Spectral confocal microscopy using white light supercontinuum sources

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.

The capabilities of confocal microscopes have been limited by the need to use lasers as light sources. This has meant that only a small number of illumination wavelengths were usually available. Furthermore, spectral resolution in detection has been similarly limited: a small number of wavelengths could be separated using dichroic filters; recent commercial systems have used different methods to extend this to a few spectral channels. The capabilities of the confocal microscope could be considerably extended if the spectral resolution of both the illumination and detection could be increased. The recent advances in commercially available white light supercontinuum sources have paralleled the development of fast, inexpensive spectral detectors. We propose to take advantage of these two recently matured technologies in order to develop a new direction for microscopy: spectral confocal microscopy. We will undertake a pilot investigation to construct and apply a confocal microscope employing spectral resolution for both illumination and detection. The illumination will be provided by a supercontinuum photonic crystal fibre source and specific wavelengths will be selected using a digital mirror device. Detection will be implemented with a high speed spectrometer. The new microscope will be used to perform three-dimensional imaging in both reflection mode, to produce reflection/absorption images of specimens, and in fluorescence mode, to generated detailed emission spectra and excitation/emission maps. The technology will be demonstrated through imaging of biological specimens provided by our collaborators. We will also identify other potential application areas with a view to submitting a full funding proposal before the end of this pilot project.