Fluorescence Microscopy (eBook)

Super-Resolution and other Novel Techniques

P. Michael Conn, Anda Cornea (Herausgeber)

eBook Download: PDF | EPUB

2014 | 1. Auflage
260 Seiten
Elsevier Science (Verlag)
978-0-12-416713-1 (ISBN)

Fluorescence Microscopy: Super-Resolution and other Novel Techniques delivers a comprehensive review of current advances in fluorescence microscopy methods as applied to biological and biomedical science. With contributions selected for clarity, utility, and reproducibility, the work provides practical tools for investigating these ground-breaking developments. Emphasizing super-resolution techniques, light sheet microscopy, sample preparation, new labels, and analysis techniques, this work keeps pace with the innovative technical advances that are increasingly vital to biological and biomedical researchers. With its extensive graphics, inter-method comparisons, and tricks and approaches not revealed in primary publications, Fluorescence Microscopy encourages readers to both understand these methods, and to adapt them to other systems. It also offers instruction on the best visualization to derive quantitative information about cell biological structure and function, delivering crucial guidance on best practices in related laboratory research. - Presents a timely and comprehensive review of novel techniques in fluorescence imaging as applied to biological and biomedical research - Offers insight into common challenges in implementing techniques, as well as effective solutions

Fluorescence Microscopy: Super-Resolution and other Novel Techniques delivers a comprehensive review of current advances in fluorescence microscopy methods as applied to biological and biomedical science. With contributions selected for clarity, utility, and reproducibility, the work provides practical tools for investigating these ground-breaking developments. Emphasizing super-resolution techniques, light sheet microscopy, sample preparation, new labels, and analysis techniques, this work keeps pace with the innovative technical advances that are increasingly vital to biological and biomedical researchers. With its extensive graphics, inter-method comparisons, and tricks and approaches not revealed in primary publications, Fluorescence Microscopy encourages readers to both understand these methods, and to adapt them to other systems. It also offers instruction on the best visualization to derive quantitative information about cell biological structure and function, delivering crucial guidance on best practices in related laboratory research. - Presents a timely and comprehensive review of novel techniques in fluorescence imaging as applied to biological and biomedical research- Offers insight into common challenges in implementing techniques, as well as effective solutions

Chapter 1

Evanescent Excitation and Emission

DanielAxelrod Departments of Physics, Biophysics, and Pharmacology, University of Michigan, Ann Arbor, Michigan, USA

Abstract

Evanescent light—light that does not propagate but instead decays in intensity over a subwavelength distance—plays a role in fluorescence microscopy in both excitation (i.e., total internal reflection, or TIR) and emission (i.e., supercritical angle fluorescence). This chapter describes the physical connection between these two forms as a consequence of geometrical compression of wavefront spacing and describes newly established or speculative applications and combinations of the two. In particular, each form can be used in analogous ways to produce surface-selective images, to examine the thickness and refractive index of films (such as lipid multilayers or protein layers) on solid supports, and to measure the absolute distance of a fluorophore to a surface. In combination, the two forms can further increase selectivity and reduce background scattering in surface images. The polarization properties of each lead to more sensitive and accurate measures of fluorophore orientation and membrane micromorphology. The phase properties of evanescent excitation lead to methods of creating a submicroscopic area of TIR illumination or enhanced-resolution structured illumination. Analogously, the phase properties of evanescent emission lead to a method of producing a smaller point spread function, in a technique called virtual supercritical angle fluorescence. This chapter emphasizes the concepts and theory (rather than experimental protocols and results) of evanescence for both excitation and emission, as well as the theory of its many existing and possible future applications.

Keywords

microscope imagingnear fieldpoint spread functionpolarizationsupercritical anglesuper-resolutiontotal internal reflection

Introduction

Some of the various super-resolution microscopy techniques share a common feature in that they attempt to exceed the standard light microscope resolution limit by employing “evanescent” light that decays in at least one direction in a distance much shorter than the wavelength. This group includes total internal reflection fluorescence microscopy (TIRFM; covered in another chapter in this book), near-field scanning optical microscopy (NSOM), and virtual supercritical angle fluorescence (vSAF) microscopy.1–4 In some cases, evanescence is in the excitation light, in other cases it is in the emission, and in some it is in both. This chapter explores the physical concepts that these techniques share and points toward some established and more speculative possible directions for future work in evanescence-based super-resolution. The effect of evanescence on the use and detection of polarization, in both excitation and emission, is covered in considerable detail.

Evanescence in both excitation and emission can be understood as a response to geometrical compression, or “squeezing,” of wavefront spacing in at least one dimension. Evanescent light can be converted to or from propagating light traveling at supercritical angles relative to a nearby interface. Evanescence has numerous applications in fluorescence microscopy. Here is a preview of those to be discussed in this chapter:

• On the excitation side

• Supercritical excitation (TIRF) is commonly used for selective excitation of surface-proximal molecules, cell/substrate contact regions, and membrane-proximal cytoplasmic organelles.

• Variable angle TIRF has been used to deduce the concentration of fluorophores as a function of distance from the substrate.

• TIRF intensity vs. incidence angle on film-coated surfaces can display a resonance behavior that may measure the thickness, refractive index and possible lateral heterogeneities of surface-supported multilayer lipid or protein coatings.

• TIRF on film-coated surfaces can enhance the evanescent intensity by at least an order of magnitude.

• Polarized excitation TIRF can highlight submicroscopic irregularities in the plasma membrane of living cells and orientation of single molecules.

• Intersecting TIRF beams can extend the super-resolution of structured illumination.

• Radially polarized ring TIR illumination at the back focal plane (BFP) can produce a uniquely small illumination volume, possibly useful for fluorescence correlation spectroscopy and high-resolution scanning.

• The evanescent field at an NSOM tip facilitates the mapping of distances to fluorophores and surface topology.

• On the emission side

• The emission intensity pattern in the supercritical zone of the BFP reports the fluorophore concentration profile as a function of distance to the surface.

• The ratio of emission power in the supercritical vs. subcritical BFP zone can sensitively report absolute distance of a fluorophore to the surface to an accuracy of tens of nanometers.

• Taking into account the interaction of the fluorophore near field with a surface alters the predicted depolarization induced by high-aperture observation.

• For a film-coated surface (such as a lipid multilayer), the emission intensity pattern in the supercritical zone of the BFP is uniquely sensitive to film thickness.

• On both excitation and emission sides

• By combining the vSAF emission image protocol with standard TIRF excitation, an even higher degree of surface selectivity should be attainable than from either individually, with much less scattering background intensity.

Evanescence in General

For propagating light, the shortest spacing (λm) between each traveling wavefront (i.e., the periodic locus of points of equal phase) for propagating light is simply given as λm = λo/nm, where λo is the light wavelength in vacuum, and nm is the refractive index of medium m (m = 1, 2, 3 will be used here for a system of stratified planar layers). The wavefronts propagate through medium m with a speed of c/nm.

There are situations, however, when the wavelength spacing can be forced to be smaller than λo/nm. Such special geometrical situations include the lower refractive index side of an interface at which total internal reflection (TIR) occurs; they also include confinement of the light source to a region smaller than its wavelength, such as very near an excited molecule or the tip of a fine optical fiber. In these cases, light cannot freely propagate and instead becomes exponentially decaying in at least one dimension.

The fundamental physical processes are related among these situations and can be understood most easily by considering the electric field of plane waves and then generalizing to other wavefront shapes. Plane-wave light propagating in a medium of refractive index nm is characterized by a wave vector, km, pointing in the direction of its propagation:

(1.1)

The orientation of the (x, y, z) axes can be arbitrary but is chosen based on the geometry of optical surfaces nearby. The amplitude of km, called the wavenumber, is

(1.2)

where ω is the angular frequency of the particular color of the light, and c is the speed of light in vacuum. Frequency ω is equal everywhere in the optical system, regardless of refractive index.

Because of the constancy of ω and c, the wavenumber, km, and its corresponding wavelength, λm, are real numbers that are fixed for any light in medium m, regardless of direction or proximity to interfaces. For freely propagating light, the spacing between wavefronts along any direction x, y, z of propagation is

(1.3)

The position-dependent part of the plane wave electric field has the form exp(ikm·r), where position vector . Therefore, the spatial variation of the electric field in, say, the z direction is the sinusoidal function exp(ikmzz).

The relevant feature of Equation (1.2) is that the sum of the squares of the components kmx, kmy, and kmz in any medium must exactly equal for that medium. What happens if the...

Erscheint lt. Verlag	24.2.2014
Sprache	englisch
Themenwelt	Naturwissenschaften ► Biologie ► Biochemie
	Naturwissenschaften ► Biologie ► Genetik / Molekularbiologie
	Naturwissenschaften ► Biologie ► Zellbiologie
	Technik
ISBN-10	0-12-416713-6 / 0124167136
ISBN-13	978-0-12-416713-1 / 9780124167131

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