By Michiel Mueller
This booklet presents a complete account of the idea of picture formation in a confocal fluorescence microscope in addition to a realistic guide to the operation of the software, its obstacles, and the translation of confocal microscopy facts. The appendices supply a short connection with optical conception, microscopy-related formulation and definitions, and Fourier theory.
- Symbols and abbreviations
- Confocal fluorescence microscopy
- functional limits
- Miscellaneous topics
- Appendix A: parts of optical theory
- Appendix B: formulation, kinfolk and definitions
- Appendix C: Fourier theory
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Additional info for Introduction to Confocal Fluorescence Microscopy, Second Edition
Perpendicular to the optical axis, that coincides with the geometrical focal point. The right panel shows an axial plane centered on the optical axis. Logarithmic contrast stretching equivalent to that in Fig. 7 has been applied to enhance the visibility of any side lobe structures. detection pinhole suppresses virtually the entire lateral side lobe structure, whereas some axial contributions remain. Image formation in a confocal microscope follows the same principle as for the wide-field microscope.
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. Microscope objectives are generally corrected for either two (achromats) or three (apochromats) wavelengths (see Sec. 1). This means that for these particular wavelengths, the aberrations are optimally balanced and minimized. Between these wavelengths, the microscope objective generally still performs well, but outside this wavelength region the aberrations usually grow exponentially, particularly towards the shorter wavelengths.
2, the speed of image acquisition in a confocal fluorescence microscope can be increased considerably by using slit excitation and detection. However, this compromises the axial resolution. Wilson (1995) calculated the axial response to an infinitely thin fluorescing layer as a function of the width, vw , of the detection slit (Fig. 9). 4. 6 when compared to pinhole detection with a pinhole diameter equal to the slit width. 4 Fluorophores Confocal fluorescence microscopy relies on the use of fluorophores to provide image contrast.
Introduction to Confocal Fluorescence Microscopy, Second Edition by Michiel Mueller