Fluorescence microscope applies high-intensity light to illuminate the substance that emits fluorescence light. The present chapter describes the basic principle and applications of fluorescence microscopy. There are two types of fluorescence microscope: transmitted fluorescent microscope and incident fluorescent microscope. The working principles of both these types of microscopes are described. The confocal fluorescence microscopy (CFM) provides three-dimensional optical resolution. In CFM, at one time we see the image of the particular depth of the object at a small point. All the out-of-focus light is eliminated by passing the light through the pinhole. Multiple images at different depth are accumulated and then reconstructed to provide a three-dimensional image. The working principle and applications of CFM are discussed in the present chapter.
Fluorescence microscope Fluorochrome dye Transmitted fluorescent microscope Incident fluorescent microscope Dichroic mirror Confocal microscopy Fluorescence recovery after photobleaching Green fluorescence protein
This is a preview of subscription content, log in to check access.
Weichselbaum M, Everett AW, Sparrow MP. Mapping the innervation of the bronchial tree in fetal and postnatal pig lung using antibodies to PGP 9.5 and SV2. Am J Respir Cell Mol Biol. 1996;15(6):703–10.CrossRefPubMedGoogle Scholar
Cook RJ, Azzopardi A, Thompson ID, Watson TF. Real-time confocal imaging, during active air abrasion–substrate cutting. J Microsc. 2001;203(Pt 2):199–207.CrossRefPubMedGoogle Scholar
Jensen E. Technical review: colocalization of antibodies using confocal microscopy. Anat Rec (Hoboken). 2014;297(2):183–7.CrossRefGoogle Scholar
Patel DV, McGhee CN. Quantitative analysis of in vivo confocal microscopy images: a review. Surv Ophthalmol. 2013;58(5):466–75.CrossRefPubMedGoogle Scholar
Nwaneshiudu A, Kuschal C, Sakamoto FH, Anderson RR, Schwarzenberger K, Young RC. Introduction to confocal microscopy. J Invest Dermatol. 2012;132(12):e3.CrossRefPubMedGoogle Scholar
Diaspro A, Chirico G, Collini M. Two-photon fluorescence excitation and related techniques in biological microscopy. Q Rev Biophys. 2005;38(2):97–166.CrossRefPubMedGoogle Scholar
Hell S, Stelzer E. Properties of a 4Pi confocal fluorescence microscope. J Opt Soc Am. 1992;A9(12):2159–66.CrossRefGoogle Scholar
Gustafsson MG. Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc Natl Acad Sci U S A. 2005;102(37):13081–6.CrossRefPubMedPubMedCentralGoogle Scholar