激光共聚焦显微镜原理与应用详细资料.docx

1、激光共聚焦显微镜原理与应用详细资料Confocal MicroscopyConfocal Microscopy: Basic ConceptsConfocal microscopy offers several advantages over conventional optical microscopy, including shallow depth of field, elimination of out-of-focus glare, and the ability to collect serial optical sections from thick specimens. In

2、the biomedical sciences, a major application of confocal microscopy involves imaging either fixed or living cells and tissues that have usually been labeled with one or more fluorescent probes.When fluorescent specimens are imaged using a conventional widefield optical microscope, secondary fluoresc

3、ence emitted by the specimen that appears away from the region of interest often interferes with the resolution of those features that are in focus. This situation is especially problematic for specimens having a thickness greater than about 2 micrometers. The confocal imaging approach provides a ma

4、rginal improvement in both axial and lateral resolution, but it is the ability of the instrument to exclude from the image the out-of focus flare that occurs in thick fluorescently labeled specimens, which has caused the recent explosion in popularity of the technique. Most current confocal microsco

5、pes are relatively easy to operate and have become part of the basic instrumentation of many multi-user imaging facilities. Because the resolution possible in the laser scanning confocal microscope (LSCM) is somewhat better than in the conventional widefield optical microscope, but still considerabl

6、y less than that of the transmission electron microscope, it has in some ways bridged the gap between the two more commonly used techniques. Figure 1 illustrates the principal light pathways in a basic confocal microscope configuration.In a conventional widefield microscope, the entire specimen is b

7、athed in light from a mercury or xenon source, and the image can be viewed directly by eye or projected directly onto an image capture device or photographic film. In contrast, the method of image formation in a confocal microscope is fundamentally different. The illumination is achieved by scanning

8、 one or more focused beams of light, usually from a laser, across the specimen (Figure 2). The images produced by scanning the specimen in this way are called optical sections. This terminology refers to the noninvasive method by which the instrument collects images, using focused light rather than

9、physical means to section the specimen.The confocal approach has facilitated much more useful imaging of living specimens, enabled the automated collection of three-dimensional (z-series) data, and improved the images obtained of specimens using multiple labeling. Figure 3 presents a comparison of a

10、 conventional epifluorescence image with a confocal image of similar regions of a whole mount of a butterfly pupal wing epithelium stained with propidium iodide. There is a striking improvement of resolution of nuclei in the LSCM image due to elimination of out-of-focus fluorescence flare.The laser

11、scanning confocal microscope (LSCM) is currently the most widely used confocal variation for biomedical research applications. Emphasis is placed on the LSCM in this introduction, since it is the design most likely to be encountered by the novice user. Other alternative designs of the instruments ar

12、e favored in specific niches within the field of biological imaging. Most of the protocols for specimen preparation can be used, with minor modification, for any of the confocal instrument variants, as well as for other methodologies for producing optical sections such as deconvolution techniques an

13、d multiple-photon imaging.Evolution of Confocal MicroscopyThe invention of the confocal microscope is usually attributed to Marvin Minsky, who produced a working microscope in 1955. The development of the confocal approach was largely driven by the desire to image biological events as they occur in

14、living tissue (in vivo), and Minsky had the goal of imaging neural networks in unstained preparations of living brains. The principle of confocal imaging advanced by Minsky, and patented in 1957, is employed in all modern confocal microscopes. Figure 1 illustrates the confocal principle, as applied

15、in epifluorescence microscopy, which has become the basic configuration of most modern confocal systems used for fluorescence imaging. Minskys original configuration used a pinhole placed in front of a zirconium arc source as the point source of light.The point of light was focused by an objective l

16、ens at the desired focal plane in the specimen, and light that passed through it was focused by a second objective lens at a second pinhole having the same focus as the first pinhole (the two were confocal). Any light that passed the second pinhole struck a low-noise photomultiplier, which generated

17、 a signal that was related to the brightness of the light from the specimen. The second pinhole prevented light originating from above or below the plane of focus in the specimen from reaching the photomultiplier. The use of spatial filtering to eliminate out-of-focus light or flare, in specimens th

18、at are thicker than the plane of focus, is the key to the confocal approach. In Minskys writings he also described a reflected light version of the microscope that used a single objective lens and a dichromatic mirror arrangement that became the basis for the systems currently in use.In order to bui

19、ld an image using the confocal principle, the focused spot of light must be scanned across the specimen in some way. In the original instrument built by Minsky the beam was kept stationary and the specimen itself was moved on a vibrating stage. This arrangement has the advantage that the scanning be

20、am is held stationary on the optical axis of the microscope, which can eliminate most lens defects that would affect the image. For biological specimens, however, movement of the specimen can cause wobble and distortion, resulting in a loss of resolution in the image. Furthermore, it is impossible t

21、o perform various manipulations on the specimen such as microinjection of fluorescently labeled probes when the stage and specimen are moving.Regardless of the means by which the illuminating beam is scanned across the specimen, an image of the specimen must be produced. A real image was not formed

22、in Minskys original design, but instead the output from the photomultiplier was translated into an image on the screen of a military surplus long persistence oscilloscope that had no provision for recording. Following the debut of his invention, Minsky later wrote that the image quality in his micro

23、scope was not very impressive because of the quality of the oscilloscope display and not because of poor resolution achieved by the microscope itself. It is now clear that the technology was not available to Minsky in 1955 to fully demonstrate the potential of the confocal approach, especially for i

24、maging biological structures. He stated that this is possibly a reason that confocal microscopy was not immediately embraced by the biological community, who were, and still are, a highly demanding group concerning the quality of their images. At the time, they had available light microscopes with e

25、xcellent optics, and could easily view and photograph their brightly stained and colorful histological tissue sections onto high-resolution color film. In todays confocal microscopes, the image is serially built up from the output of a photomultiplier tube or captured using a digital camera incorpor

26、ating a charge-coupled device, directly processed in a computer imaging system, displayed on a high-resolution video monitor, and output on a hard copy device, with outstanding results. The information flow in a modern laser scanning confocal microscope is diagramed in Figure 4.The basic optics of t

27、he optical microscope have remained fundamentally unchanged for decades because the final resolution achieved by the instrument is governed by the wavelength of light, the objective lens, and the properties of the specimen itself. The dyes used to add contrast to specimens, and other technology asso

28、ciated with the methods of optical microscopy, have improved significantly over the past 20 years. The growth and refinement of the confocal approach is a direct result of a renaissance in optical microscopy that has been fueled largely by advancements in modern technology. A number of major technol

29、ogical advances that would have been a benefit to Minskys confocal design have gradually become available (or more affordable) to biologists and other microscopists. Among these are stable multiwavelength lasers for improved point light sources, improved dichromatic mirrors, sensitive low-noise phot

30、odetectors, fast microcomputers with image processing capabilities enhanced by availability of affordable large-capacity memory chips, sophisticated image analysis software packages, and high-resolution video displays and digital image printers.These technologies were developed independently, and si

31、nce 1955, have been gradually incorporated into confocal imaging systems. As one example, digital image processing techniques were first applied effectively in the early 1980s by researchers at the Woods Hole Oceanographic Institute. Using what they termed video-enhanced microscopes they were able t

32、o image cellular structures such as microtubules, which are just beyond the theoretical resolution of the optical microscope. The apparent increase in resolution was enabled by digital enhancement of images that were captured using a low light level silicon intensified target (SIT) video camera conn

33、ected to a digital image processor. The cellular structures were imaged using differential interference contrast (DIC) optics, and the images were further enhanced using digital processing methods.The classification of confocal microscope designs is usually done on the basis of the method by which the specimens are scanned. The two fundamental means o