Basics of Fluorescence, Fluorophores, Detectors, and Light Microscopy

Methods in Cellular Imaging 
Oxford University Press

Edited by 
Ammasi Periasamy, Ph.D.


Basics of Fluorescence, Fluorophores, Detectors, and Light Microscopy


As every new student in biology learns Antoni van Leeuwenhoek observed protozoa, bacteria, spermatoza, and red blood cells using a simple microscope with a high quality lens in the 1670’s. Since then the microscope has been an essential tool found in virtually every biological laboratory. The ability to study the development, organization, and function of unicellular and higher organisms and to investigate structures and mechanism on the micron size has allowed scientists to better grasp the relationship between microscopic and macroscopic behavior. Further, the modern microscope preserves temporal and spatial relationships that are frequently lost in traditional biochemical techniques and gives two- or three-dimensional resolution that other laboratory methods cannot.

The benefits of fluorescence microscopy techniques are numerous. Some advantages are the inherent specificity and sensitivity of fluorescence; the high temporal, spatial, and three-dimensional resolution; and the enhancement of contrast resulting from detection of an absolute rather than a relative signal. Additionally, the plethora of well-described spectroscopic techniques that provide different types of information, and the commercial availability of fluorescent probes, many of which exhibit an environment- or analytic-sensitive response, broaden the range of possible applications in the biomedical sciences. Recent advances in available light sources; detection systems; data acquisition methods; and image enhancement, analysis, and display methods have further broadened the applications in which fluorescence microscopy can be successfully implemented for various biological applications.

            Chapter 1 explains the basic principles of fluorescence excitation and emission, and the various phenomena involved, such as absorption, emission, quenching, quantum yield, anisotropy, and photo bleaching.  A basic understanding of the principles of fluorescence allows informed selection of fluorophores for fluorescence labeling, imaging and quantification of cellular events. The second chapter (Chapter 2) describes the fluorophores that can be used for measuring different ion concentrations, membrane potential, and pH. Chapter 2 not only provides information on how to load the cells with different fluorophore molecules, but it also provides a list of probes used for different cellular function studies with appropriate excitation and emission wavelengths. The selection of detectors for acquiring fluorescence signals is an important aspect of the fluorescence microscopy. Chapter 3 describes detector characteristics, such as sensitivity, signal-to-noise (S/N) ratio, and different types of detectors, including charge-coupled device (CCD) cameras, photodiodes, and photomultiplier tubes (PMT).  Different cameras that are selected for the various microscopic techniques are described in Chapter 4. In Chapter 4, the authors also address how the biologist or physiologist can choose a microscope to monitor or image the cellular activity using several examples. Also, the authors provide useful information such as important microscope terminology and definitions and information regarding the sources of different parts of the imaging system, such as lenses, cameras, software etc.

            The wide-field microscope image includes information above and below the focal plane. This information can be obtained and utilized by digital deconvolution of the optical-sectioned images. The digital deconvolution method cannot monitor cell signaling in real time, but Chapter 5 describes the laser scanning confocal microscope, which rejects out-of-focus information by using a pinhole in front of the detector and does offer real time imaging.  Chapter 5 explains the basic principles involved in confocal microscopy and how to implement ratio imaging using confocal microscopy for calcium, pH, and membrane potential in cardiac myocytes. Chapter 6 focuses on mitochondria responses to cell signaling, both physiological and pathophysiological by monitoring the mitochondrial potential, redox state, and calcium handling using epi-fluorescence microscopic imaging.

Chapter 7 describes the fluorescence recovery after photobleaching (FRAP) method and its instrumentation and data acquisition for biological samples using a conventional fluorescence microscopy configuration. This FRAP method provides fundamental information about the dynamics of biologically important solutes and macromolecules in cell membrane and aqueous compartments.  It is important to understand in a wired on-line society how best to present digital images acquired from light microscopy. Chapter 8 gives step-by-step information on using Adobe Illustrator on converting files to different digital formats such as Tiff and JPEG, using Macromedia Fireworks for graphic manipulations, and presenting microscopic images on a website.