Influence of optical properties on two-photon fluorescence imaging in turbid samples

Dunn, Andrew K.; Wallace, Vincent P.; Coleno, Mariah L.; Berns, Michael W.; Tromberg, Bruce J.

doi:10.1364/ao.39.001194

Cited by 182 publications

(179 citation statements)

References 18 publications

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“…Fields of view of 150, 200, or 300 m were scanned at 10 or 20 Hz using a combination of a resonant scanner (CRS-Series, GSI) and a galvanometric scanner (M-Series, 9 ϫ 20 mm, GSI). To improve the imaging depth while preserving a high optical axial sectioning, an effective numerical aperture (NA) smaller than the objective full NA was used (Dunn et al, 2000;Helmchen and Denk, 2005). The beam diameter at the objective (Olympus XLum 20ϫ, NA 0.95) back focal plane was adjusted to obtain a fill factor of its back aperture of ϳ0.7 and a resulting effective NA of ϳ0.65 (Helmchen and Denk, 2005).…”

Section: Methodsmentioning

confidence: 99%

Late Emergence of the Vibrissa Direction Selectivity Map in the Rat Barrel Cortex

Kremer

Léger²,

Goodman³

et al. 2011

Journal of Neuroscience

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In the neocortex, neuronal selectivities for multiple sensorimotor modalities are often distributed in topographical maps thought to emerge during a restricted period in early postnatal development. Rodent barrel cortex contains a somatotopic map for vibrissa identity, but the existence of maps representing other tactile features has not been clearly demonstrated. We addressed the issue of the existence in the rat cortex of an intrabarrel map for vibrissa movement direction using in vivo two-photon imaging. We discovered that the emergence of a direction map in rat barrel cortex occurs long after all known critical periods in the somatosensory system. This map is remarkably specific, taking a pinwheel-like form centered near the barrel center and aligned to the barrel cortex somatotopy. We suggest that this map may arise from intracortical mechanisms and demonstrate by simulation that the combination of spike-timing-dependent plasticity at synapses between layer 4 and layer 2/3 and realistic pad stimulation is sufficient to produce such a map. Its late emergence long after other classical maps suggests that experience-dependent map formation and refinement continue throughout adult life.

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Section: Methodsmentioning

confidence: 99%

Late Emergence of the Vibrissa Direction Selectivity Map in the Rat Barrel Cortex

Kremer

Léger²,

Goodman³

et al. 2011

Journal of Neuroscience

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“…aging because it offers label-free deep optical sectioning capabilities even inside optically turbid samples [4,5]. Non-linear optical processes play a crucial role in terms of achievable penetration depth [6][7][8], spatial resolution [9,10], and excitation capability [11][12][13].…”

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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“…The higher power (and longer wavelength) permits deeper optical penetration, particularly in turbid media such as cartilage. 25 Further, the two-photon excitation spectra of most flurophores are broader and flatter than their single photon counterparts. 8 The average power after the Ti:Sapphire laser is controlled using neutral density filters.…”

Section: Two-photon Excitation Laser Scanning Microscopymentioning

confidence: 99%

Two-Photon Excitation Laser Scanning Microscopy of Human, Porcine, and Rabbit Nasal Septal Cartilage

et al. 2001

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Two-photon excitation laser scanning microscopy (TPM) was used to image human, porcine, and rabbit nasal septal cartilage. TPM provides optical sections of thick tissue specimens in situ without the use of exogenous dyes or need for tissue fixation. The cartilage tissue was imaged using near-infrared light generated by a mode-locked titanium/sapphire laser that was raster-scanned and coupled to an inverted microscope. Absorption of two photons by endogenous molecules and subsequent fluorescence was filtered to specific spectral bandwidths and detected with photomultiplier tubes. Two-photon stimulated fluorescence was detected with photomultiplier tubes optimized to specific spectral bandwidths. Signal intensity corresponds to the concentration of fluorophores, principally NADH, NADPH, and flavoproteins hence providing a means of redox imaging the cellular metabolic state. Specimens were scanned from the surface to a depth of about 150 mm. Image size was 50 3 50 mm with a diffraction limited pixel size of 0.4 mm. Cell membranes, nuclei, and matrix structures were identified in human, pig, and rabbit tissues. TPM provides a means to study three dimensional chondrocyte structure and matrix organization in situ at substantial depths, and permits longitudinal examination of cultured tissue explants without the need for exogenous dyes, tissue preparation, or fixation.

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Influence of optical properties on two-photon fluorescence imaging in turbid samples

Cited by 182 publications

References 18 publications

Late Emergence of the Vibrissa Direction Selectivity Map in the Rat Barrel Cortex

Late Emergence of the Vibrissa Direction Selectivity Map in the Rat Barrel Cortex

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

Two-Photon Excitation Laser Scanning Microscopy of Human, Porcine, and Rabbit Nasal Septal Cartilage

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