By Michiel Mueller
This booklet offers a accomplished account of the speculation of photograph formation in a confocal fluorescence microscope in addition to a pragmatic guide to the operation of the tool, its barriers, and the translation of confocal microscopy info. The appendices offer a brief connection with optical concept, microscopy-related formulation and definitions, and Fourier theory.
- Symbols and abbreviations
- Confocal fluorescence microscopy
- functional limits
- Miscellaneous topics
- Appendix A: components of optical theory
- Appendix B: formulation, kin and definitions
- Appendix C: Fourier theory
Read Online or Download Introduction to Confocal Fluorescence Microscopy, Second Edition PDF
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Extra info for Introduction to Confocal Fluorescence Microscopy, Second Edition
Artificial contrast stretching has been applied to counteract the sharp drop-off in OTF amplitude near the cut-off frequency. Confocal Fluorescence Microscopy 13 that, for instance, the in-focus position of a thin, homogeneously fluorescent layer cannot be determined in a wide-field fluorescence microscope. Nor can its position be determined when it is part of a stack of such layers with a certain axial spacing between them. Only if the object contains some lateral structure does the wide-field fluorescence microscope provide a certain amount of axial resolution.
In this case, the converging spherical wavefront propagates undistorted from the microscope objective to the desired focal position. Since the refractive index of the cover glass is perfectly matched to that of the immersion oil, the propagating light does not encounter any interface and the thickness of the cover glass is not relevant. Light propagating from all directions will arrive at the focal point in-phase, providing a diffraction-limited focal-field distribution PSF. However, optical aberrations are induced when a change in refractive index occurs in moving from the cover glass (refractive index, n1 ) to the mounting medium (refractive index n2 ), as shown in Fig.
Generally, however, two causes of aberration are present: chromatic aberrations and aberrations induced by a refractive index mismatch. 1 Chromatic aberration A lens or imaging system that has chromatic aberration possesses imaging properties that vary with wavelength. Generally, this appears to be a dependence of Confocal Fluorescence Microscopy 23 the focal length (chromatic focal shift) and lateral magnification (lateral color) on the wavelength. , when multiple labeled specimens are used. The degree to which chromatic aberrations influence the imaging properties strongly depends on the microscope objective and the range of wavelengths used in excitation and detection.