臺灣博碩士論文加值系統

在這篇論文中，我們建立了一套多模態雷射掃瞄顯微系統，並使用反射式共焦顯微術、二倍頻、三倍頻及雙光子螢光顯微術來作為影像模態。共焦的技術和非線性光學的效應都可以達到顯微鏡應用所需的光學切片特性。在結合一個工作在於生物穿透較深波段的近紅外光飛秒雷射光源和一個高數值孔徑的物鏡後，這個多模態雷射掃瞄顯微術可以提供超過１公厘的穿透深度及次微米的空間解析度並在生物樣本中有著較少的光破壞及光毒性現象。利用這個多模態雷射掃瞄顯微術我們可以在活體生物取得充足的影像資訊來幫助在生物學上的研究並且和鈦藍寶石雷射為主的技術比起來可以擁有較低程度的侵入性破壞。
這樣的顯微系統可以「放大」我們在組織學和發展生物學上的視野，這些我們在後面的章節會展示。作為一個充分發展的皮膚光學切片工具，正常皮膚的反射式共焦影像和傳統組織學的圖像是非常吻合的。將反射式共焦模態和倍頻的模態結合在一起，這個顯微鏡工具可以提供比傳統的共焦雷射掃瞄顯微術更豐富的的影像資訊並有著較好的空間解析度及多模態影像，這些功能對皮膚學上的研究是非常有幫助的。
利用反射式共焦顯微術和三倍頻顯微術所取得的人皮膚影像亦提供了非常有用的證據來證明當細胞的小胞器在細胞中的體積比例增加時，光在細胞中的散射機制會是由萊利散射所主導。這說明了反射共焦信號和三倍頻信號所提供的細胞影像亮區是包含了許多小胞器如粒腺體之類的細胞質影像。我們也發現了在皮膚深的信號強度衰減是由於焦平面的擴大所導致的，這也是為什麼在皮膚深層每個影像模態的解析度都會明顯變差的原因。
在利用基因轉殖技術的協助下，使用螢光蛋白標定特定組織這個方法在發展生物學上已經成為一個強而有力的工具，因為螢光蛋白質在空間上的表現可以顯現出帶有這個基因的表現型態。這樣的基因轉殖的螢光蛋白質再搭配雙光子螢光顯微術，我們可以藉其非線性光學的效應得到擁有高三維解析度的影像。有著基因轉殖技術的輔助及利用在於生物穿透較深波段的近紅外光飛秒雷射光源，我們結合了雙光子螢光顯微術和倍頻光學顯微術來取得生物組織中的分子影像、型態學及有結構的蛋白質資訊。包含了光學切片的特性、高穿透深度、及較少量的光破壞，這個多模態的方法可以提供極佳的顯影能力，對未來要動態的研究脊椎動物胚胎的發育會是一個非常有用的工具。
多模態雷射掃瞄顯微術所提供的反射式共焦信號和非線性光學信號，讓我們可以在活體生物中利用反射共焦信號和三倍頻信號取得型態學上的資訊、利用二倍頻信號取得有結構的蛋白質資訊並利用雙光子螢光信號取得分子影像的資訊。這樣的顯微鏡系統將可以解決未來特定生物學上的問題，並在臨床醫學上能夠有非常有用的幫助。

In this thesis, a multi-modality laser scanning microscopy system is build based on a modified optical scanning microscope and a Cr:forsterite laser. Reflection confocal, second harmonic generation (SHG), third harmonic generation (THG), and 2-photon fluorescence (2PF) have been used as imaging modalities. Both confocal techniques and nonlinear natures can achieve the optical sectioning property desirable for microscopy applications. Combined with the near-infrared femtosecond source working in the biological penetration window and a high numerical aperture (NA) objective, multi-modality laser scanning microscopy can offer >1-mm penetration depth and sub-micron spatial resolution in biological samples with much-reduced photodamage and phototoxicity. Multi-modality laser scanning microscopy can thus provide sufficient information deep inside a live biological specimen for biological studies less invasively than Ti:sapphire based techniques.
Such a microscopy system would “enlarge” the field of view on histology and developmental biology researches, as presented in the following chapter. As a well-developed optical biopsy tool, reflection confocal images of normal skin correlate very well with images from conventional histology. Combining reflection confocal imaging modality with SHG and THG imaging modalities, this microscopic tool can provide much more information than traditional confocal laser scanning microscopy with better spatial resolution and multiple imaging modalities that are very useful for dermatology studies.
The images in human skin taken from reflection confocal and THG microscopy provided very helpful evidences that the light scattering from cells is dominated by Rayleigh scattering when the volume fraction of organelles increases, which confirms the bright reflection confocal and THG signals from cytoplasm that containing multiple organelles such as mitochondria. The signal intensity decay in deeper parts of human skin was found to be mainly caused by the focal plane broadening which was also the reason of the resolution degradation of each imaging modality.
With the aid of transgenic techniques, specific tissues tagged by fluorescent protein have become a powerful tool in developmental biology studies because the spatial expression of that fluorescent protein enables to encapsulate the expression pattern of endogenous genes. Based on a 2PF microscope, images high 3-dimensional (3D) resolution can be obtained due to its nonlinear nature. We have combined 2PF microscopy with higher-harmonic optical microscopy based on the femtosceond Cr:forsterite laser with the aid of new transgenic lines tagged with HC-red fluorescent protein to obtain molecular, morphological, and structural protein information in biological tissues. With its optical sectioning property, high penetration depth, and much-reduced photodamages, this multi-modal method provides superb imaging capability for dynamic developmental studies of vertebrate embryos in the future.

謝誌 I
Abstract IV
摘要 VI
Contents VIII
Publication list X

Chapter 1. Introduction 1
1.1 General introduction to multi-modality laser scanning microscopy 1
1.2 Purpose of this thesis 9
Chapter 2. Basic principles 12
2.1 Basic concepts of microscopy resolution 12
2.2 Confocal microscopy 17
2.3 Nonlinear microscopy 20
2.3.1 Second harmonic generation (SHG) 22
2.3.2 Third harmonic generation (THG) 26
2.3.3 Two-photon fluorescence (2PF) 32
2.4 Resolution comparison 35
Chapter 3. Multi-modality laser scanning microscopy 37
3.1 Laser source selection 37
3.2 Multi-modality laser scanning microscopy setup 45
3.2.1 Multi-modality laser scanning microscopy with reflection confocal modality 45
3.2.2 Multi-modality laser scanning microscopy with 2PF modality 47
Chapter 4. Applications of multi-modality laser scanning microscopy 50
4.1 Preliminary studies of dermatology 51
4.1.1 Multi-modality laser scanning imaging in human skin 56
4.1.2 Optical properties in human skin 60
4.1.3 Discussion 71
4.2 In vivo studies of the heart development in zebrafish embryos 74
4.2.1 Plasmid construction and germ-line transmission of transgenic
Zebrafish 77
4.2.2 In vivo observation of the zebrafish embryos 79
4.2.3 Two-photon fluorescent signal from zebrafish line tagged by far-Red
81
4.2.4 Heart images of live zebrafish embryos 85
4.2.5 Three-dimensional heart imaging of zebrafish embryos 89
4.2.6 Discussion 91
Chapter 5. Summary 93
Reference 98

Anderson RR, Parish JA (1981) The optics of human skin. J Invest Dermat 77:13
Armstrong JA, Bloembergen N, Ducuing J, Pershan PS (1962) Interactions between light
waves in a nonlinear dielectric. Phys Rev 127:1918
Barad Y, Eisenberg H, Horowitz M, Silberberg Y (1997) Nonlinear scanning
laser microscopy by third harmonic generation. Appl Phys Lett 70:922
Beyer H (1985) Handbuch der Mikroskopie, 2nd Edition, VEB Verlag Technik Berlin, DE
Birge RR (1983) in Ultrasensitive Laser Spectroscopy, ed. D. S. Kliger, Academic, NY, USA
Blab GA, Lommerse PHM, Cognet L, Harms GS, Schmidt T (2001) Two-photon excitation
action cross-section of the autofluorescent proteins. Chem. Phys. Lett. 350: 71
Born M, Wolf E (1999) Principle of Optics, 7th ed., Cambridge University Press, Cambridge,
UK
Bouma BE, Tearney GJ, Bilinsky IP, Golubovic B, Fujimoto JG (1996) Self phase-modulated
Kerr-lens mode locked Cr:forsterite laser source for optical coherence tomography. Opt Lett
21:1839
Boyd RW (1992) Nonlinear Optics. Academic Press, San Diego, CA, USA
Campagnola PJ, Wei MD, Lewis A, Loew LM (1999) High-resolution nonlinear optical
imaging of live cells by second harmonic generation. Biophys J 77:3341
Campagnola PJ, Millard AC, Terasaki M, Hoppe PE, Malone CJ, Mohler WA (2002)
Three-dimensional high-resolution second-harmonic imaging of endogenous protein
structural proteins in biological tissues. Biophy J 82:493
Canioni L, Rivet S, Sarger L, Barille R, Vacher P, Viosin P (2001) Imaging of Ca2+
intracellular dynamics with a third-harmonic generation microscope. Opt Lett 26:515
Chen IH, Chu SW, Sun CK, Cheng PC, Lin BL (2002) Wavelength dependent cell damages
in multi-photon confocal microscopy. Opt Quan Electron 34:1251
Chen IH, Chu SW, Bresson F, Tien MC, Shi JW, Sun CK (2003) Three-dimensional electric
field visualization utilizing electric-field-induced-second-harmonic-generation in nematic
99
liquid crystals. Opt Lett 28:1338
Cheng JX, Xie XS (2002) Green’s function formulation for third-harmonic generation
microscopy. J Opt Soc Am 19:1604
Cheng PC, Pan SJ, Shih A, Kim KS, Liou WS, Park MS (1998) Highly efficient upconverters
for multiphoton fluorescence microscopy. J Microscopy 189:199
Cheng PC, Lin BL, Kao FJ, Gu M, Xu MG, Gan X, Huang MK, Wang YS (2001)
Multi-photon fluorescence microscopy – the response of plant cells to high intensity
illumination.Micron 32:661
Chou CY, Horng LS, Tsai HJ (2001) Uniform GFP expression in transgenic medaka (Oryzias
latipes) at the F0 generation. Transgenic Res. 10:303
Chu SW, Chen IH, Liu TM, Cheng PC, Sun CK, Lin BL (2001) Multimodal nonlinear
spectral microscopy based on a femtosecond Cr:forsterite laser. Opt Lett 26:1909
Chu SW, Chen IS, Liu TM, Sun CK, Lee SP, Lin BL, Cheng PC, Kuo MX, Lin DJ, Liu HL
(2002) Nonlinear bio-photonic crystal effects revealed with multi-modal nonlinear
microscopy. J Microscopy 208:190
Chu SW, Liu TM, Sun CK, Lin CY, Tsai HJ (2003A) Real-time second-harmonic-generation
microscopy based on a 2-GHZ repetition rate Ti: sapphire laser. Opt Express 11:933
Chu SW, Chen SY, Tsai TH, Liu TM, Lin CY, Tsai HJ, Sun CK (2003B) In vivo
developmental biology study using noninvasive multi-harmonic generation microscopy. Opt
Express 11:3093
Chu SW, Chen SY, Chern GW, Tsai TH, Chen YC, Lin BL, Sun CK (2004) Studies of χ(2)/ χ(3)
tensors in submicron-scaled bio-tissues by polarization harmonics optical microscopy.
Biophys J 86:1
Chung VQ, Dwyer PJ, Nehal KS, Rajadhyaksha M, Menaker GM, Charles C, Jiang SB (2004)
Use of ex vivo confocal scanning laser microscopy during Mohs surgery for nonmelanoma
skin cancers. Dermatol Surg 30:1470-1478
Denk W, Strickler JH, Webb WW (1990) Two-photon laser scanning fluorescence microscopy.
Science 248:73
Dombeck DA, Kasischke KA, Vishwasrao HD, Hyman BT, Webb WW (2003) Uniform
100
polarity microtubule assemblies imaged in native brain tissue by second-harmonic generation
microscopy. Proc Natl Acad Sci USA 100:7081
Dombeck DA, Blanchard-Desce M, Webb WW (2004) Optical recording of action potentials
with second-harmonic generation microscopy. J Neurosci 24:999
Dunn A, Richards-Kortum R (1996) Three-dimensional computation of light scattering from
cells, IEEE J Sel Top Quant 2:898
Elias H, Pauly JE, Burns ER (1978) Histology and Human Microanatomy, 4th Edition, Piccin
Medical Books, Padova, Italy
Franken PA, Hill AE, Peters CW, Weinreich G (1961) Generation of optical harmonics. Phys
Rev Lett 7:118
Freund I, Deutsch M (1986A) 2nd harmonic microscopy of biological tissue. Opt Lett 11:94
Freund I, Deutsch M, Sprecher A (1986B) Connective tissue polarity. Optical
second-harmonic microscopy, crossed- beam summation, and small-angle scattering in
rat-tail tendon. Biophys J 50:693
Gannaway JN, Sheppard CJR (1978) Second harmonic imaging in the scanning optical
microscope. Opt Quantum Electron 10:435
González S, Tannous Z (2002) Real-time, in vivo confocal reflectance microscopy of basal
cell carcinoma. J Am Acad Dermatol 47:869
Goldgeier M, Fox CA, Zavislan JM, Harris D, Gonzalez S (2003) Noninvasive imaging,
treatment, and microscopic confirmation of clearance of basal cell carcinoma. Dermatol Surg
29:205
Göppert –Mayer M (1931) Ueber Elementarakte mit zwei Quanenspruengen. Ann Phys 9:273
Guo Y, Ho PP, Tirksliunas A, Liu F, Alfano RR (1996) Optical harmonic generation from
animal tissues by the use of picosecond and femtosecond laser pulses. Appl Opt 35:6810
Guo Y, Ho PP, Savage H, Harris D, Sacks P, Schantz S, Liu F, Zhadin N, Alfano RR (1997)
Second-harmonic tomography of tissues. Opt Lett 22:1323
Gurskaya NG, Fradkov AF, Terskikh A, Matz MV, Labas YA, Martynov VI, Yanushevich YG,
Lukyanov KA, Lukyanov SA (2001) GFP-like chromoproteins as a source of far-red
101
fluorescent proteins. FEBS Lett. 507:16
Haus HA (1984) Waves and fields in optoelectronics, Prentice-Hall Inc., Englewood Cliffs,
New Jersey.
Helmchen F, Waters J (2002) Ca2+ imaging in the mammalian brain in vivo. Eur J Pharmacol
447:119
Hsiao CD, Hsieh FJ, Tsai HJ (2001) Enhanced expression and stable transmission of
transgenes flanked by inverted terminal repeats from adeno- associated virus in zebrafish.
Dev. Dynam. 220:323
Huang D, Swanson EA, Lin CP, Cschuman JS, Stinson WG, Chang W, Hee MR, Flotte T,
Gregory K, Puliafito CA, Fujimoto JG (1991) Optical coherence tomography. Science
254:1178
Huang CJ, Tu CT, Hsiao CD, Hsieh FJ, Tsai HJ (2003) Germ-Line Transmission of a
Myocardium-Specific GFP Transgene Reveals Critical Regulatory Elements in the Caradiac
Myosin Light Chain 2 Promoter of Zebrafish. Dev. Dynam. 228:30
Jacques SL, McAuliffe DJ, Blank IH, Parish JA (1987) Controlled removal of human stratum
corneum by pulsed laser. J Invest Dermatol 88:88
Kaiser W, Garrett CGB(1961) Two-Photon Excitation in CaF2: Eu2+. Phys Rev Lett 7:229
Kogelnik H, Li T (1966) Laser beams and resonators. Appl Opt 5:1550
König K, So PTC, Mantulin WW, Gratton E (1997) Cellular response to near-infrared
femtosecond laser pulses in two-photon microscopes. Opt Lett 22:135
König K, Riemann I, Fritzsche W (2001) Nanodissection of human chromosomes with
near-infrared femtosecond laser pulses. Opt Lett 26:819
Lin BL, Cheng PC, Sun CK (2001) Absorption and multiphoton excited fluorescent
properties of maize tissues. Maize Genetics Cooperation News Letters 75:61
Liu TM, Chu SW, Sun CK, Lin BL, Cheng PC, Johnson I (2001) Multi-photon confocal
microscopy using a femtosecond Cr:forsterite laser. Scanning 23:249
Maiman TH (1960) Stimulated optical radiation in ruby lasers. Nature 187:493
Masters BR, So PTC (2001) Confocal microscopy and two-photon excitation microscopy of
102
human skin in vivo. Optics Express 8:2
McDonald DM, Choyke PL (2003) Imaging of angiogenesis: From microscope to clinic. Nat
Med 9:713
Mertz J, Moreaux L (2001) Second harmonic generation by focused excitation of
inhomogeneously distributed scatterers. Opt Commun 196:325
Millard AC, Jin L, Lewis A, Loew LM (2003) Direct measurement of the voltage sensitivity
of second-harmonic generation from a membrane dye in patch-clamped cells. Opt. Lett.
28:1221
Minsky M (1957) Microscopy apparatus. U.S. Patent 03013467.
Moreaux L, Sandre O, Mertz J (2000A) Membrane imaging by second-harmonic generation
microscopy. J Opt Soc Am B 17:1685
Moreaux L, Sandre O, Blanchard-Desce M, Mertz J (2000B) Membrane imaging by
simultaneous second-harmonic and two-photon microscopy. Opt Lett 25:320
Moreaux L, Sandre O, Charpak S, Blanchard–Desce M, Mertz J (2001) Coherent scattering
in multi-harmonic light microscopy. Biophys J 80:1568
Mohler W, Millard AC, Campagnola PJ (2003) Second harmonic generation imaging of
endogenous structural proteins. Methods 29:97
Müller M, Squier J, Wilson KR, Brakenhoff GJ (1998) 3D-microscopy of transparent objects
using third-harmonic generation. J Microscopy 191:266
Peleg G, Lewis A, Linial M, Loew LM (1999) Non-linear optical measurement of membrane
potential around single molecules at selected cellular sites. Proc Natl Acad Sci USA 96:6700
Piston DW, Masters BR, Webb WW (1994) Three-dimensionally resolved NAD(P)H cellular
metabolic redox imaging of the in situ cornea with two-photon excitation laser scanning
microscopy. J Microsc 178:20
Pluta M (1988) Advanced Light Microscopy, Elsevier, Amsterdam, NL
Povazay B, Bizheva K, Unterhuber A, Hermann B, Sattmann H, Fercher AF, Drexler W,
Apolonski A, Wadsworth WJ, Knight JC, Russell P St J, Vetterlein M, Scherzer E (2002)
Submicrometer axial resolution optical coherence tomography. Opt Lett 27:1800
103
Prasad PN (2003) Introduction to Biophotonics, John Wiley & Sons, Hoboken, NJ, USA
Rajadhyaksha M, Grossman M, Esterowitz D, Webb RH, Anderson RR (1995) In vivo
confocal scanning laser microscopy of human skin: melanin provides strong contrast, J Invest
Dermatol 104:946
Rajadhyaksha M, González S, Zavislan JM, Anderson RR, Webb RH (1999) In vivo confocal
scanning laser microscopy of human skin II: Advances in instrumentation and comparison
with histology. J Invest Dermatol 113:293-303
Saidi I, Jacques S, Kittel F (1995) Mie and Rayleigh modeling of visible-light scattering in
neonatal skin. Appl Opt 34:7410
Sauter EG (1996) Nonlinear optics, John Wiley & Sons, Hoboken, NJ, USA
Schiebener P, Straub J, Levelt Sengers JMH, Gallagher JS (1990) Refractive index of water
and steam as function of wavelength, temperature and density. J Phys Chem Ref Data 19:677
Schönle A, Hell SW (1998) Heating by absorption in the focus of an objective lens. Opt. Lett.
23:325
Selkin B, Rajadhyaksha M, González S, Langley RG (2001) In vivo confocal microscopy in
dermatology. Dermatol Clin 19369–77:ix–x
Shen YR (1989) Surface properties probed by second-harmonic and sum-frequency
generation. Nature 337:519
Shen YR (2002) The Principles of Nonlinear Optics, John Wiley & Sons, Hoboken, NJ, USA
Sheppard CJR, Shotton DM (1997) Confocal Laser Scanning Microscopy, BIOS Scientific
Publisher, Oxford, UK
Shreve AP, Trautmen JK, Owens TG, Albrecht AC (1990) Two-photon excitation
spectroscopy of thylakoid mem- branes from phaeodactylum tricornutum: evidence for an in
vivo two-photon allowed carotenoid state. Chem Phys Lett 170:51
Singh S, Bradley LT (1964) Three-photon absorption in napthalene crystals by laser
excitation. Phys Rev Lett 12:612
So PTC, Kim H, Kochevar IE (1998) Two-photon deep tissue ex vivo imaging of mouse
dermal and subcutaneous structures. Optics Express 3:339
104
Squier JA, Müller M, Brakenhoff GJ, Wilson KR (1998) Third harmonic generation
microscopy. Opt Express 3:315
Squirell JM, Wokosin DL, White JG, Bavister BD (1999) Long-term two-photon fluorescence
imaging of mammalian embryos without compromising viability. Nat Biotechnol 17:763
Squier J, Müller M (2001) High resolution nonlinear microscopy: A review of sources and
methods for achieving optimal imaging. Rev Sci Instrum 72: 2855
Stainier DYR (2001) Zebrafish genitics and vertebrate heart formation. Nat. Rev. Genet. 2:39
Sun CK, Chu SW, Tai SP, Keller S, Mishra UK, Denbaars SP (2000) Mapping
piezoelectric-field distribution in gallium nitride with scanning second-harmonic generation
microscopy. Appl Phys Lett 77; 2331
Sun CK, Chu SW, Tai SP, Keller S, Abare A, Mishra UK, Denbaars SP (2001) Mapping
piezoelectric-field distribution in gallium nitride with scanning second-harmonic generation
microscopy. Scanning 23:182
Sun CK, Chen CC, Chu SW, Tsai TH, Chen YC, Lin BL (2003) Multi-harmonic generation
biopsy of skin. Opt Lett 28:2488
Sun CK, Chu SW, Chen SY, Tsai TH, Liu TM, Lin CY, Tsai HJ (2004) Higher harmonic
generation microscopy for developmental biology. J Struct Bio 147:19
Tannous Z, Torres A, González S (2003) In vivo real-time confocal reflectance microscopy: a
noninvasive guide for Mohs micrographic surgery facilitated by aluminum chloride, an
excellent contrast enhancer. Dermatol Surg 29:839
Tearney GJ, Brezinski ME, Bouma BE, Boppart SA, Pitris C, Southern JF, Fujimoto JG
(1997) In vivo endoscopic optical biopsy with optical coherence tomography. Science
276:2037
Tirlapur UK, König K, Peuckert C, Krieg R, Halbhuber JJ (2001) Femtosecond near-infrared
laser pulses elicit generation of reactive oxygen species in mammalian cells leading to
apoptosis-like death. Exp Cell Research 263:88
Tirlapur UK, König K (2002) Femtosecond near-infrared laser pulses as a versatile
non-invasive tool for intra-tissue nanoprocessing in plants without compromising viability.
Plant J 31:365
105
Tsang TYF (1995) Optical third-harmonic generation at interfaces. Phys Rev A 52:4116
Udvadia AJ, Linney E (2003) Windows into development: historic, current, and future
perspectives on transgenic zebrafish. Dev. Biol. 256:1
Veiro JA, Cummins PG (1994) Imaging of skin epidermis from various origins using
confocal laser scanning microscopy. Dermatology 189:16
Vogel A, Noack J, Hüttmann G, Paltauf G (2002) Femtosecond-laser-produced low-density
plasmas in transparent biological media: A tool for the creation of chemical, thermal and
thermomechanical effects below the optical breakdown threshold,” Proc. SPIE 4633A:1
Ward JF, New GHC (1969) Generation of high-order harmonics from solid surfaces by
intense femtosecond laser pulses. Phys. Rev 185:57
Watanabe W, Arakawa N, Matsnaga S, Higashi T, Fukui K, Isobe K, Itoh K (2004)
Femtosecond laser disruption of subcellular organelles in a living cell. Opt Express 12:4203
Yamashita T, Kuwahara T, Gunzales S, Takahashi M (2005) Non-invasive visualization of
melanin and melanocytes by reflectance-mode confocal microscopy. J Invest Dermotal
124:235
Yanik MF, Cinar H, Cinar HN, Chisholm AD, Jin Y, Bem-Yakar A (2004) Functional
regeneration after laser axotomy. Nature 432:822
Yasuno Y, Makita S, Sutoh Y, Itoh M, Yatagai T (2002) Birefringence imaging of human skin
by polarization-sensitive spectral interferometric optical coherence tomography. Opt Lett
27:1803
Yelin D, Silberberg (1999) Laser scanning third-harmonic-generation microscopy in biology.
Opt Express 5:169
Yelin D, Silberberg Y, Barad Y, Patel JS (1999B) Depth-resolved imaging of nematic liquid
crystals by third harmonic microscopy. Appl Phys Lett 74:3107
Yelin D, Oron D, Korkotian E, Segal M, Silberberg Y (2002) Third-harmonic microscopy
with a titanium-sapphire laser. Appl Phys B 74:S97
Yelin D, Oron D, Thiberge S, Moses E, Silberberg Y (2003) Multiphoton plasmon-resonance
microscopy. Opt Express 11:1385
106
Yuste R, Denk WW (1995) Dendritic spines as basic functional units of neuronal integration.
Nature 375:682
Zoumi A, Yeh A, Tromberg BJ (2002) Imaging cells and extracellular matrix in vivo by using
second-harmonic generation and two-photon excited fluorescence. Proc Natl Acad Sci USA
99:11014
Zipfel WR, Williams RM, Christie R, Nikitin AY, Hyman BT, Webb WW (2003B) Live
tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and
second harmonic generation. Proc Natl Acad Sci USA 100:7075
“BD Living Colors HcRed,” Clontechniques 17, 12-13 (2002).