High-Resolution Nonlinear Optical Imaging of Live Cells by Second Harmonic Generation

Campagnola, Paul J.; Wei, Meide; Lewis, Aaron; Loew, Leslie M.

doi:10.1016/s0006-3495(99)77165-1

Cited by 477 publications

(353 citation statements)

References 38 publications

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“…The image-forming signal in MPM arises from the simultaneous interaction of two or more photons with the sample. Two-photon interactions in MPM result in SHG and twophoton excited fluorescence (TPF) [12][13][14][15]. In TPF, two near-infrared (NIR) wavelength photons are simultaneously absorbed, resulting in the emission of a longer wavelength photon [16].…”

Section: Mpm and Second Harmonic Generation (Shg) Imagingmentioning

confidence: 99%

In vivo multiphoton fluorescence imaging: A novel approach to oral malignancy

et al. 2004

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Background and Objective: Current techniques for oral diagnosis require surgical biopsy of lesions, and may fail to detect early malignant change. Non-invasive, sensitive tools providing early detection of oral cancer and a better understanding of malignant change are needed. These studies evaluated in vivo multiphoton excited fluorescence (MPM) techniques to (1) map epithelial and subepithelial changes through out oral carcinogenesis and (2) serve as an effective diagnostic modality. Study Design/Materials and Methods: In the hamster model (n ¼ 70), epithelial and subepithelial change was imaged in vivo throughout carcinogenesis. MPM-and histopathology-based diagnoses on a scale of 0 (healthy)-6 (squamous cell carcinoma [s.c.c.]) were scored by two prestandardized investigators. Results: Collagen matrix and fibers, cellular infiltrates, blood vessels, and microtumors were clearly visible. MPM agreed with the histopathology for 88.6% of diagnoses. Conclusions: In vivo MPM images provide (1) high resolution information on specific components of the carcinogenesis process (2) an excellent basis for oral diagnostics.

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Section: Mpm and Second Harmonic Generation (Shg) Imagingmentioning

confidence: 99%

In vivo multiphoton fluorescence imaging: A novel approach to oral malignancy

et al. 2004

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“…Although second-harmonic generation (SHG) in biological tissues was first demonstrated two decades ago (6)(7)(8), SHG has only recently been used for biological imaging applications (9)(10)(11)(12). Because TPEF and SHG involve different contrast mechanisms, they can be used in tandem to provide complementary information regarding tissue structure and function.…”

mentioning

confidence: 99%

“…Recently, a combination of TPEF and SHG has been implemented for the study of cells stained with exogenous probes possessing large molecular anisotropy and second-order nonlinearity (10,13,(19)(20)(21). Combined TPEF͞SHG has been demonstrated for thin tissue sections (22), but not for the more practical case of thick, unstained living specimens because, although TPEF is measured in epi-illumination geometry, the forward propagating nature of the phase-coherent SHG signal seems to restrict SHG microscopy to a transmission mode of detection.…”

mentioning

confidence: 99%

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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“…The chirality of the staining dyes plays a significant role in contrast generation (46). Because SHG is a two photon process, higher spatial resolution is possible (since the signal is proportional to the laser intensity squared, the beam diameter is narrowed upon signal generation).…”

Section: Nonlinear Optical Spectroscopymentioning

confidence: 99%

The Physical Chemistry of Chirality

Hicks

2002

ACS Symposium Series

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High-Resolution Nonlinear Optical Imaging of Live Cells by Second Harmonic Generation

Cited by 477 publications

References 38 publications

In vivo multiphoton fluorescence imaging: A novel approach to oral malignancy

In vivo multiphoton fluorescence imaging: A novel approach to oral malignancy

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

The Physical Chemistry of Chirality

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