur where that particular wavelength is reflected; areas where the image appears dark are where the specimen absorbs the light. By using a white light source and colour sensitive, spectral detection, we will be able to produce three-dimensionally resolved, colour images. Since absorption and reflection properties depend upon the chemical make-up, these images will provide information about the component substances of the specimen. We will work with biologists in order to develop the techniques and investigate applications for the new microscope. In the first instance, we will use the microscope in reflection mode to investigate the spectral properties of natural optical structures, for example the iridescent scales of butterfly wings. In fluorescence mode, the microscope will be applied to the investigation of naturally occurring fluorescent structures. We will also perform imaging of cells labelled with a combination of fluorescent proteins in order learn about the cellular processing of DNA and RNA. It is expected that this new approach will provide more detailed and reliable information than is obtained using present microscopes. We will identify other, new application areas that will benefit from the use of the spectral confocal microscope.

共聚焦显微镜是一种强大的成像工具，广泛应用于生物科学。它的优势在于它能够在三维中以高分辨率对样品进行成像，而不是传统光学显微镜提供的二维图像。利用这种三维分辨率，人们可以产生细胞结构和过程的图像，这些图像可以揭示许多生物过程。共聚焦显微镜是特别有用的，当用于图像的荧光，可以是自然发生的或可以故意引入，以标记一个特定的部分标本。荧光涉及吸收一种波长的光，随后发射不同的更长波长的光。不同的荧光标记通常吸收和发射不同波长的光。因此，荧光显微镜通常需要一个以上的光源，以激发各种荧光团，以及波长特异性的检测系统，以分离发射波长。共焦显微镜依赖于使用激光来产生明亮的点状光源。然而，可能实际用于显微镜的激光器仅在有限数量的离散波长中可用。出于这个原因，商业共焦显微镜通常只包含两个或三个激光波长。因此，只能使用某些荧光标记，并且在某些情况下只能以低效的方式使用。虽然照明光已经以这种方式被约束，但是不同波长的检测也被限制到少量通道。为了减轻这些限制，我们建议建立一个新的显微镜，将“白色光激光”和光谱分辨探测器。基于光子晶体光纤的白色光源将产生包括由标准激光器产生的照明波长的宽的连续光谱。光谱检测器将提供比先前使用的更大的波长分辨率。它们一起将允许获取详细的，高分辨率的，光谱分辨的，三维图像，将提供更多的信息，生物标本比可能使用目前的显微镜。新的显微镜还将允许“真实的颜色”共焦反射显微镜，其中图像的颜色对应于从标本反射的光。在传统的反射式共焦显微镜中，照明仅在一个波长下，只能看到样品的亮或暗区域。明亮的区域出现在特定波长被反射的地方;图像看起来很暗的区域是样本吸收光的地方。通过使用白色光源和对颜色敏感的光谱检测，我们将能够产生三维分辨率的彩色图像。由于吸收和反射特性取决于化学组成，这些图像将提供有关样品组成物质的信息。我们将与生物学家合作，开发新显微镜的技术并研究其应用。首先，我们将使用反射模式的显微镜来研究自然光学结构的光谱特性，例如蝴蝶翅膀的彩虹色鳞片。在荧光模式下，显微镜将被应用于自然发生的荧光结构的调查。我们还将对荧光蛋白组合标记的细胞进行成像，以了解DNA和RNA的细胞加工。预计这种新方法将提供比使用现有显微镜获得的更详细和可靠的信息。我们将确定其他新的应用领域，将受益于光谱共聚焦显微镜的使用。