2.2 Confocal Microscopy

Amos, Brad; McConnell, Gail; Wilson, Tony

doi:10.1016/b978-0-12-374920-8.00203-4

Cited by 26 publications

(27 citation statements)

References 25 publications

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“…660, Invitrogen P7220) provided an FWHM of 1.1 µm laterally and 6.2 µm axially. This compares to theoretical values of (0.51*λ) / ((√2)*NA) = 484 nm laterally and (0.64* λ) / (n-√(n2-NA2)) = 1.7 µm axially [21], [22]. Latex beads did not generate a suitable THG signal.…”

Section: Methodscontrasting

confidence: 66%

Label-Free 3D Visualization of Cellular and Tissue Structures in Intact Muscle with Second and Third Harmonic Generation Microscopy

et al. 2011

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Second and Third Harmonic Generation (SHG and THG) microscopy is based on optical effects which are induced by specific inherent physical properties of a specimen. As a multi-photon laser scanning approach which is not based on fluorescence it combines the advantages of a label-free technique with restriction of signal generation to the focal plane, thus allowing high resolution 3D reconstruction of image volumes without out-of-focus background several hundred micrometers deep into the tissue. While in mammalian soft tissues SHG is mostly restricted to collagen fibers and striated muscle myosin, THG is induced at a large variety of structures, since it is generated at interfaces such as refraction index changes within the focal volume of the excitation laser. Besides, colorants such as hemoglobin can cause resonance enhancement, leading to intense THG signals. We applied SHG and THG microscopy to murine (Mus musculus) muscles, an established model system for physiological research, to investigate their potential for label-free tissue imaging. In addition to collagen fibers and muscle fiber substructure, THG allowed us to visualize blood vessel walls and erythrocytes as well as white blood cells adhering to vessel walls, residing in or moving through the extravascular tissue. Moreover peripheral nerve fibers could be clearly identified. Structure down to the nuclear chromatin distribution was visualized in 3D and with more detail than obtainable by bright field microscopy. To our knowledge, most of these objects have not been visualized previously by THG or any label-free 3D approach. THG allows label-free microscopy with inherent optical sectioning and therefore may offer similar improvements compared to bright field microscopy as does confocal laser scanning microscopy compared to conventional fluorescence microscopy.

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Section: Methodscontrasting

confidence: 66%

Label-Free 3D Visualization of Cellular and Tissue Structures in Intact Muscle with Second and Third Harmonic Generation Microscopy

et al. 2011

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“…Fluorescence labelling combined confocal microscopy has become a powerful tool in daily use in bioscience laboratories throughout the world (see, e.g. Wilson & Sheppard, 1984; Wilson, 1990; Pawley, 1995; Diaspro, 2001; Amos et al , 2011). The main reason for the popularity of these instruments is that the confocal microscope, unlike its conventional counterpart, has the ability to image well only detail that arises from the regions of the specimen which lie close to the focal plane.…”

Section: Introductionmentioning

confidence: 99%

Resolution and optical sectioning in the confocal microscope

Wilson¹

2011

Journal of Microscopy

Self Cite

186

145

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SummaryIn this short review, we present a self-contained discussion of the image formation properties of the fluorescent confocal microscope. The optical sectioning or depth discrimination property is discussed in detail and new analytic formulae are presented, which relate the optical sectioning strength to the wavelength, numerical aperture and pinhole aperture size in a particularly simple fashion.

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“…As a result, aberrations do not negatively affect the axial resolution within uncertainty limits. In order to evaluate the HiLo microscope against state-of-the-art techniques, the axial resolution of classical confocal and widefield microscopy are calculated according to [28] …”

Section: Spatial Resolutionmentioning

confidence: 99%

Volumetric HiLo microscopy employing an electrically tunable lens

et al. 2016

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Electrically tunable lenses exhibit strong potential for fast motion-free axial scanning in a variety of microscopes. However, they also lead to a degradation of the achievable resolution because of aberrations and misalignment between illumination and detection optics that are induced by the scan itself. Additionally, the typically nonlinear relation between actuation voltage and axial displacement leads to over- or under-sampled frame acquisition in most microscopic techniques because of their static depth-of-field. To overcome these limitations, we present an Adaptive-Lens-High-and-Low-frequency (AL-HiLo) microscope that enables volumetric measurements employing an electrically tunable lens. By using speckle-patterned illumination, we ensure stability against aberrations of the electrically tunable lens. Its depth-of-field can be adjusted a-posteriori and hence enables to create flexible scans, which compensates for irregular axial measurement positions. The adaptive HiLo microscope provides an axial scanning range of 1 mm with an axial resolution of about 4 μm and sub-micron lateral resolution over the full scanning range. Proof of concept measurements at home-built specimens as well as zebrafish embryos with reporter gene-driven fluorescence in the thyroid gland are shown.

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2.2 Confocal Microscopy

Cited by 26 publications

References 25 publications

Label-Free 3D Visualization of Cellular and Tissue Structures in Intact Muscle with Second and Third Harmonic Generation Microscopy

Label-Free 3D Visualization of Cellular and Tissue Structures in Intact Muscle with Second and Third Harmonic Generation Microscopy

Resolution and optical sectioning in the confocal microscope

Volumetric HiLo microscopy employing an electrically tunable lens

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