REVIEW ARTICLES ACTA ANAESTHESIOL TAIWANICA 42:33-40, 2004

Confocal Laser Scanning Microscopy: I. An Overview of Principle and Practice in Biomedical Research Jui-Tai Chen, Ruei-Ming Chen, Yi-Ling Lin, Huai-Chia Chang Yu-Hua Lin, Ta-Liang Chen, Tyng-Guey Chen Department of Anesthesiology, Wan-Fang Hospital and Taipei Medical University Hospital, Taipei Medical University, Taipei, Taiwan, R.O.C.

Evolving from conventional microscopic technologies, confocal microscopy has proved itself to play an important role in the biomedical research during the past decade. Confocal microscope has many advantages over traditional microscope including the ability to look deeply into inside cells with less photodamage and photobleach, reconstruct three-dimensional images, and chart intracellular dynamic events in the living cells. With these remarkable properties and the availability of fluorescent dyes for living cells, the confocal microscopy has been widely used in solving many unknown questions in biological and pharmacological fields. In clinics, confocal microscope has also served as an important tool to observe the living cells in skins and eyes. For anesthesiologists, confocal microscope has made possible novel experimental approaches for the effects of multiple anesthetic agents on cells. Furthermore, the technology of fibreoptical confocal endomicroscopy is now on its way of maturation. It will soon be the era for confocal microscopy to explore the "cell behavior" inside of intact living tissues.

Key words : Microscopy, confocal.

cientists had theoretically developed the optic concept of microspectrometry about half century ago to analyze nucleic acid.1 In 1957, Minsky added a scanning stage to construct image in microscopy and thus the first confocal microscope in the world was created.2 Although there was great advance in microscopic technology thereafter, it was not until 1970s that the practical confocal microscopy was to come of age. With the invention of laser, the commercial models were available in the mid-1980's. In recent years, technological improvements contributed much to the development of modern digital imaging methods, laser technology, and availability of fluorescent probes. Such great improve-

ments make new approaches for multiple labeled fluorescence looking into inside of cells and tissues, and real time image of three-dimensional morphology and dynamic events possible.3-5

S

The Principle of Confocal Microscope According to Minsky's patent of confocal microscope in 1957, penetrating a pinhole from a zirconium arc light source produces the point source of light.6 Then the objective lens focuses this light into specimen at the exact focal plane of the objective lens. The light reflecting back from the illuminated spot on the specimen is collected by the same lens and directed by a beam splitter to a pinhole in front of the detector, as shown in Fig. 1. Only the in-focus spot light will pass to the detector and light from out-of-focus planes will be rejected by the detector pinhole, therefore not contributing to final image formation. Because the same single lens is used as both condenser and objective folding optical path, we can make sure their focal points exactly coincide all the time.

Received: April 4, 2003. Revised version received: October 24, 2003. Accepted for publication: October 28, 2003. Address correspondence and reprint requests: Dr. Tyng-Guey Chen, Department of Anesthesiology, Taipei Medical University Affiliated Wan-Fang Hospital, No. 111, Sec. 3, Hsing -Lung Rd. Taipei, 116, Taiwan, R.O.C. Tel: +886-2-29307930 ext. 2156 Fax: +886-2-89315940 E-mail: [email protected]

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movement and fail to stand for a longer time. Thereafter, scanning beam method becomes the mainstream in the world of confocal microscopy. A variety in basic principles of confocal microscopy was introduced, and finally two configurations had been chosen for popular use by the end of last decade.

Disk Scanning Method

Fig. 1. Diagram of structures of the confocal laser scanning microscope. Laser beam is focused to a spot in the specimen by the objective lens. Fluorescent light emitted from this spot is focused, via the confocal pinhole, onto a detector (photomultiplier). Light from other spots in the specimen is rejected by the confocal pinhole.

As illumination and detection system are at the same focal plane, they are termed as "confocal". By scanning the specimen successively with point illumination, a plane of image can be formed. In this way we can get a crisp, blur-free, image with high quality. Taking a series of confocal images within successive planes also can create a true three-dimensional image for specimen and assembling them by the computer, which is far above the analytic power of traditional light microscopes.

The Development of Confocal Microscope Scattered or aberrant light contributes much to the background haze that might spoil the image of interest. It is important to illuminate only one spot on the specimen at a time. To achieve the purpose this way, most of extra light should be avoided to obtain a better image. As one can speculate, laser beam provides an ideal solution to serve as the point source of light. Thus the single-point illumination is enabled to measure one point image at a time. By scanning the specimen point by point, the obtainment of a confocal microscopy becomes too timeconsuming. In order to acquire the speed for real-time analysis, scanning method plays the role.

Stage Scanning Method When designing the confocal microscope, Prof. Marvin Minsky was confronted with the difficulty of choosing between moving the specimen and the beam light. Finally he selected the stage scanning method due to its easier optical alignment. But it was slow and impractical should the specimen be easily disturbed by

Nipkow disk microscope employs a uniform light source and combines with a spinning Nipkow disk in his original design.7 A Nipkow disk contains thousands of pinholes arranged in a spiral tract. When the disk is spinning, the moving pinholes could cover the whole field of view. By spinning thousands of points simultaneously, the Nipkow disk can achieve real-time (video-rate) confocal images. For example, QLC 100 from Yokogawa company based on Nipkow disk technology can capture 360 frames per second of two- dimension confocal image. Moreover, Nipkow disk microscopy can use stationary white light allowing live viewing in real time. However, these systems own a poor light budget, and only 0.25%-1% of available light passes through the pinholes. Most of light has stopped before entering and thus the system is lesser sensitive. If the samples are strong reflectors, the loss of incident light would cause no adverse sequence. So Nipkow disk microscopes have been widely used in inspection and metrology of semiconductor industry. Nowadays, a second wheel carrying a microlens array in front of Nipkow disk to converge incident light is introduced by the Yokogawa Company in Japan. In this way, 60 – 70% incoming light can pass through the pinholes. Thus the optical efficiency is much improved by a thousand fold and starts to attract the biological world again.

Point Beam Scanning Method The confocal laser scanning microscope (CLSM), using laser beam as point light source instead of a lamp with illumination pinhole, is now the widely used format for confocal microscopes in the biological field.8 In modern times, CLSM typically utilizes either movable scanning mirrors or an acousto-optic deflector to direct laser beam scanning across the specimen. 9,10 The excited fluorescent signals travel the reverse pathway first, and then are separated from the laser beam by beam-splitter dichroic mirrors. Finally the fluorescent signals are detected and amplified by photomutiplier tube (PMT) and built into image by a computer. Thus the combination of laser illumination, fluorescent probes, and the processing power of computer makes CLSM a powerful tool of great importance in the modern biological world. Relatively slow acquisition of speed is still the

JUI-TAI CHEN et al.

major problem of CLSM. When trying to work on living cells or analyzing the dynamic intracellular events, the video-rate (30 frame/sec) scanning speed helps a lot. The limitation is now in the scanning mirrors. Many different approaches are made and some of them obtain successful results. Olympus Company has offered 'Fluoview FV500', which uses a pair of fast-type galvanometer mirrors controlled by microprocessor to speed up the scanning. One galvanometer mirror scans in x direction and the other in y direction. With the good cooperation and quick motions of galvano-type mirrors, Fluoview FV500 can deliver flare-free, high contrast images up to 2048×2048 pixel resolution with wide dynamic range of 4096 gradations. The high-speed image capture mode provides 4 confocal frames (512× 512) per second. One manufacturer, Bio-Rad, creates a highly innovative scanning method with fixed optic alignment. Both x and y scan motions are produced by fast galvano-type mirrors, the same as other confocal microscopes. The secret of this unique optic design lies in two additional concave mirrors, which are arranged so as to image one galvanotype mirror on the other. This produces rotation about a point on the second mirror and thus speed up the scanning process. In this way, Bio-Rad CLSM can provide up to 100 frames per second with uncompromised image quality. Noran Instruments integrated a special acousto-optic deflector (AOD) with an ultra-fast piezoelectric objective lens translator. AOD utilizes a high frequency sound wave through a special crystal to create a diffraction grafting that deflects laser light beam. By changing the frequency of sound wave, AOD can adjust the angle of laser beam and makes specimen scanning more quickly. Actually with a scan range that starts at 30 frames per second and extends as high as 480 frames per second, Noran has produced the fastest point-scanning confocal microscope available now.11

Confocal Microscope Vs. Traditional Light Microscope Confocal microscopy is a relatively new light microscopical imaging technique. Comparing with traditional ones, confocal microscope provides a modest increase in optical resolution in both lateral (focal plane, x-y-axis) and axial (depth plane, z-axis) aspects. The image of the point source of light is actually a diffraction-limited light spot, which is regarded as Airy Disk. The Airy disk is defined as the intensity of light with a function of radius. Two point objects are said to be just resolved when the center of one Airy Disk falls on the first minimum of the other Airy Disk. And radius could be given by: airy

= 1.22 / NA

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where = wavelength of light and NA = numeric aperture of the objective lens. In ideal situation, ordinary light microscope and confocal microscope will obtain the same theoretical lateral resolution of 0.14 m and axial resolution of 0.23 m with a objective lens of 1.4 NA with white light. In practice, with the optimal size of pinholes (about 0.8 airy ), confocal microscope can get lateral resolution of 0.2 m and an axial resolution of 0.5 m that are better than ordinary light microscope due to its prevention for out-of-focus light by pinholes.12 In addition, the confocal microscope can look deeper into intact tissue, which is a great benefit to the study of biological science. In fact, ordinary light microscope can also sense light signals of the same depth as confocal microscope. The problem is that ordinary light microscope cannot offer an image as clear as CLSM does because the whole tissue is illuminated evenly, including above and below the focal plane. This ability of confocal microscope to look deeper creates a valuable concept of "optical section", which is an image from a small depth of field of one focal plane inside the intact tissue. By the power of modern computers, we can analyze and process optical sections and therefore reconstruct the three-dimensional image of specimen.

Multiphoton Fluorescence Microscope Conventional confocal microscopes use single laser penetrating into the specimen to produce fluorescent signals. Single high-energy photon hits and promotes the fluorescent molecule from the ground state to the excited state, thus producing fluorescent signals. Therefore conventional confocal microscopes are ranked as single photon fluorescence excitation. In single photon confocal microscope, the average intensity of excitation beam is approximately uniform above and below the focal plane. This results in generating out-of-focus fluorescent signals and thus jeopardizing the image quality. It will be greatly advantageous if we can exactly excite the plane of interest while silence the other regions. The method of multiphoton excitation (MPE) can achieve this goal. MPE, theoretically predicted in 1931 by GoppertMayer, is based on the probability that multiple low energy photons arrive simultaneously at a fluorophore and excite it. 13 This idea was first applied to microscopic technology in 1978. It was not until early 1990s, after the development of robust ultra-fast (10 -14 sec/pulse) laser, that a working two-photon excitation microscope was shown in the world and began to take part in the biological world.14 In MPE, the focal plane gets the strong possibility to receive multiple photons simultaneously and thus emits the fluorescent light. The intensity of emitted

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PRINCIPLES OF CONFOCAL MICROSCOPE AND ITS APPLICATIONS

fluorescent signals decays quickly from the focal plane, because the possibility of excitation is highly related to the power n, where n stands for the number of simultaneously absorbed photons. Due to the slim possibility of out-of-focus excitation, MPE is highly restricted to the submicron-sized volume at the focus plane. Because background scattered light rejection is unnecessary, MPE confocal microscope can maintain many desirable qualities of conventional confocal microscopes without the need for confocal pinholes. 15 Theoretically MPE confocal microscope has the same lateral and axial resolution as conventional confocal microscope. MPE exhibited a number of clear advantages over single photon excitation. Due to the scattering light from above or below the focal plane along the z-axis, the penetration depth of conventional confocal microscope is limited to a range of 100 – 200 m. On the contrary, MPE confocal microscope only excites exactly the focal plane and usually uses far-red or infrared light as illumination; it offers clear images at least 2 – 3 times deeper than conventional confocal microscope. Photobleaching above or below the plane of focus is also eliminated. The excitation power used in MPE confocal microscope is enormous. By using ultra-fast pulsed laser, the heating effect on specimen is less when compared with the conventional confocal microscope. Furthermore, the light with longer wavelength does less harm to cells. For these reasons, MPE confocal microscope has advantages of imaging deep into tissues and observing cells for longer periods. For example, neurons can be imaged deep inside and the intact brain mitochondria membrane potential can be measured in living cells for extended periods. 16

vantages with MPE microscope, such as intrinsic threedimensionality as well as the ability to section deeply within tissues. Furthermore, SHG does not arouse an absorptive process, the photodamage is much more reduced. Both SHG and MPE microscopes could image cell membrane labeled with a potential-sensitive dye such as styryl dye. After serious comparisons, it is found that SHG microscope can image membrane with excellent specificity and sensitivity that are even better than MPE microscopes.18 Furthermore, it is well-known that the biological structure protein arrays consist largely of collagen, myosin and tubulin and their associated proteins. 19 These structure protein arrays that are highly ordered and birefringent can produce large SHG signals without the need of any exogenous labels. This makes the SHG method an excellent choice for in vivo image approaches. Deng et al. have measured the SHG signals of fresh-frozen hyperplastic parenchyma and stroma in malignant human prostate tissue under a femtosecond pulsed laser with a wavelength-tuning range from 730 – 870 nm. The results in this two extreme regions were considerably different and could possible be an indicator for identifying or distinguishing normal or malignant tissue. 20 Due to so many advantages in living cell imaging, it is expected that SHG microscope will be as widely used as MPE microscope in the future.

Applications of Confocal Microscopy With the ability to construct accurate three-dimensional images and chart time-lapse dynamic events, confocal microscope is highly expected to fare well as it has appeared in the marketplace and now been utilized in various areas.

Second Harmonic Generation (SHG) Biological Research In 1962, Kleinmen demonstrated that when the pulse of deep red ruby laser light is directed through quartz, ultraviolet light could be produced and known as second harmonic light.17 The mechanism for second harmonic generation lies in that light can be thought as an electromagnetic wave and electric field will propagate while light is moving. When intense light is traveling through a structure, the distribution of internal electric charges in molecules will change with electric fields. The energy resulting from redistribution of internal electric charges will consequently give rise to an additional electric field. If the structure is orderly but not centrosymmetric, there is chance to generate second harmonic incident light. Microscopic imaging based on SHG is now on the marketplace. Because SHG is a non-linear optical phenomenon, SHG microscope shares several common ad-

CLSM is enthusiastically received in the biological fields. Accompanied by the availability of fluorescent dyes for living cells, CLSM enables the biologists to learn more detailedly about cell physiology. Indeed, the introduction of confocal microscope has opened many novel experimental territories.

Green Fluorescent Protein (GFP) GFP is a naturally occurring protein from the jellyfish Aequorea Victoria producing fluoresces when excited by UV or blue light. Prasher et al. first demonstrated that expression of the gene in other organisms generating fluorescence gave the clues to the utility of GFP. 21 By tagging the GFP gene to the gene of interest, either stably or transiently expressed in the living cells, we can get

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images with no need for further fluorescence-labeling. With the help of CLSM, the biologists can more accurately identify the location tagged with GFP, study the structure of tissue, and even the dynamic events within living cells.

Fluorescence Recovery Time after Photobleaching (FRAP) FRAP technology is based on the principle of observing the rate of recovery of fluorescence due to the movement of fluorescent molecules. When applying laser light to irradiate or photobleach a region to become nonfluorescent, the subsequent redistribution of fluorescent molecules from other regions within cell occurs. The time for redistribution of fluorescent molecules across plasma membrane, defined as recovery time, varies from seconds to micro- or milli-second. FRAP was first proposed in the 1970s by Axelord and coworkers and initially utilized to study the plasma membrane of living cells.22 By measuring the recovery time, we can learn more about the membrane properties and the mobility of fluorescence-tagged proteins. By FRAP technology in combination with CLSM, Klein et al. measured the lateral diffusion of a fluorescent analogue of sphingomyelin to study the membrane perturbing effect, which has been described in neurodegenerative process such as Alzheimer's disease. 23 In many fields of biology, FRAP is also applied to determine the dynamics and interaction of GFP-tagged proteins in living cells.24 The introduction of CLSM really extends the topics of study, where FRAP can be applied to the inside of living cells.

Fluorescence Loss in Photobleaching (FLIP) FLIP, a variation of FRAP, was initially used to determine whether there is communication between two subcellular compartments. A compartment within cell is bleached with low intensity laser and then the presumable connected compartments are imaged. The loss or reduction in fluorescent signals indicates a possible connection between compartments. One can also measures the residence time of molecules for specific accumulation such as motility of the alternative splicing factors through the nucleoplasm.25 CLSM is favorable for this situation to image subcellular compartment and trace the dynamic events.

Fluorescence Resonance Energy Transfer (FRET) Due to the availability of different fluorescent proteins like GFP and its derivatives, the FRET technology is popular recently in biological world.26 This technology provides the information about distance between

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molecules. Two molecules are labeled with different fluorescent dyes, which the emission spectrum of one must overlap the absorption spectrum of the other. Two molecules must be close enough or even intact with each other to allow non-radiative energy transfer. The calmeleon dye is one of the classic examples. Cameleon is a molecule consisting of two different GFP derivatives at each end, cyan fluorescent protein and yellow fluorescent protein for example. It is linked by calcium-mediated contractile protein calmodulin. In usual situation, dye with short wavelength will be dominant. When calmodulin binds with calcium and two dyes are brought close enough FRET occurs. Energy from short wavelength dye is transferred to the dye with longer wavelength, and the fluorescence emitted is then detected. In addition, the entire construct gene can be incorporated into the genome of organism for functional study of living cells. Besides, the relationship between ligand and receptor is always the focus in pharmacology and FRET technology is now increasingly used in this pursuit. The advent of GFP has further extended the applications of FRET-based imaging.27

Bio-luminance Resonance Energy Transfer (BRET) BRET is another form of radiative-free energy transfer that energy moves from donor to acceptor and is similar to FRET.28 Difference lies at the donor, which is bio-luminescent for BRET and fluorescent for FRET. BERT is a naturally occurring phenomenon in several marine animals such as jellyfish Aequorea Victoria and in plants Arabidopsis Thaliana. A prerequisite for BRET is that donor and receptor must be brought into molecular proximity that makes it a system of choice to monitor protein-protein interactions in living cells.

Clinical Research In pharmacology, confocal microscope plays a major role in recording the interactions of chemical agent on living cells. Through the introduction of confocal microscope, vital fluorescent dyes have been important tools for pharmacologists and now become more popular than ever. Many of these dyes are ratiometric and can emit two different specific fluorescent wavelengths according to different physiological conditions. The ratio between two different wavelengths can give an absolute measurement, irrespective of the absolute concentration of the dye. One of a target molecule for ratiometric dye is calcium. Calcium is an important secondary messenger and signaling in almost all cells. With the CLSM, the ratiometric calcium imaging therefore can accurately detect the change in intracellular calcium concentration after adding some pharmacological agents. Furthermore,

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PRINCIPLES OF CONFOCAL MICROSCOPE AND ITS APPLICATIONS

membrane potential is an equally important physiological parameter revealing cell-to-cell communication and, as in mitochondria, an indicator for cell viability. By the power of confocal microscope, one can examine the impact of interested drugs, e.g. propofol, onto the morphology and functions of mitochondria and capture the intracellular calcium shift within cell to further explain its pharmacodynamic events. 29 The information about calcium release from intracellular store is more detailed by the ability to image release across cells, to identify the source of release or/and to investigate the trigger factor for release.30 The powerful ability of confocal microscope also gives the clinicians an important new tool. For ophthalmologists, confocal microscope can produce high contrast reflected light image or optical sections through the deep living ocular tissue. By the processing of computer, confocal microscope can even provide threedimensional imagines of cornea, ocular lens, retina, and optical nerve.31 For example, by real-time confocal microscopy, ophthalmologists can carefully observe the healing process after cryo-ablation surgery of cornea that is impossible for slit lamp and the spectrum microscope to offer due to much haze produced by edema fluid. 32 Application in studies of living skin tissue is also popular. Dermatologists utilized confocal microscope to produce thin section of human skin and allowed visualization of cellular and nuclear details in vivo. With the high-resolution and non-invasive imagining, dermatologists now focus on the research of identification of benign or/and malignant pigmented skin lesions, non-melanoma skin cancer, inflammatory skin conditions, and dynamic skin healing process.33 The development of fiber-optic CLSM is another advance in clinical diagnostic application and makes possible the examination of hollow organ perforation via the intraluminal endoscopic access. Bulky components including the light source, scanning systems, and detection systems are separated from objective with a linkage by a flexible optic fiber.34 The remote objective is incorporated into a 10-mm imagine head with a confocal endomicroscope for in vivo imagine being built. Although there are still problems in limitation of depth and immobilization, many studies have been done including normal and abnormal in vivo histology of colonic mucosa and inspection of nerves and vessels of mesenteric plexus via serosal approach.35 In clinics, with properties such as being capable of imaging human tissues in nearly realtime and minimally invasive optical biopsy for the early detection of tumor cells, fiber-optic confocal microscope seems to be a useful and powerful tool in the future.36

Confocal microscope does not remain static since its inception. For the years, major improvements have been made at all stages of imaging chains and become more speedy, friendly, and being capable of providing a wide range of image modalities. With the diverse fluorescent probes and multiple-labeling technique, confocal microscope has become a tool of choice for study in living cells. Since technology never stops, in vivo and in situ measurements seem soon to be the next stage in the evolution of confocal microscope. The day is anticipated when we can see the "cell behavior" in its vital context instead of being removed from tissues.

Conclusion

17. Ramazza PL, Ducci S, Zava tta A, Bellini M, Arecchi FT:

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Second-harmonic generation from a picosecond Ti: Sa laser in LBO: Conversion efficiency and spatial properties. Appl Phys B 75:53-58, 2002. 18. Campagnola PJ, Clark HA, Mohle r WA, Le wis A, Loew LM: Second-harmonic imaging microscopy of living cells. J Biomed Opt 6:277-286, 2001. 19. Mohler W, M illard AC, Campagnola PJ: Second harmonic generation imaging of endogenous structural proteins. Methods 29:97-109, 2003. 20. Deng X, Williams ED, Thompson EW, Gan X, Gu M: Secondharmonic generation from biological tissues: Effect of excitation wavelength. Scanning 24:175-178, 2002. 21. Prasher D, McCann RO, Cormier MJ: Cloning and e xpression of the cDNA coding for aequorin, a bioluminescent calciumbinding protein. Biochem Biophys Res Commun 126:12591268, 1985. 22. Axelrod D, Koppel DE, Schlessinger J, Elson E, Webb WW: Mobility mea surement by analysis of fluorescence photobleaching recovery kinetics. Biophys J 16:1055-1069, 1976. 23. Klein C, Pillot T, Chambaz J, Drouet B: Determina tion of plasma membrane fluidity with a fluorescent ana logue of sphingomyelin by FRAP measurement using a standard confoca l microscope. Brain Res Protoc 11:46-51, 2003. 24. White J, Stelzer E: Photobleaching GFP reve als protein dynamics inside live cells. Trends Cell Biol 9:61-65, 1999. 25. Kruhlak M J, Lever MA, Fischle W, Verdin E, Bazett-Jones DP, Hendzel MJ: Reduced mobility of the a lternate splicing factor (ASF) through the nucleoplasm and steady state speckle compartments. J Cell Biol 150:41-51, 2000. 26. Heyduk T: Measuring protein conformational changes by FRET/LRET. Curr Opin Biotechnol 13:292-296, 2002.

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27. Tsien RY: The gree n fluorescent protein. Annu Rev Biochem 67:509-544, 1998. 28. van Roe ssel P, Brand AH: Imaging into the future: visualizing gene expression and protein interactions with fluorescent proteins. Nat Cell Biol 4:E15-E20, 2002. 29. Chang HC, Tsai SY, Wu GJ, Lin YH, Chen RM, Chen TL: Effects of propofol on mitochondrial function and intrace llular ca lcium shift in bovine aortic endothelial model. Acta Anaesthesiol Sin 39:115-122, 2001. 30. Tai YT, Wu CC, Wu GJ, Chang HC, Chen TG, Chen RM , Chen TL: Study of propofol in bovine aortic endothelium: I. Inhibitory effect on bradykinin-induced intracellular calcium immobilization. Acta Anaesthesiol Sin 38:181-186, 2000. 31. M asters BR, Bohnke M: Three -dimensional confocal microscopy of the living human eye. Ann Review Biomed Engineering 4:69-91, 2002. 32. Abramovits W, Goldstein AM, Gonzalez S: Confocal microscopy oriented cryosurge ry. Int J Dermatol 41:284-285, 2002. 33. Selkin B, Rajadhyaksha M, Gonzalez S, Langley RG: In vivo confocal microscopy in dermatology. Dermatol Clin 19:369377, 2001. 34. Delaney PM, King RG, Lambert JR, Harris MR: Fibre optic confocal imaging (FOCI) for subsurface microscopy of the colon in vivo. J Anat 184:157-160, 1994. 35. Papworth GD, Delaney PM, Bussau LJ, Vo LT, King RG: In vivo fibre optic confocal ima ging of microvasculature and nerves in the rat vas deferens and colon. J Anat 192:489-495, 1998. 36. Krohne I, Pfeifer T, Zacher M, Depiereux F, Stepp H: New concept for the development of a confocal endomicroscope. Biomed Tech 47:206-208, 2002.

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共軛焦雷射掃瞄顯微鏡於生物醫學研究 之 原 理 與 應 用 (I) 陳 睿 泰，

陳 瑞 明，

林 鈺 樺，

林 怡 伶，

陳 大 樑，

張 懷 嘉

陳 廷 貴

共 軛焦顯微鏡是利用 一般點光源或雷射 ，在光學基礎上採 用光源焦平面與觀 察者焦平面共軛 成 相的原理，並利用電 腦對所掃描分析 的對象進行數位圖 像處理的觀察和分 析系統。與傳統 光 學顯微鏡相較，共 軛焦顯微鏡具有許 多優點，如較高的 水平解析度、能提 供組織深部清晰 的 二維影像、可依時 序擷取細胞內部動 態變化、並利用電 腦分析可重建三維 影像。配合多種 螢 光染色的技術，使得 共軛焦顯微鏡在 生物細胞及藥學界 被廣泛使用。臨床 上共軛焦顯微鏡 亦 可應用在對皮膚、 角膜、水晶體、視網 膜等的觀察。與內 視鏡配合使用也 獲得初步成功的 結 果。未來共軛焦顯微 鏡的發展可能朝 向應用在活組織上 觀察細胞的變化， 於生物醫學之研 究 ，包括麻醉醫學藥物作 用於各類細胞之研究， 提供一探討的嶄新途徑 。 關 鍵詞 ：共軛焦顯微鏡。

臺 北醫學大學醫 學院醫學系麻醉 學科暨市立萬 芳醫院麻醉科 。