Confocal Microscopy

• Published in: Fluorescence Microscopy p. 1
• Main Authors: Naredi‐Rainer, Nikolaus, Prescher, Jens, Hartschuh, Achim, Lamb, Don C
• Format: Book Chapter
• Language: English
• Published: Germany John Wiley & Sons 2017; John Wiley & Sons, Incorporated; Wiley‐VCH Verlag GmbH & Co. KGaA
• Edition: 2nd Edition
• Subjects: Biochemistry, Biology & Biotechnology; Biology & Microbiology; Burst analysis; confocal microscopy; Engineering Principles; fluorescence correlation spectroscopy; General Engineering & Project Administration; multiparameter fluorescence detection; number and brightness; pulsed interleaved excitation; raster image correlation spectroscopy; spinning disk confocal microscope
• ISBN: 3527338373; 9783527338375
• DOI: 10.1002/9783527687732.ch5

In chapter 5, we discuss the fundamentals of confocal microscopy. After a brief introduction, we introduce the theory for confocal microscopy and discuss the influence the pinhole has on the axial and lateral resolution of the system. We discuss the optimal configuration of the system depending on the desired signal and resolution. A second element of the confocal microscopy that is essential for imaging is scanning. Hence, we discuss the different scanning methods: stage scanning, laser scanning and spinning disk confocal microscopy and the advantages and drawbacks of the different approaches. We also briefly describe confocal deconvolution, an approach that can further enhance the resolution of confocal images. After covering the fundamentals of confocal microscopy, we discuss several applications. Imaging is the most obvious application. However, confocal microscopy has played a major role in non‐imaging applications as well such as fluorescence correlation spectroscopy (FCS). We provide the basics of FCS and fluorescence cross‐correlation spectroscopy and the advantages of pulsed interleaved excitation. Confocal microscopy is also an important tool for single‐molecule Förster Resonance Energy Transfer experiments. Therefore, we discuss burst analysis experiments along with multiparameter fluorescence detection. In the last section, we bring together the scanning functionalities of confocal microscopy with FCS and discuss two recent correlation methods that are interesting for cellular imaging, the number and brightness analysis and raster image correlation spectroscopy.