Three-dimensional Coherent Transfer Function in Reflection-mode Confocal Microscopy Using Annular Lenses

Cryo-electron microscopy combined with single-particle reconstruction techniques has allowed us to form a three-dimensional image of the Escherichia coli ribosome.In the interior, we observe strong density variations which may be attributed to the difference in scattering density between ribosomal RNA (rRNA) and protein. This identification can only be tentative, and lacks quantitation at this stage, because of the nature of image formation by bright field phase contrast. Apart from limiting the resolution, the contrast transfer function acts as a high-pass filter which produces edge enhancement effects that can explain at least part of the observed variations. As a step toward a more quantitative analysis, it is necessary to correct the transfer function in the low-spatial-frequency range. Unfortunately, it is in that range where Fourier components unrelated to elastic bright-field imaging are found, and a Wiener-filter type restoration would lead to incorrect results. Depending upon the thickness of the ice layer, a varying contribution to the Fourier components in the low-spatial-frequency range originates from an “inelastic dark field” image. The only prospect to obtain quantitatively interpretable images (i.e., which would allow discrimination between rRNA and protein by application of a density threshold set to the average RNA scattering density may therefore lie in the use of energy-filtering microscopes.

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Laser Scanning Confocal Microscopy and Three-Dimesnsional Reconstruction of the Mitotic Spindles of Unequally Dividing Embryonic Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100158376 ◽

1990 ◽

Vol 48 (3) ◽

pp. 166-167

Author(s):

J. Holy ◽

G. Schatten

Keyword(s):

Confocal Microscopy ◽

Mitotic Spindle ◽

Laser Scanning ◽

Three Dimensional ◽

Laser Scanning Confocal Microscopy ◽

Two Dimensions ◽

Embryonic Cells ◽

Mitotic Spindles ◽

Cleavage Division ◽

Scanning Confocal Microscopy

One of the classic limitations of light microscopy has been the fact that three dimensional biological events could only be visualized in two dimensions. Recently, this shortcoming has been overcome by combining the technologies of laser scanning confocal microscopy (LSCM) and computer processing of microscopical data by volume rendering methods. We have employed these techniques to examine morphogenetic events characterizing early development of sea urchin embryos. Specifically, the fourth cleavage division was examined because it is at this point that the first morphological signs of cell differentiation appear, manifested in the production of macromeres and micromeres by unequally dividing vegetal blastomeres.The mitotic spindle within vegetal blastomeres undergoing unequal cleavage are highly polarized and develop specialized, flattened asters toward the micromere pole. In order to reconstruct the three-dimensional features of these spindles, both isolated spindles and intact, extracted embryos were fluorescently labeled with antibodies directed against either centrosomes or tubulin.

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Two-Photon Excitation Imaging of Glucose Metabolism in Living Tissue

Microscopy and Microanalysis ◽

10.1017/s1431927600008412 ◽

1997 ◽

Vol 3 (S2) ◽

pp. 305-306

Author(s):

David W. Piston

Keyword(s):

Confocal Microscopy ◽

Collection Efficiency ◽

Three Dimensional ◽

Spatial Filter ◽

Input Power ◽

Photon Absorption ◽

Focal Volume ◽

Two Photon ◽

Photon Excitation ◽

Two Photon Excitation

Two-photon excitation microscopy (TPEM) provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging and photochemistry. It provides three-dimensional resolution and eliminates background equivalent to an ideal confocal microscope without requiring a confocal spatial filter, whose absence enhances fluorescence collection efficiency. This results in inherent submicron optical sectioning by excitation alone. In practice, TPEM is made possible by the very high local instantaneous intensity provided by a combination of diffraction-limited focusing of a single laser beam in the microscope and the temporal concentration of 100 femtosecond pulses generated by a mode-locked laser. Resultant peak excitation intensities are 106 times greater than the CW intensities used in confocal microscopy, but the pulse duty cycle of 10−5 limits the average input power to less than 10 mW, only slightly greater than the power normally used in confocal microscopy. Because of the intensity-squared dependence of the two-photon absorption, the excitation is limited to the focal volume.

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