Quantitative fluorescence imaging

Quantitative fluorescence imaging techniques and FLIM in particular are becoming increasingly important in biological and biomedical sciences. Knowledge of instrumentation and data analysis is required to avoid misinterpretation of the experimental results and to exploit the wealth of information provided by these techniques. 

Quantitative Fluorescence Imaging Techniques for the Study of Organization and Signaling Mechanisms in Cells... 

Fluorescence microscopy, which has been applied by Jain and co-workers in their studies of interstitial diffusion [20, 21] and lymphatic flow, can be used for measurements within the tissue of a living animal, provided that the tissue can be accessed by light. This access can sometimes be obtained by installing window chambers in the tissue [22]. Multi-photon fluorescence imaging, an important new technique introduced by Webb and colleagues [23-25], promises to broaden the applications of this technique, since quantitative fluorescence imaging can be performed in three-dimensional specimens, even specimens that scatter light. 

FIGURE 14.7 Quantitative fluorescence imaging method for the measurement of solute permeability in a rat pial microvessel. The images were collected during the in vivo experiments and the fluorescence intensity was analyzed off-line. When the fluorescence labeled test solute was injected into the carotid artery, the pial microvessel lumen filled with fluorescent solute (red frame in b), producing AJ . With continued perfusion, the measured fluorescence intensity increased indicating further transport of the solute out of the microvessel and into the surrounding tissue. The initial solute flux into the tissue was measured from the slope (dJ/dt)o (a). The solute permeability P was calculated by P= 1/Afo (dl/dt) rl2. Here, r is the microvessel radius. The scale bar in (b) is 50 Um. (Redrawn from Yuan W et al. 2009. Microvasc Res. 77 166.)... 

Villoing A, Ridhoir M, Cinquin B, Erard M, Alvarez L, Vallverdu G, Pemot P, Grailhe R, Merola F, Pasquier H (2008) Complex fluorescence of the cyan fluorescent protein comparisons with the FU48D variant and consequences for quantitative cell imaging. Biochemistry 47 12483-12492... 

Sanders, R., Draaijer, A., Gerritsen, H. C., Houpt, P. M. and Levine, Y. K. (1995). Quantitative pH imaging in cells using confocal fluorescence lifetime imaging microscopy. Anal. Biochem. 227, 302-8. 

BRET [31, 32]), lock-in detection techniques exploiting optical switches [33], and schemes for alternating D/A excitation (ALEX [34]). The increased attention to quantitative FRET imaging encompasses the use of polarization [35-39], the perennial issue of calibration and standards [40-44], and practical guides to operational principles and protocols ([45, 46] and other references above). The fundamental distinctions between the requirements for live and fixed cell imaging cannot be overemphasized, as is exemplified in a report of erroneous FRET determinations with visible fluorescent proteins (VFPs) in fixed cells [47],... 

Xu X, Brzostowski JA, Jin T (2006) Using quantitative fluorescence microscopy and FRET imaging to measure spatiotemporal signaling events in single living cells. Methods Mol Biol 346 281-96... 

Fluorescence microscopy is routinely used to study the location and movement of intracellular species. In general, the fluorescence image reflects the location and concentration of the probe, or that amount of probe remaining in a photobleached sample (Figure 1.8, lower left). Consequently, quantitative fluorescence microscopy... 

Based on the qualitative and quantitative analyses of a two-dimensional fluorescent image scan using a low laser power, areas within the field can be chosen for rescanning at a higher killing laser power. This results in the selective cell death of those cells that meet a specific fluorescence-labeling criteria, i.e., selecting for those cells that are either above or below a certain fluorescence-intensity threshold value (see Fig. 5). 

Fluorescence images at the start and end of the micro-bead zone reveal that within only a short distance after entering the micro-bead bed, fluorescence is observed, whereas there is no fluorescence before [162], A quantitative fluorescence analysis confirms this visual observation. 

Wang, Y.-L, and Taylor, D.L. (1989b). Fluorescence Microscopy of Living Cells in Culture. Part B Quantitative Fluorescence Microscopy- Imaging and Spectroscopy. Academic Press, New York. 

This technique also has important applications in medicine. For example, it can be used for the spectral classification of a normal human liver cell versus a cancerous liver cell as shown in Figure 4.14. Both the normal human liver cell (E) and the cancerous liver cell (F) contain three dominant types of spectra, each of which is displayed as a distinct colour. However, when the two cells are compared quantitatively, as shown in the histogram area measurements, the abnormalities in the cancerous cell can be quantified objectively. Another example of the use of fluorescence microscopy in medicine is illustrated in Figure 4.15. Using confocal fluorescence imaging, the uptake and distribution of drug (in this case an anthracycline) can be profiled in tumour cells. 

R. Sanders, A. Draaijer, H.C. Gerritsen, P.M. Houpt, Y.K. Levine, Quantitative pH Imaging in cells using confocal fluorescence lifetime imaging microscopy, Analytical Biochemistry 227, 302-308 (1995)... 

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