ABSTRACT
Nicola Green and John W. Haycock 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.1.1 What Is Fluorescence Microscopy? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 2.1.2 Fluorescence Microscopy in Regenerative Medicine . . . . . . . . . . . . . . . . 30
2.1.2.1 Advantages and Disadvantages of Fluorescent Microscopy in Regenerative Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.1.3 Fluorescence Excitation and Emission . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 2.1.4 Fluorescence Microscope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.1.4.1 Epiuorescence Microscope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 2.1.4.2 Confocal Laser Scanning Microscope . . . . . . . . . . . . . . . . . . . . . 33
2.1.5 Two-Photon Excitation Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
2.2 Fluorescence Microscopy for Clinical and Biological Measurements . . . . . . . . 37 2.2.1 Noninvasive Imaging for Continuous Study of Live Cells . . . . . . . . . . . . 37
2.2.1.1 Short-Term Imaging of Live Cells . . . . . . . . . . . . . . . . . . . . . . . . . 37 2.2.1.2 Extended Imaging of Live Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
2.3 Technical Implementation of Fluorescence Microscopy . . . . . . . . . . . . . . . . . . . 39 2.3.1 Imaging Living Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
2.3.1.1 Important Considerations for Live Cell Imaging . . . . . . . . . . . . . 40 2.3.2 Labeling of Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
2.3.2.1 Labeling Fixed Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 2.3.2.2 Labeling Live Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 2.3.2.3 Dealing with Autouorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
2.3.3 Two-Photon Excitation Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
2.4 Example Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 2.4.1 Characterization of Stem Cell Differentiation . . . . . . . . . . . . . . . . . . . . . . 52 2.4.2 Developing Vascularized Tissue-Engineered Constructs . . . . . . . . . . . . 53
2.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
2.1.1 What Is Fluorescence Microscopy? Fluorescence microscopy is an imaging technique that relies upon the phenomenon that when certain molecules are exposed to light of a specic wavelength, those molecules will emit light of a lower energy and increased wavelength. is emitted signal is separated from the light used to illuminate the sample using optical lters and the signal collected to generate the image. Generally, uorescent dyes-also referred to as uorophores-are used to stain particular components of a sample, with the location of the uorescence indicating the location of the constituent of interest. Many biological molecules are also intrinsically uorescent, and this property can be exploited for imaging purposes without the need for additional uorophores.
