Optical Imaging Techniques in Cell Biology, Second Edition
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Write a product review. Most helpful customer reviews on Amazon. Shipping worked very well! This is a wonderfully rich, yet compact, description of light microscopy techniques and the physics underlying them. Any research lab with a microscope should have a copy nearby! Get to Know Us. Delivery and Returns see our delivery rates and policies thinking of returning an item? See our Returns Policy.
Visit our Help Pages. Audible Download Audio Books. Thus, as we move forward in our understanding of the processes occurring in the cell, it is crucial to reflect on how much of the cell biophysics remains unexplained or unknown. A ubiquitous observation in cell biology is that the translational motion of molecules within the intracellular environment is strongly suppressed as compared to that in dilute solutions.
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By contrast, molecular rotation is not affected by the same environment, indicating that the close proximity of the molecule must be aqueous. Theoretical models provide explanations for this apparent discrepancy pointing to the presence of macromolecular intracellular crowding, but with expectations that depend on the nanoscale organization assigned to crowding agents.
A satisfactory experimental discrimination between possible scenarios has remained elusive due to the lack of techniques to explore molecular diffusion at the appropriate spatiotemporal scale in the 3D-intracellular environment. In one approach, lipids with specific fluorescent markers are incorporated in the cell membranes. The advantage of this approach is that it is possible to study the membrane distribution of specific lipids.
However, when the aim of the study is to detect membrane microdomains independently of their lipid composition, it is more useful to use a single probe that can report on the specific properties of the membrane icrodomains, independently of the probe segregation in one specific domain and location in the cell. One fluorescent probe that has been successfully used for this purpose is the lipophilic probe Laurdan 2-dimethylaminolauroylnaphthalene , originally synthesized by Weber and Farris.
The sensitivity of the emission spectrum However, most of the FCS studies done so far are based on the original idea of measuring temporal correlation at a single point in the membrane. Measuring a single location in the membrane is restrictive since the temporal fluctuations at one point cannot reveal local microstructures or the anisotropic molecular transport in membranes.
The Laurdan spectral phasor method to explore membrane micro-heterogeneity and lipid domains in live cells. In this method paper we describe the spectral phasor analysis applied to Laurdan emission for the assessment of the fluidity of different membranes in live cells. We first introduce the general context and then we show how to obtain the spectral phasor from data acquired using a commercial microscope. The phasor approach to fluorescence lifetime imaging: The phasor approach to fluorescence lifetime imaging FLIM is emerging as a practical method for data visualization and analysis.
These methods are based on correlation of fluorescence intensity fluctuations from microscope images that can be measured in a conventional laser-scanning confocal microscope. In this chapter, we discuss the mathematical framework used for data analysis as well as the parameters need for data acquisition. Three-dimensional particle tracking in a laser scanning fluorescence microscope.
Single-particle and single molecules techniques have become essential tools in the fields of Biophysics and Cell Biology. One of the main reasons of the strong impact of these techniques is that they provide crucial information that is average out in traditional ensemble methods. Amongst these new techniques, single particle tracking SPT constituted a remarkable new tool to study dynamics of biological processes. In this chapter, we briefly describe most common techniques used for tracking particles and focus in recent advances in the field of microscopy that resulted in an improvement of these methods.
We discuss different strategies followed to obtain information regarding the axial position of the particle in image-based tracking approaches and describe in detail a routine we have designed to achieve three dimensional 3D tracking with a laser scanning microscope. Finally, we show the application of this technique to the study of chromatin dynamics in interphase cells to There is a series of outstanding questions regarding the detection of brain activity in measurements made at the head surface and regarding the origin of the observed changes in the optical parameters of tissues.
In this chapter we review the information generated so far and discuss the evidence available about the origin of the effects observed. Because the field is still controversial and rapidly advancing, we mainly focus on our own work and a few other studies that we chose to illustrate our opinions Laurdan is a fluorescent molecule that detects changes in membrane phase properties through its sensitivity to the polarity of the environment in the bilayer.
Polarity changes are shown by shifts in the Laurdan emission spectrum, which are quantified by calculating the generalized polarization GP. This technique was originally developed to be used in a cuvette fluorometer.
Optical Imaging Techniques in Cell Biology by Guy Cox (Paperback, 2017)
With the development of twophoton microscopy, Laurdan GP has evolved into a technique capable of spatially resolve micro-domains of different solvent penetration. We discuss here the basic concepts, instrumentation and experimental considerations when transferring the GP technique from the cuvette to the microscope. We also discuss examples of Laurdan GP in membrane model systems using both cuvette and microscope approaches to compare the two methodologies.
Fluorescence microscopy to study pressure between lipids in giant unilamellar vesicles. The authors developed a technique to apply high hydrostatic pressure to giant unilamellar vesicles and to directly observe the consequent structural changes with two-photon fluorescence microscopy imaging using high numerical aperture oil-immersion objectives. The data demonstrate that high pressure has a dramatic effect on the shape of the vesicles, and both fluidity and homogeneity of the membrane.
Book Chapters - Laboratory for Fluorescence Dynamics
Single point fluctuation correlation spectroscopy FCS is an established technique to study diffusion and chemical equilibria in solution. It has limitations when applied to the cell interior. A major difficulty is that the movements of the entire cell or of cellular components are difficult to separate and filter out from the fast dynamics of the molecules moving in the cell. It is the study of these fast dynamics that helps us in understanding molecular interactions.
Scanning FCS, in which the laser beam is moved in a circular orbit, provides the fluctuation amplitude and dynamics at many points simultaneously. It can be used to infer cell movement, but has limitations in the time scales accessible. Image correlation spectroscopy, an alternative technique in which the entire field of view is analyzed at once, has the potential to provide detailed maps of the dynamics in a cell, but it suffers from limitations imposed Laser assisted confocal microscopy has made a lot of progress over the past few years.
Laser systems have become more modular and compact. There is an ever-increasing number of available laser excitation lines as well as an improvement in user friendliness and ease of use. At the same time, expansion of web resources has provided easy access to a wealth of information. Our goal is both to aid the experienced and novice microscopist in quickly locating and sorting through the relevant laser information and to provide a means of avoiding common problems and pitfalls in the use of laser excitation in the various fluorescence techniques such as fluorescence correlation spectroscopy FCS , fluorescence lifetime imaging microscopy FLIM , fluorescence loss in photobleaching FLIP , fluorescence recovery after photobleaching FRAP , optical coherence tomography OCT , second harmonic generation SHG , single molecule detection SMD , and single particle tracking SPT.
In this chapter we describe the characteristic Fluctuation correlation spectroscopy in cells: When the ethydium dye binds to DNA its fluorescence quantum yield changes by a large factor. It is essentially not fluorescent when free in solution and it becomes strongly fluorescent when bound to double strand DNA. Although the processes are very different in nature, the instrumentation used for the FCS experiment is derived from dynamic light scattering. There are however major differences between dynamic light scattering and FCS Real-time fluorescence lifetime imaging and FRET using fast gated image intensifiers.
Several reviews are already available Clegg and Schneider, ; [Clegg, et al, ] and [Clegg, et al, ]; Periasamy et al.
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However, we feel that the general reader and aspiring user of FLI with applications to FRET would benefit from a concise, coherent presentation of fundamental aspects of these measurements and interpretations of the data. We will not include particular results from a biological system, nor specifics of new instrumentation. Instead, we focus on basic descriptions of the photophysical measurements and the general characteristics The applications of Fluorescence Resonance Energy Transfer FRET have expanded tremendously in the last 25 years, and the technique has become a staple technique in many biological and biophysical fields.
Our understanding of photosynthesis is tightly coupled to our understanding of the transfer of captured energy from the absorption of photons, and following the energy flow through the complex maize of chemical reactions utilizing this energy. Many of these steps involve resonance energy transfer. In this chapter, we have examined some general salient features of resonance energy transfer by stressing the kinetic competition of the FRET pathway with all other pathways of de-excitation.
This approach emphasizes many of the biotechnological and biophysical uses of FRET, as well as emphasizing the important competing processes and biological functions of FRET in photosynthesis. Application of fluorescence correlation spectroscopy to hapten-antibody binding. Two-photon fluorescence correlation spectroscopy 2P-FCS has received a large amount of attention over the past ten years as a technique that can monitor the concentration, the dynamics, and the interactions of molecules with single molecule sensitivity.
In this chapter, we explain how 2P-FCS is carried out for a specific ligand-binding problem. We briefly outline considerations for proper instrument design and instrument calibration. General theory of autocorrelation analysis is explained and straightforward equations are given to analyze simple binding data. Specific concerns in the analytical methods related to IgG, such as the presence of two equivalent sites and fractional quenching of the bound hapten-fluorophore conjugate, are explored and equations are described to account for these issues.
We apply these equations to data on two antibody-hapten pairs: Digoxin and digitoxin are important cardio glycoside drugs, toxic at higher levels, and their blood concentrations This chapter describes several approaches to the optical study of biological tissue using reflectance and transmittance spectroscopy. This topic has spurred significant research efforts as a result of the relevant physiological and metabolic information provided by the optical data, in conjunction with the safe, non-invasive, and costeffective optical approach to the study of tissue.
We give a brief historical introduction in Section The latter model is commonly employed in time-domain and frequency-domain techniques, which Two-photon fluorescence imaging and reactive oxygen species detection within the epidermis. Two-photon fluorescence microscopy is used to detect ultraviolet-induced reactive oxygen species ROS in the epidermis and the dermis of ex vivo human skin and skin equivalents.
Skin is incubated with the nonfluorescent ROS probe dihydrorhodamine, which reacts with ROS such as singlet oxygen and hydrogen peroxide to form fluorescent rhodamine Unlike confocal microscopic methods, two-photon excitation provides depth penetration through the epidermis and dermis with little photodamage to the sample. This method also provides submicron spatial resolution such that subcellular areas that generate ROS can be detected.
In addition, comparative studies can be made to determine the effect of applied agents drugs, therapeutics upon ROS levels at any layer or cellular region within the skin.
Optical Imaging Techniques in Cell Biology
We give a brief historical introduction in Section 1 1. Three different methods of fluorescence-lifetime imaging for microscopy are presented along with some examples of their use. All three methods use the frequency-domain heterodyning technique to collect lifetime data. Because of the nature of the data collection technique, these instruments can measure the correct lifetime even when the sample undergoes strong photobleaching.
Each instrument has unique capabilities that complement the others. The first microscopic-lifetime imaging technique uses a fast charge-coupled device CCD camera and a gated image intensifier. The camera system collects an entire lifetime image in parallel in only a few seconds. This microscope is well suited to kinetic studies of intracellular lifetime changes. The other two techniques use scanned laser source to collect sequential lifetime information pixel by pixel.
One microscope uses two-photon excitation to achieve three-dimensional, confocal-like imaging without using detection pinholes. Two-photon excitation also limits the effects of out-of-plane photodamage of the sample. The second laser-scanning microscope The global analysis of fluorescence intensity and anisotropy decay data: The aim of this chapter is to describe, in detail, the design, application, and "philosophy" behind the current generation of global analysis programs. The sections of this chapter can be summarized as follows: Introduction to time-resolved fluorescence data, some experimental techniques, and some typical examples of how previous works have benefitted from global analysis.
Historical overview of how the emphasis of global analysis has evolved from one of multi-dimensional curve fitting to that of multi-dimensional physical model evaluation. General elements required to perform a global analysis of distributed and discrete models.
The basic equations of the compartmental approach are examined and the systems theory view of photophysical events is described. In-depth example of the "inner-workings" of the general purpose global analysis program developed at the Laboratory for Fluorescence Dynamics LFD. Spontaneous, microscopic fluctuations are an integral part of every fluorescence measurement and add a noise component to the observed fluorescence signal.
Fluorescence correlation spectroscopy FCS extracts information from this noise and characterizes the kinetic processes that are responsible for the signal fluctuations. For instance, the dynamic equilibrium between a fluorescent and a nonfluorescent state of a fluorophore introduces fluctuations. Another example is Brownian motion, which leads to the stochastic appearance and disappearance of fluorescent molecules in a small observation volume.
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FCS characterizes any kinetic process that leads to changes in the fluorescence, because the spontaneous fluctuations at thermodynamic equilibrium are governed by the same laws that describe the kinetic relaxation of a system to equilibrium. Thus, FCS offers a very convenient method for determining kinetic properties at equilibrium without requiring a physical perturbation of the sample. This is especially important for systems in which the use of perturbation techniques in Giant vesicles, Laurdan, and two-photon fluorescence microscopy: We have given an overview of what one can gain by lifetime-resolved imaging and reviewed the major issues concerning lifetime-resolved measurements and FLI instrumentation.
Instead of giving diverse selected examples, we have discussed the underlying basic pathways of deexcitation available to the molecules in the excited state. It is by traversing these pathways that compete kinetically with the fluorescence pathway of deactivation—and therefore affect the measured fluorescence lifetime—that we gain the information that lifetime-resolved fluorescence provides. It is hoped that being aware of the diversity, of pathways available to an excited fluorophore will facilitate potential users to recognize the value of FLI measurements and inspire innovative experiments using lifetime-resolved imaging.
FLI gives us the ability within a fluorescence image of measuring and quantifying dynamic events taking place in the immediate surroundings of fluorophores as well as locating the fluorescent components within the image. Just as measurements in cuvettes, lifetime-resolved imaging