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Light Microscopy Techniques

Figure 2 : TIRF image of fl uorescently labeled actin from chicken muscle. Dennis Breitsprecher, Hannover Medical School, Institute of Biophysical Chemistry, Hannover, Germany. Honorable Mention, 2010 Olympus BioScapes Digital Imaging Competition.

adheres. Total internal refl ection fl uorescence (TIRF) imaging ( Figure 2 ) requires a research-grade fluorescence microscope, a specialized prism, and a laser. At a specifi c angle, the beam of laser light passing through the objective is totally refl ected at the glass/specimen interface, rather than passing through; this is called the critical angle. T is angle is very shallow relative to the interface and requires the objective lens to have a very high numerical aperture (greater than 1.45). Refl ection at the critical angle produces an evanescent wave, which propagates into the aqueous medium. This evanescent wave has an identical wavelength to that of the incident light. T e critical angle can vary depending on the materials involved, but the strength of this evanescent wave decreases exponentially as it extends through the second medium [ 2 ].

TIRF microscopy is a powerful way of elucidating processes occurring at the cell surface, such as membrane adhesion and movement, cell interactions, protein secretion, and adsorption. The shallow penetration depth of the evanescent wave allows only those fluorophores near the surface of a specimen to become excited, creating a thin optical section that can readily be imaged [ 3 ]. Although it is only applicable at shallow depths (to 200 nm), TIRF is an excellent technique for obtaining high-contrast images with excellent signal-to-noise ratios because light penetration outside the evanescent fi eld is minimal.

Confocal Microscopy

Thick, living tissue can sometimes scatter so much light that very little specimen-emitted fluorescence reaches the detector, and the challenges of photobleaching and phototoxicity remain daunting. As specimens become more three-dimensional and regions of interest demand extreme depths, traditional fl uorescence techniques reach their limit of eff ectiveness.

Confocal microscopy removes out-of-focus light originating from outside the focal plane, permitting thin optical sectioning

2016 May •

Figure 3 : Confocal image of neurofi laments in coronal rat brain section. Image courtesy of the late Michael W. Davidson, Florida State University, Tallahassee, FL.

( Figure 3 ). T is makes confocal imaging ideal for live-cell fl uores- cence imaging at a multitude of depths. Confocal systems are widely used in life science laboratories because they allow scientists to study biological processes in real time. Also, by imaging stacks of individual sections created from optical slices, researchers can create three-dimensional images with good resolution in each dimension.

But even confocal systems have their limits; excitation light generates fl uorescence throughout the entire depth of the sample, including all the areas above and below the region of interest, oſt en photobleaching and damaging the entire volume of a specimen [ 4 ]. Also, collecting the emitted signal can be complex. Emitted photons from the focal plane must travel from the sample through the microscope’s optical system and then pass through a small slit or pinhole, called the confocal aperture, before arriving at the detector. Any signal that comes from outside the focal plane is removed by the confocal aperture. Scattering or deflection of the fluorescence as it passes through the sample can easily result in the light being removed by the confocal aperture. Photons traveling from deep within the specimen experience higher levels of scattering and rejection compared to shallower objects, making deep imaging a challenge.

Multiphoton Microscopy

Where confocal imaging falls short, multiphoton systems excel. Multiphoton systems allow imaging at much deeper levels than their confocal relatives, typically up to about 700–1,000 nm below the surface. Typically longer wavelengths of light are used in multiphoton systems because these wavelengths scatter less when moving through tissue and are not as harmful to living cells.

In multiphoton imaging, multiple long-wavelength, low- energy excitation photons are absorbed simultaneously by a single fl uorophore at a specifi c point location in x , y and z [ 5 ]. When this happens, the photons combine their energies as


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