Quantitative fluorescence imaging technique

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. 

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],... 

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. 

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