Spectral confocal microscopy using white light supercontinuum sources

共焦显微镜是一种功能强大的成像工具，广泛应用于生物科学领域。它的优势在于它能够以高分辨率对样品进行三维成像，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。共焦显微镜在用于成像荧光时特别有用，这种荧光可以是自然发生的，也可以是故意引入的，以便标记样本的特定部分。荧光包括吸收一种波长的光，然后发射另一种更长波长的光。不同的荧光标记通常吸收和发射不同波长的光。因此，一个荧光显微镜通常需要一个以上的光源来激发各种荧光团，并且需要一个特定于波长的检测系统来分离发射波长。共焦显微镜依靠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只在有限的离散波长下可用。出于这个原因，商业共焦显微镜通常只包含两到三个激光波长。因此，只能使用某些荧光标记，然后在某些情况下，只能以低效的方式使用。虽然照明光以这种方式受到限制，但对不同波长的检测也被限制在少量的通道中。为了缓解这些限制，我们建议建造一种新的显微镜，它包括一个‘白光激光器’和一个光谱分辨探测器。白光光源以光子晶体光纤为基础，将产生包括标准激光产生的照明波长的广泛连续光谱。光谱探测器将提供比以前使用的更高的波长分辨率。它们一起将允许获取详细的、高分辨率、光谱分辨率的三维图像，这些图像将提供关于生物标本的更多信息，而不是使用目前的显微镜。这种新的显微镜还将允许“真彩色”共聚焦反射显微镜，即图像的颜色与从标本反射的光相对应。在传统的反射共聚焦显微镜中，照明只有一个波长，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；图像看起来较暗的区域是样品吸收光线的地方。通过使用白光源和颜色敏感的光谱探测，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成分，这些图像将提供有关样品的组成物质的信息。我们将与生物学家合作，以开发新显微镜的技术并研究其应用。首先，我们将在反射模式下使用显微镜来研究自然光学结构的光谱特性，例如蝴蝶翅膀的虹彩鳞片。在荧光模式下，显微镜将应用于研究自然产生的荧光结构。我们还将对用荧光蛋白组合标记的细胞进行成像，以了解DNA和RNA的细胞处理过程。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更可靠的信息。我们将确定其他将从光谱共聚焦显微镜的使用中受益的新应用领域。

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.