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Microscopy Techniques

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Advanced Imaging Techniques in Clinical Pathology

Part of the book series: Current Clinical Pathology ((CCPATH))

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Abstract

The microscope is certainly the first equipment that was used by biologists; its invention dates back 400 years, and it has remained an essential tool in biomedical research. Over these four centuries, the microscope has undergone significant improvements: several lenses have been added along the optical path to increase magnification; lens with fewer aberration defects have been produced; and August Köhler developed a technique enabling the proper illumination of a specimen. This kind of illumination generates an even illumination of the sample and prevents the illumination source (lamp filament) from being visible in the specimen image. Modern microscopes are composed of two-stage magnifying systems comprising a separate lens, the objective, and the ocular. The objective is the most important component of an optical microscope because it determines the quality of the final image, and a wide range of objectives are produced to satisfy a large number of applications. An objective is characterized by its numerical aperture, which determines its resolving power. Microscopes are now designed to investigate molecular mechanisms in living cells; for this reason, highly corrected water immersion objectives have been produced. The following microscopy techniques are described in this chapter:

Phase-Contrast Microscopy

Phase-contrast microscopy is a technique that can be applied to unstained biological specimens because it improves the contrast images of transparent specimens without affecting the resolution. It is mainly used to examine dynamic events in living cells. The technique is based on an optical mechanism that converts light phase variations into changes in amplitude, which can be visualized as differences in image contrast.

Differential Interference Contrast

Differential interference contrast is a mechanism for enhancing contrast in transparent specimens; it produces contrast by visually showing the refractive index gradients of different areas of a specimen. The light beam is polarized and then split into two separate beams, the distance of which is equal to the resolution of the objective lens. One beam path is directed through the specimen and the other acts as a reference beam; the two beams are then combined. Since different parts of the specimen have different refractive indices, when the beams are gathered by the second polarizing filter, the vibrational planes of the beams are restored, causing variations in amplitude that are visualized as differences in brightness.

Wide-Field Fluorescence

Wide-field fluorescence or epi-fluorescence microscopy is the most popular technique to acquire both topographical and dynamic information from a specimen. It is based on the irradiation of the whole sample with a light of a specific wavelength. The weaker emitted fluorescence is then separated from the stronger excitation light. The microscope is configured in such a way that only the emission light can reach the detector or eye. The resulting fluorescent image is superimposed with high contrast against a black background. The limits of detection are generally regulated by the contrast between the fluorescent image and the darkness of the background.

Confocal Microscopy

Confocal microscopy can produce images of a reduced degradation because most out-of-focus light from the specimen is removed. To do this, the confocal microscope is equipped with a pinhole located just before the emission filter and the detector in such a way that the irradiated surface of the specimen is on the same focal plane as the pinhole. Therefore, fluorescent light coming from specimen regions other than the focal plane is excluded, increasing image resolution. When the focus changes to a new specimen region along the z axis, the new region becomes confocal to the observed region on the detector. Thus, images of different specimen slices along the z axis can be obtained, and a 3-dimensional image can be reconstituted using appropriate software.

Total Internal Reflection Fluorescence

Total internal reflection fluorescence is based on an optical effect produced by a light beam passing at a high incident angle through glass (i.e., a coverslip) or plastic (i.e., a Petri dish). The difference between refractive indexes between the glass and the water determines the amount of refraction or reflection light at the interface as a function of the beam incident angle. At a specific angle of the glass (or plastic)–water interface, the light beam is totally reflected per a phenomenon described by Snell. The reflected light generates an electromagnetic field with the same frequency as the incident light, called an evanescent wave. Since this wave is produced by a very small (about 200 nm) electromagnetic field, and its intensity decreases exponentially with distance, only fluorophores located near the glass–liquid interface can be excited. This effect produces high-contrast images of the specimen surface with a considerable increase in signal-to-background ratio compared with classic wide-field microscopy.

Förster Resonance Energy Transfer

Förster resonance energy transfer is used to study inter- and intramolecular interactions in living cells. It is based on the transfer of non-radiative energy from a donor to an accepting fluorophore. To obtain a transfer, two essential requisite must exist: (1) the emission spectrum of the donor must overlap the excitation spectrum of the accepting fluorophore (the larger the overlapping area, the larger the transfer); and (2) the distance between the two fluorophores must be in the range of 10 nm as the transfer depends upon the molecular distance at an inverse sixth power. If these conditions are present, the experiment can be designed by setting the excitation of the donor and acquiring the fluorescence at the wavelength emission of the acceptor.

Fluorescence Recovery After Photobleaching

Photobleaching is generally an unwanted phenomenon in fluorescence microscopy because it reduces the intensity of the probe fluorescence. Photobleaching occurs after lengthy irradiation of molecules and seems to be due to the interaction of oxygen with an excited fluorophore. The oxygen radical reacts with the more reactive excited fluorophore and thus quenches the fluorescence. However, this quenching can be used in fluorescence recovery after photobleaching investigations to determine the kinetics of diffusion in living cells. In fact, if a small portion of the cell is subjected to long irradiation, the fluorescence in that area is completely quenched; the diffusion or active movement of molecules within the cell then replaces the bleached fluorophore with unbleached molecules that were located in a different part of the cell, thus restoring fluorescence.

Fluorescence Lifetime Imaging Microscopy

The fluorescence lifetime can be defined as the average time a molecule spends in its excited singlet state before spontaneous emission occurs. Fluorescence lifetime imaging microscopy (FLIM) has some advantages in terms of conventional fluorescence microscopy, because each fluorescent dye has its own lifetime in the excited state. Thus, by detecting differences in lifetimes, it is possible to distinguish dyes with overlapping fluorescent wavelengths or autofluorescence, which can be undistinguishable when using conventional fluorescence microscopy based on spectral characteristics. In addition, FLIM finds its most significant applications in visualizing the environmental changes of a probe in a living cell. Lifetime is independent of dye concentration, photobleaching, light scattering, and excitation light intensity, but the lifetime of a fluorophore can change with solvent change; therefore, fluorescence lifetime imaging enables accurate ion concentration measurement and FRET analysis.

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Authors and Affiliations

  1. Department of Biology, University of Naples “Federico II”, Via Cinthia, 2, 80126, Naples, Italy

    Antonia Feola

  2. INT-CROM, “Pascale Foundation” National Cancer Institute-Cancer Research Center, 83013, Mercogliano, Italy

    Letizia Cito

  3. Department of Medico Surgical Sciences and Biotechnologies, University of Rome “Sapienza”, Corso della Repubblica, 79, 04100, Latina, Italy

    Angelina Di Carlo

  4. Department of Biochemistry, Biophysics and General Pathology, Seconda Università degli Studi di Napoli, Via de Crecchio, 7, 80138, Naples, Italy

    Alfonso Giovane & Marina Di Domenico

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  1. Antonia Feola
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  2. Letizia Cito
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  3. Angelina Di Carlo
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  4. Alfonso Giovane
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  5. Marina Di Domenico
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Correspondence to Antonia Feola .

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  1. College of Science and Technology, Temple University, Philadelphia, Pennsylvania, USA

    Francesco M. Sacerdoti

  2. College of Science and Technology, Temple University, Philadelphia, Pennsylvania, USA

    Antonio Giordano

  3. NAPLAB, IRCCS SDN NAPLAB, Naples, Italy

    Carlo Cavaliere

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Feola, A., Cito, L., Di Carlo, A., Giovane, A., Di Domenico, M. (2016). Microscopy Techniques. In: Sacerdoti, F., Giordano, A., Cavaliere, C. (eds) Advanced Imaging Techniques in Clinical Pathology. Current Clinical Pathology. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3469-0_4

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