FLIM

The markets for polyetherimides arise to an extent from stricter regulations concerning flammability and smoke evolution coupled with such features as high strength, toughness and heat resistance. Application areas include car under-the-bonnet uses, microwave equipment, printed circuit boards and aerospace (including carbon-fibre-reinforced laminated materials). The polymer is also of interest in flim, fibre and wire insulation form. 

FIGURE I2.3B Flim-devetoping machine and a laboratory model. 

On September 13 2005 I received the invitation from professor P.C. van der Vliet (the editor of the Laboratory Techniques in Biochemistry and Molecular Biology Series) to become the editor of a new volume in the series on FRET and FLIM . In the letter it was mentioned that in view of the rapid developments in single cell technology, we feel that a book on imaging techniques in living cells, such as FRET and FFIM, is appropriate and timely . 

Hence, for modern FRET and FLIM techniques in Molecular Biology and Biochemistry it is important to keep the enthusiasm for the in situ technique, yielding unprecedented rich information on molecular states in live cells, and to keep the advantages of easy labeling techniques, modern microscopes and automated data processing. However, we need to educate the new generations of FRET scientists in the theoretical background of the technique, how it should be done correctly, and what the sources of errors are. Only then it will be clear that FRET-(FLIM) is a very direct, robust, extremely sensitive, and reliable technique. 

The second chapter by Peter Verveer and Quentin Hanley describes frequency domain FLIM and global analysis. While the frequency domain technique for fluorescence lifetime measurement is sometimes counterintuitive, the majority of the 10 most cited papers using FLIM have taken advantage of the frequency domain method as stated by these authors. The global analysis of lifetime data in the frequency domain, resolving both E and /d has contributed significantly to this advantage. 

The third chapter by Alessandro Esposito et al., describes the time domain counterpart of FLIM. When photon economy and... 

The fourth chapter by James McGuinty et al. describes the more advanced forms of time-domain FLIM. While not immediately available on commercial instruments this chapter should give the reader an idea what the current state-of-the-art is in terms of FLIM instrumentation, and perhaps what to expect on future commercial instruments. Real-time FLIM, combined FLIM-spectral imaging, hyperspectral FLIM-imaging, combined lifetime-anisotropy imaging and some of their applications are covered here. 

While VFPs have boosted the applications of FRET-FLIM, chemical FRET probes should not be dismissed. The advantage of chemical probes is that they are much smaller in size and that they often have much better spectral readout than VFP probes. In Chapter 6, Amanda Cobos Correa and Carsten Schultz highlight the various small molecule-based FRET probes and their use in bioimaging. 

For many scientists dedicated FLIM instruments are too expensive and/or too complicated to work with. Therefore, Chapter 7 by Jacco van Rheenen and Kees Jalink is included dealing with low budget but high quality Filter FRET. Filter FRET has the advantage that it is fast, sensitive, direct and inexpensive. However, if you want to do it quantitatively and without errors, you need to... 

In Chapter 11, by Phill Jones et al., biomedical FRET-FLIM applications are reviewed and illustrated. The molecular background of a variety of diseases (e.g., Alzheimer s disease) can be uncovered by using FRET-FLIM. In this major funding area, the killer -applications of the technology are and will be found, leading to a further boost of the implementation and commercial availability of high-end microscopes with automated acquisition and standardized analysis features. 

Laboratory Techniques in Biochemistry and Molecular Biology, Volume 33 FRET and FLIM Techniques T. W. J. Gadella (Editor)... 

Chen, Y., Mills, J. D. and Periasamy, A. (2003). Protein localization in living cells and tissues using FRET and FLIM. Differentiation 71, 528-41. 

Gadella, T., Jovin, T. and Clegg, R. (1994). Fluorescence lifetime imaging microscopy (FLIM) Spatial resolution of microstructures on the nanosecond time scale. Biophys. Chem. 48, 221-39. 

Wallrabe, H. and Periasamy, A. (2005). Imaging protein molecules using FRET and FLIM microscopy. Curr. Opin. Biotechnol. 16, 19 27. 

Frequency domain FLIM theory, instrumentation, and data analysis... 

While publications on fluorescence lifetime imaging microscopy (FLIM) have been relatively evenly divided between time and frequency domain methods, a majority of the 10 most highly cited papers using FLIM have taken advantage of the frequency domain method [1, 2-9]. Both techniques have confronted similar challenges as they have developed and, as such, common themes may be found in both approaches to FLIM. One of the most important criteria is to retrieve the maximum information out of a FLIM... 

In the next section of this chapter, we will review a variety of instrumentation approaches to the FLIM experiment. In particular, we describe conventional systems as well as those designed to observe variation in a, and systems designed for the collection of multifrequency data. In this context, we will also look at data collection strategies and the subsequent first pass analysis of the acquired... 

Although LEDs are emerging as a likely dominant light source for FLIM, there are some regions of the spectrum... 

FLIM systems can be purchased as an add-on for a standard fluorescence microscope. Such a system will consist of a CCD camera coupled to a modulatable image intensifier, an LED light source, and driver electronics. This system will modulate the LED and image intensifier while shifting the phase between them as it takes a series of images (Fig. 2.1). 

An alternative to purchasing a turnkey system is to construct a similar system from components. CCD cameras are available from a wide range of suppliers. For FLIM applications, the CCD need not have particularly high QE or exceptionally low noise. 

Examination of Eqs. (2.9-2.11) suggests that having frequency domain lifetimes measured at a variety of frequencies is desirable, as it will allow a mixture of fluorophores to be determined. With this in mind, two approaches may be taken to obtain multifrequency results. The first of these is simply to make a series of FLIM measurements while stepping through a predetermined set of frequencies. In practice, this is of limited utility for biological systems because of photo-induced damage to the specimen. 

Spectral FLIM involves measuring the apparent lifetimes in a preparation at many wavelengths with the assistance of a spectrograph or a series of filters (see also Chapter 4, Figs. 4.7 and 4.8 depicting hyperspectral FLIM in the time domain). The goal of the measurement is similar to that of the multifrequency approach ... 

Instrumentally, spectral FLIM generates a spectrally resolved set of lifetimes by either introducing filters to provide spectral resolution or a spectrograph between the sample and image intensifier. The first such system was created for looking at the long lifetimes of lanthanide dyes [37]. Later, a spectral FLIM system was described for measuring from a two-dimensional (2D) area of a microscope field... 

Introducing the spectrograph is relatively straightforward compared with the difficulty of assembling and programming a FLIM system and may be completed at reasonable cost (Fig. 2.2). 

Introduction of metalized neutral density filters in FLIM systems should be done cautiously as artifacts have been observed due to single or multiple reflections between pairs of ND filters. 

Once familiar with methods for calibrating the FLIM system, it is worthwhile to verify the range over which a given FLIM system performs well. This is particularly useful for persons new to the method to... 

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