Two-Photon Excitation Fluorescence Microscopy

Diaspro, Alberto; Schneider, Marc; Bianchini, Paolo; Caorsi, Valentina; Mazza, Davide; Pesce, Mattia; Testa, Ilaria; Vicidomini, Giuseppe; Usai, Cesare; Bos, A. van den

doi:10.1007/978-0-387-49762-4_11

Cited by 13 publications

(25 citation statements)

References 80 publications

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“…short and long) may exist for the fluorophore. Additionally, SHG signals can be distinguished from fluorophores with FLIM as SHG has no fluorescent lifetime proportional to the molecular cross-section and the square of excitation intensity [18,24]. It is this quadratic dependence on excitation intensity that is responsible for the optical sectioning effect, abrogating the need for a confocal aperture.…”

Section: A Brief Description Of Multiphoton-laser Scanning Microscopymentioning

confidence: 99%

“…Following multiphoton excitation the resulting fluorescence emission is produced at the same wavelength as the corresponding single photon excitation (half of the MPE excitation wavelength for the case of 2-photon excitation; see Fig. 2), but with less scattering and a quadratic dependence for fluorescent intensity, which can be described formally as [18,23,24]:…”

Section: A Brief Description Of Multiphoton-laser Scanning Microscopymentioning

confidence: 99%

“…emission collected before passing back to the scanning mirror along the original light path) is commonly used with multiphoton microscopy. Non-descanned detection in MPLSM is advantageous since it allows for more efficient collection References: PET [11,12]; whole body bioluminescence/fluorescence [5,9,11]; wide-field epifluorescence [15]; confocal microscopy [15,19]; multiphoton microscopy [15,18,24] Clin Exp Metastasis (2009) 26:357-370 359 of scattered photons emitted following deep tissue imaging. Use of a non-descanned light path at the bottom or side ports of the microscope gives the shortest possible light path with minimal optical components between the specimen and the photomultiplier tube (PMT) providing the most efficient collection of emitted light irrespective of the optical configuration of the scan head.…”

Section: A Brief Description Of Multiphoton-laser Scanning Microscopymentioning

confidence: 99%

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Multiphoton microscopy and fluorescence lifetime imaging microscopy (FLIM) to monitor metastasis and the tumor microenvironment

Provenzano

Eliceiri

Keely

2008

Clin Exp Metastasis

183

169

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Cancer metastasis involves complex cell behavior and interaction with the extracellular matrix by metabolically active cells. To observe invasion and metastasis with sub-cellular resolution in vivo, multiphoton microscopy (MPM) allows imaging more deeply into tissues with less toxicity, compared with other optical imaging methods. MPM can be combined with second harmonic generation (SHG), fluorescent lifetime imaging microscopy (FLIM), and spectral-lifetime imaging microscopy (SLIM). SHG facilitates imaging of stromal collagen and tumor-stroma interactions, including the architecture and remodeling of the tumor microenvironment. FLIM allows characterization of exogenous and endogenous fluorophores, such as the metabolites FAD and NADH to score for metabolic state and provide optical biomarkers. SLIM permits additional identification and separation of endogenous and exogenous fluorophores by simultaneously collecting their spectra and lifetime, producing an optical molecular "fingerprint". Both FLIM and SLIM also serve as an improved method for the assessment of Förster (or fluorescence) resonance energy transfer (FRET). Hence, the use and further development of these approaches strongly enhances the visualization and quantification of tumor progression, invasion, and metastasis. Herein, we review recent developments of multiphoton FLIM and SLIM to study 2D and 3D cell migration, invasion into the tumor microenvironment, and metastasis.

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Section: A Brief Description Of Multiphoton-laser Scanning Microscopymentioning

confidence: 99%

Section: A Brief Description Of Multiphoton-laser Scanning Microscopymentioning

confidence: 99%

Section: A Brief Description Of Multiphoton-laser Scanning Microscopymentioning

confidence: 99%

See 1 more Smart Citation

Multiphoton microscopy and fluorescence lifetime imaging microscopy (FLIM) to monitor metastasis and the tumor microenvironment

Provenzano

Eliceiri

Keely

2008

Clin Exp Metastasis

183

169

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“…where δ 2 is the two photon cross section for the fluorophore, η is the quantum yield of the fluorophore. P ave is the average power of the excitation beam, τ P is the pulse width of the excitation pulses, f P is the repetition rate of the laser, NA is the numerical aperture of the objective, h and c are Planck's constant and the speed of light respectively, and λ exc is the wavelength of the excitation light (Diaspro et al, 2006). In fact, the probability of two-photon absorption decreases as the fourth power of distance away from this focal region along the Z-axis (as can be seen by the NA dependence in Eqn.…”

Section: Two-photon Fluorescence Microscopy (Tpfm)mentioning

confidence: 99%

Fluorescence Microscopy: A Concise Guide to Current Imaging Methods

2017

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The field of fluorescence microscopy is rapidly growing and offers ever more imaging capabilities for biologists. Over the past decade, many new technologies and techniques have been developed that allow for combinations of deeper, faster, and higher resolution imaging. These have included the commercialization of many super‐resolution and light sheet fluorescence microscopy techniques. For the non‐expert, it can be difficult to match the best imaging techniques to biological questions. Picking the most appropriate imaging modality requires a basic understanding of the underlying physics governing each of them, as well as information comparing potentially competing imaging properties in the context of the sample to be imaged. To address these issues, we provide here concise descriptions of a wide range of commercially available imaging techniques from wide‐field to super‐resolution microscopy, and provide a tabular guide to aid in comparisons among them. In this manner we provide a concise guide to understanding and matching the correct imaging modality to meet research needs. © 2017 by John Wiley & Sons, Inc.

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“…In confocal microscopy, a fluorophore is excited by the absorption of one photon of relatively high energy in the visible or ultraviolet spectrum. Multiphoton excitation, however, is a nonlinear process in which a fluorophore is excited by two or more photons simultaneously of lower energy and longer wavelength in the infrared region [1]. Lower energy input, hence a reduced phototoxicity, and higher penetration of the excitation light are the distinct advantages over conventional LSM [2].…”

Section: Introductionmentioning

confidence: 99%

Depth profiling of gold nanoparticles and characterization of point spread functions in reconstructed and human skin using multiphoton microscopy

Hampel

Thude

Reutlinger

et al. 2011

Journal of Biophotonics

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Multiphoton microscopy has become popular in studying dermal nanoparticle penetration. This necessitates studying the imaging parameters of multiphoton microscopy in skin as an imaging medium, in terms of achievable detection depths and the resolution limit. This would simulate real-case scenarios rather than depending on theoretical values determined under ideal conditions. This study has focused on depth profiling of sub-resolution gold nanoparticles (AuNP) in reconstructed (fixed and unfixed) and human skin using multiphoton microscopy. Point spread functions (PSF) were determined for the used water-immersion objective of 63×/NA = 1.2. Factors such as skin-tissue compactness and the presence of wrinkles were found to deteriorate the accuracy of depth profiling. A broad range of AuNP detectable depths (20-100 μm) in reconstructed skin was observed. AuNP could only be detected up to ∼14 μm depth in human skin. Lateral (0.5 ± 0.1 μm) and axial (1.0 ± 0.3 μm) PSF in reconstructed and human specimens were determined. Skin cells and intercellular components didn't degrade the PSF with depth. In summary, the imaging parameters of multiphoton microscopy in skin and practical limitations encountered in tracking nanoparticle penetration using this approach were investigated.

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Two-Photon Excitation Fluorescence Microscopy

Cited by 13 publications

References 80 publications

Multiphoton microscopy and fluorescence lifetime imaging microscopy (FLIM) to monitor metastasis and the tumor microenvironment

Multiphoton microscopy and fluorescence lifetime imaging microscopy (FLIM) to monitor metastasis and the tumor microenvironment

Fluorescence Microscopy: A Concise Guide to Current Imaging Methods

Depth profiling of gold nanoparticles and characterization of point spread functions in reconstructed and human skin using multiphoton microscopy

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