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. 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. 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.
Optical Imaging Techniques in Cell Biology: 2nd Edition (Paperback) - Routledge
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. 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.
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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 Phospholipase A2 PLA2 catalyzes hydrolysis of the sn-2 acyl chain of phospholipids. Physiologically, it appears to be involved in diverse functions such as digestion, membrane homeostasis, production of precursors for synthesis of several lipid mediators, defense against bacteria, clearing of dead or damaged cells, and as ligands for receptors.
Three basic types have been identified: Previous fluorescence investigations of EB-tRNA interactions, carried out for more than 30 years, have indicated a "strong" binding site with a lifetime near 26 ns and one or more "weak, non-specific" binding sites with reduced lifetimes. In our study, under specific conditions in which only one EB is bound, a fluorescence lifetime of 27 ns was obtained.
Global Analysis of the lifetime data was consistent with a model in which the second EB molecule bound has a lifetime of only 5. Global Analysis also indicated that this second binding event leads to a reduction in the lifetime of the first EB bound, namely from 27 ns to The lifetime decrease associated with the "strong" binding site could be Assessment of membrane fluidity in individual yeast cells by Laurdan Generalized Polarization and multi-photon scanning fluorescence microscopy.
Here we describe techniques that we developed for monitoring membrane fluidity of individual yeast cells during environmental adaptation and physiological changes. Multi-photon scanning fluorescence microscopy using laurdan as a membrane probe enables determination whether fluidity changes seen by spectroscopy reflect universal responses or changes only of sub-populations.
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Yeast membranes are a primary site of environmental response and adaptation. To determine whether such responses reflect all cells we conducted the first multi-photon scanning fluorescence microscopy study of yeasts, measuring laurdan GP. We assessed membrane fluidity responses of individual yeasts related to growth phase, heat stress and ethanol stress.
Average fluidity decreased as cultures aged, however the decreased fluidity was due in some cases to an increasing proportion of uniformly Biological applications of time-resolved, pump-probe fluorescence microscopy and spectroscopy in the frequency domain. In biological applications of optical microscopy, technical developments often lead to novel imaging modalities with significant applications. For example, the development of confocal microscopy led to microscopic imaging with enhanced contrast see Chapter 5 , and the more recent development of two-photon fluorescence microscopy revolutionized fluorescence microscopy by providing an imaging modality capable of high image contrast, reduced photodamage, and exciting possibilities in controlling localized photochemical reactions in three dimensions see Chapters Two-photon excitation microscopy for image spectroscopy and biochemistry of tissues, cells, organelles and lipid vesicles under physiological conditions.
Two-photon excitation spectroscopy has inherent 3-D resolution with excitation volumes as small as 0. The fluorescence fluctuations within the small excitation volume provide, via fluorescence correlation spectroscopy FCS , a unique way to study biological phenomena.
In this contribution, we outline the instrumentation of two-photon fluorescence correlation spectroscopy and highlight technical details of our experimental setup.
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We discuss the autocorrelation analysis for single and multiple species with emphasis on the normalized fluctuation amplitude G 0. Furthermore, we revisit another data analysis technique called moment analysis. This method was originally introduced 10 years ago and provides a very fast and convenient characterization of the fluctuation amplitude. We compare experimental results from moment analysis with that of another data analysis method, the photon counting histogram PCH , and discussed differences between these two methods.
In the macroscopic world fluctuations are typically exceedingly small and beyond the resolution power of most experiments. Only in special circumstances, such as near the critical point of a liquid, do fluctuations become visible to the naked eye. However, the importance of fluctuations increases once the macroscopic world is left behind and one starts to consider mesoscopic or microscopic systems, where fluctuation phenomena are readily observed.
Fluctuation spectroscopy exploits this source of information [ Kinetics of binding of Hoechst dyes to DNA studied by stopped-flow fluorescence techniques. Practical aspects of fluorescence resonance energy transfer FRET and its applications in nucleic acid biochemistry. One naturally needs to expand from classical single-point measurement to simultaneous multiple-point measurement for the formation of an image.
This technical expansion raises a new challenge, namely, the ability to acquire images with fluorescence lifetime information within a reasonable time frame and with similar accuracy and precision comparable to that of single-point spectroscopy measurement. This goal is the main scope of this chapter, which provides an overview of the different techniques used in fluorescence lifetime imaging microscopy FLIM. This chapter begins with an overview of two-photon microscopy relevant to applications in deep-tissue imaging. Basic principles, deep-tissue models, and instrumentation are discussed.
From there, recent advances in two-photon deep-tissue imaging are addressed: Multiphoton excitation microscopy and spectroscopy of cells, tissues, and human skin in vivo. This chapter describes the history, theory, instrumentation, and cell and tissue applications of non-linear multiphoton microscopy.
Optical Imaging Techniques in Cell Biology
It includes a discussion of laser sources, detectors, scanners, and microscope objectives, and experimental applications are presented to illustrate the unique capabilities of these innovative techniques. The emphasis of the chapter is on the application of multiphoton excitation microscopy to the functional imaging of in vivo human skin, and the two examples presented—of transparent and turbid media—illustrate the utility of multiphoton excitation microscopy in thick specimens. Multiphoton excitation microscopy is an important new optical technique with many applications in biology.
It has advantages over confocal laser scanning microscopy as well as wide-field microscopy. National Library of Australia. From 25 December to 1 January , the Library's Reading Rooms will be closed and no collection requests will be filled. Collection delivery service resumes on Wednesday 2 January Further information on the Library's opening hours is available at: Optical imaging techniques in cell biology. Request this item to view in the Library's reading rooms using your library card. To learn more about how to request items watch this short online video.
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