Second-harmonic tomography of tissues

Guo, Yici; Ho, P. P.; Savage, Howard E.; Harris, D.J.; Sacks, Peter G.; Schantz, Stimson P.; Liang, Feng; Zhadin, N.; Alfano, R. R.

doi:10.1364/ol.22.001323

Cited by 151 publications

(100 citation statements)

References 9 publications

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“…The transmitted SHG signals from various depths, z, exhibit interference described by the factor sin 2 (2 ⌬nL͞ ex ), where ⌬n is the dispersion of the RAFT, and ex is the fundamental wavelength (30). The distance L represents the sample scattering length, l scat , because, due to the coherence of the SHG process, the measured SHG intensity is determined by ballistic photons that carry the coherent information from the medium (28). Thus, l scat represents the mean distance through which coherently interfering SHG waves can travel before scattering.…”

Section: Resultsmentioning

confidence: 99%

“…However, the maximum imaging depth reached here is only limited by the working distance of the microscope objective and the low value of excitation power used (approximately 5 mW at the sample site). Previous studies using SHG imaging in reflection geometry to obtain structural information deep inside tissue (28,29) are characterized by long acquisition times (0.5-2.8 h) and poor resolution even for excitation powers as high as 80 mW at the sample (29).…”

Section: High-resolution Reflectance Imaging Deep Inside Tissuementioning

confidence: 99%

“…In fact, under certain conditions, a significant amount of backscattering second-harmonic light can be generated at the interfaces between nonlinear and linear media (23)(24)(25)(26)(27). Reflected SHG signals provided the basis for the development of the first in vivo SHG tissue tomography (28,29). The results obtained from this approach, however, were characterized by relatively poor resolution, lack of structural detail, and prohibitively long acquisition times (0.5-2.8 h).…”

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Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence

Zoumi

Yeh²,

Tromberg³

2002

Proc. Natl. Acad. Sci. U.S.A.

809

725

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Multiphoton microscopy relies on nonlinear light-matter interactions to provide contrast and optical sectioning capability for high-resolution imaging. Most multiphoton microscopy studies in biological systems have relied on two-photon excited fluorescence (TPEF) to produce images. With increasing applications of multiphoton microscopy to thick-tissue ''intravital'' imaging, second-harmonic generation (SHG) from structural proteins has emerged as a potentially important new contrast mechanism. However, SHG is typically detected in transmission mode, thus limiting TPEF͞SHG coregistration and its practical utility for in vivo thick-tissue applications. In this study, we use a broad range of excitation wavelengths (730 -880 nm) to demonstrate that TPEF͞SHG coregistration can easily be achieved in unstained tissues by using a simple backscattering geometry. The combined TPEF͞SHG technique was applied to imaging a threedimensional organotypic tissue model (RAFT). The structural and molecular origin of the image-forming signal from the various tissue constituents was determined by simultaneous spectroscopic measurements and confirming immunofluorescence staining. Our results show that at shorter excitation wavelengths (<800 nm), the signal emitted from the extracellular matrix (ECM) is a combination of SHG and TPEF from collagen, whereas at longer excitation wavelengths the ECM signal is exclusively due to SHG. Endogenous cellular signals are consistent with TPEF spectra of cofactors NAD(P)H and FAD at all excitation wavelengths. The reflected SHG intensity follows a quadratic dependence on the excitation power, decays exponentially with depth, and exhibits a spectral dependence in accordance with previous theoretical studies. The use of SHG and TPEF in combination provides complementary information that allows noninvasive, spatially localized in vivo characterization of cell-ECM interactions in unstained thick tissues. S ince its introduction by Denk et al.(1), two-photon excited fluorescence (TPEF) has been widely used for imaging structure and dynamic interactions in biological tissues (2-5). Although second-harmonic generation (SHG) in biological tissues was first demonstrated two decades ago (6-8), SHG has only recently been used for biological imaging applications (9-12). Because TPEF and SHG involve different contrast mechanisms, they can be used in tandem to provide complementary information regarding tissue structure and function. Specifically, SHG signals depend on the orientation, polarization, and local symmetry properties of chiral molecules, whereas TPEF results from the nonlinear excitation of molecular fluorescence.The primary tissue constituent responsible for SHG is collagen (9,13,14), which is also a well documented source of tissue autofluorescence (4,15). This fact has created uncertainty over whether the image-forming signal from two-photon excitation of collagen in biological tissues is TPEF or SHG and prompts further investigation into the precise origin of signal in the nonlinear microscopy of...

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Section: Resultsmentioning

confidence: 99%

Section: High-resolution Reflectance Imaging Deep Inside Tissuementioning

confidence: 99%

mentioning

confidence: 99%

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Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence

Zoumi

Yeh²,

Tromberg³

2002

Proc. Natl. Acad. Sci. U.S.A.

809

725

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“…SHG has already been largely used for imaging non-centrosymmetric molecules inside cells [62,63] and tissues [64]. In particular, SHG microscopy has been demonstrated extremely powerful to image collagen rich tissues [65] such as cornea [66,67], tendon [68,69], and arteries [70].…”

Section: Introductionmentioning

confidence: 99%

From molecular structure to tissue architecture: collagen organization probed by SHG microscopy

Cicchi

Vogler

Kapsokalyvas

et al. 2012

Journal of Biophotonics

171

132

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Second‐harmonic generation (SHG) microscopy is a fantastic tool for imaging collagen and probing its hierarchical organization from molecular scale up to tissue architectural level. In fact, SHG combines the advantages of a non‐linear microscopy approach with a coherent modality able to probe molecular organization. In this manuscript we review the physical concepts describing SHG from collagen, highlighting how this optical process allows to probe structures ranging from molecular sizes to tissue architecture, through image pattern analysis and scoring methods. Starting from the description of the most relevant approaches employing SHG polarization anisotropy and forward – backward SHG detection, we then focus on the most relevant methods for imaging and characterizing collagen organization in tissues through image pattern analysis methods, highlighting advantages and limitations of the methods applied to tissue imaging and to potential clinical applications. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

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“…SHG is a coherent second-order process, where two photons add together to give a photon at exactly half the wavelength, without any absorption by the medium (10). SHG microscopy (11)(12)(13)(14) and 2PEF microscopy possess similar advantages for imaging within tissues, but provide different informations. In particular, SHG scales as the square of the density of harmonophores, and nonzero macroscopic SHG can be obtained only for noncentrosymmetric organizations.…”

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confidence: 99%

Micrometer scale Ex Vivo multiphoton imaging of unstained arterial wall structure

Boulesteix

Pena

Pagès³

et al. 2005

Cytometry Pt A

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Background: We characterize the application of multiphoton microscopy to the observation of the extracellular matrix of fresh unstained vessels. Method: Combined two-photon-excited fluorescence (2PEF) and second harmonic generation (SHG) imaging of large arteries reveals the architecture of elastin and collagen fibers in the vessel wall with remarkable specificity.Results: We present elastin/collagen imaging in unstained rat vessels at both micrometer and whole vessel scales, and we characterize the optical properties of rat

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Second-harmonic tomography of tissues

Cited by 151 publications

References 9 publications

Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence

Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence

From molecular structure to tissue architecture: collagen organization probed by SHG microscopy

Micrometer scale Ex Vivo multiphoton imaging of unstained arterial wall structure

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