Ratio-imaging

How pectic signals were transduced was unknown, but information suggested that cytosolic free Ca might be involved [22]. Fluorescence ratio imaging has then been used to follow the evolution of free calcium concentrations ([Ca " ] ) after stimulation of carrot protoplasts by oligogalacturonides [23]. 

The first FRET-based biosensors employing fluorescent proteins were developed over 10 years ago. These protease sensors consisted of a BFP donor fused to a GFP acceptor by a protease-sensitive linker [44, 119]. BFP and GFP have well separated emission spectra, resulting in little fluorescence bleed-through (Figs. 5.5A and 5.6A). This facilitates data analysis for FRET ratio imaging... 

Emission ratio imaging is extremely popular due to its simplicity and speed. In essence, cells expressing donors and acceptors are illuminated at the donor wavelength and fluorescence intensity data are collected both at donor (D) and at acceptor (S) channels. Collected data may be either images, or, in case high acquisition speed is crucial and spatial information is not required, dualchannel photometer readings (see Textbox 1). S and D are not overlap-corrected and FRET is simply expressed as the ratio of intensities1 as ratio = S/D. 

Ratio imaging is particularly suited for single-polypeptide FRET sensors. In these constructs FRET changes are due to altered distance and/or orientation of the donor and acceptor, and since the fluorophores are tethered their stoichiometry is always fixed. Thus, the filterFRET problems are easier to address and, assuming full maturation of both FPs [4], it can in fact be shown that under these circumstances two images suffice to calculate FRET quantitatively (see Textbox 1 and Appendix 7.A.6). 

Ratio imaging nicely cancels out some of the main complications in the interpretation of wide-field images in that it normalizes fluorescence intensity differences caused by for example, cell height (Fig. 7.T1) as well as possible slow drift in excitation intensity. Light sources invariably are much less stable than detectors. Incidentally, for these reasons emission ratio imaging has been applied for over 3 decades by the Ca2+ imaging community. 

In general, ratio imaging is not quantitative nor is it, strictly spoken, normalized because the acquired data do not permit Problems 1-4 (see Sect. 7.1.1) to be properly addressed. One important exception is the case where donors and acceptors are present at a fixed stoichiometry. Examples of that are the popular single-polypeptide FRET sensors. In this case, the normalization problem (2) is inherently solved and the overlap- and reference-image problems (1 and 3) simplify considerably. It can be shown [1 and Appendix 7.A.6] that in that case FRET efficiency ( ) can be calculated from D and S images. 

Various optical detection methods have been used to measure pH in vivo. Fluorescence ratio imaging microscopy using an inverted microscope was used to determine intracellular pH in tumor cells [5], NMR spectroscopy was used to continuously monitor temperature-induced pH changes in fish to study the role of intracellular pH in the maintenance of protein function [27], Additionally, NMR spectroscopy was used to map in-vivo extracellular pH in rat brain gliomas [3], Electron spin resonance (ESR), which is operated at a lower resonance, has been adapted for in-vivo pH measurements because it provides a sufficient RF penetration for deep body organs [28], The non-destructive determination of tissue pH using near-infrared diffuse reflectance spectroscopy (NIRS) has been employed for pH measurements in the muscle during... 

Two objects with different spectral properties, i.e., variation in the slope of spectral reflectance curve of two bands, can be separable with the help of ratio images (Lillesand et al. 2007). In this study standard reflectance data of USGS Spectral Library and John Hopkins University spectral library (Available in ENVI) have been used. To enhance the dissimilarity between different rock types in the scene, plots with a higher reflectance were kept in the numerator and plots with low reflectance were kept in the denominator, while taking the band ratios. Using this approach, a ratio of 5/3 was taken for basalt, 7/3 for peridotite, and 4/2 for vegetation. 

Figure 3 displays a Band Ratio image (5/3, 7/3, 4/2), in which peridotite appears dark yellowish black colour. It is difficult to distinguish between gabbro and basalt, due to their similar reflectance properties. 

Fig. 3. Band ratio image of 5/3, 7/3, 4/2 as RGB false colour composite.

If we refer back to figure 2, it is clear that although particle contrasts are usefully enhanced in the ratio image, it is impossible to deduce from this image alone whether the sample is a mixture of Pt and Pd particles or comprises alloyed Pt/Pd clusters. 

Diffraction from Crystalline Supports. Although diffraction conveniently boosts particle contrast in ratio images,... 

Synthesis and Characterization of Dual-Wavelength CU-Sensitive Fluorescent Indicators for Ratio Imaging, Am.J. Physiol. 276, C747-57. 

MLR systems offer many advantages in optical, e-beam, x-ray, and ion-beam lithography. An advantage common to all imaging methods is in enhancement of resist sensitivity. As the resolution and the aspect ratio requirements are separated in an MLR system, faster resists that are usable only for low aspect ratio images can now be candidates for the top layer. Other advantages of MLR systems differ from one imaging method to the other. They will be discussed separately. 

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