Filter-FRET

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... 

Sensitized emission FRET (also called filter FRET or channel FRET)—a method that assesses the changes in donor and acceptor fluorescence intensity upon successful energy transfer [26, 48-50] (see Chapter 7) ... 

Wear In case of gouging and abrasion check source of abrasives evaluate affectiveness of lubricants Failure of seals of filters fretting induced by looseness in clamped joints subject to vibration bearing or proper design may lessen or eliminate the failure water contamination High velocities or uneven flow, cavitation... 

FLIM is not the only technique for observing FRET. If both donor and acceptor chromophores are fluorophores, FRET can also be estimated with steady state techniques, including acceptor photobleaching [102,103], spectral imaging [61,104] and ratio-imaging (or filter-FRET) [105,106] techniques. Recently all possible modes of FRET-microscopy (also including yet unexplored FRET-imaging modes) have been reviewed by Erijman and Jovin [107]. 

With each method of FRET estimation there are advantages and disadvantages. Here, specifically the three most widely used other FRET-imaging techniques besides FRET-FLIM i) acceptor-bleaching, ii) filter-FRET and iii) spectral imaging are briefly discussed in relation to FLIM. Also elsewhere, reviews and experimental studies have appeared comparing FLIM with other FRET-microscopy techniques [61,63,108]. 

Filter-FRET (or ratio-imaging) methods have also become quite popular for measiuing FRET. Usually three images with different filter sets are obtained ... 

Figure 9 illustrates the fret that the release of biocide from the carriers is a dynamic process. Here a quantity of loaded carrier was slurried with a fixed volume of water and aliquots taken after 1 hour. From previous experiments it was found that after an initial period of rapid release, a steady-state concentration of free biocide was present in the aqueous extract. To probe the effects of repetitive extraction, the carrier was filtered from the slurry, the water replenished and the process repeated. It can be seen that only after ten successive extractions does the amount of the biocide OIT released by the carrier fall below the MIC. It should be noted that the conditions employed to illustrate this continuous release are rather more severe than would be experienced when the loaded carrier is incorporated in a coating (see Section 2.5). 

The term filterFRET here refers to intensity-based methods for calculating fluorescence resonance energy transfer (FRET) from sets of images of the preparation collected at different excitation and/or emission wavelength. The term is not intended to imply that interference filters are actually present in the setup very similar considerations apply when donor- and acceptor fluorophores are spectrally resolved by other means, such as monochromators or spectral detectors. 

Note that G as derived here relates the FRET-induced sensitized emission in the S channel to the loss of donor emission in the D channel and that it is identical to the correction factor y/ [2] or G [6, 14]. Note however, that if the correction factors or 5 change, G and

correction factor C [3] is a constant that depends only on fluorophore properties and filter settings, and therefore it does not change with excitation intensity or detector gain. This is a clear advantage for confocal filterFRET. C (Eq. (7.14)) and G (Eq. (7.19)) are related as ... 

The fluorescent components are denoted by I (intensity) followed by a capitalized subscript (D, A or s, for respectively Donors, Acceptors, or Donor/ Acceptor FRET pairs) to indicate the particular population of molecules responsible for emission of/and a lower-case superscript (d or, s) that indicates the detection channel (or filter cube). For example, / denotes the intensity of the donors as detected in the donor channel and reads as Intensity of donors in the donor channel, etc. Similarly, properties of molecules (number of molecules, N quantum yield, Q) are specified with capitalized subscript and properties of channels (laser intensity, gain, g) are specified with lowercase superscript. Factors that depend on both molecular species and on detection channel (excitation efficiency, s fraction of the emission spectrum detected in a channel, F) are indexed with both. Note that for all factorized symbols it is assumed that we work in the linear (excitation-fluorescence) regime with negligible donor or acceptor saturation or triplet states. In case such conditions are not met, the FRET estimation will not be correct. See Chap. 12 (FRET calculator) for more details. 

Used to relate the loss in D due to FRET to the gain in S due to FRET independent from excitation intensity and gain and therefore constant for given filters and fluorophores negative... 

This is straightforward in case the A and S filters are identical (i.e., F =f and F% = Fd). With confocal FRET this is commonly the ease with CCD imaging, it requires matching the filters. Without this assumption, an analogous result can be obtained, although derivation is significantly more complicated. 

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