In ratio fluorometric experiments a scanning monochromator is useful for investigating intracellular cation concentrations. This article describes the various benefits of using the scanning monochromator.
Avoiding Gross Overestimation of [Ca++]1
It is important to measure the fluorescence excitation spectrum of the indicator dye in situ to prove its sufficiency for a meaningful measurement.
For instance, to examine the changes in the intracellular concentration of calcium, the membrane-permeable acetoxymethyl form of the calcium-sensitive dye Fura-2 is loaded into cells. Then, intracellular esterases cleave the acetoxymethyl group, thus enabling the chelation of Ca++.
The unhydrolyzed portion of Fura-2/AM is not sensitive to calcium and will appear as a red-shifted peak at 390-400nm. The unhydrolyzed dye significantly affects calibration.
The following analytical expression is used to change measured 340/380 nm fluorescence ratios into calcium concentrations.
[Ca++] = Kd x B x (R-Rmin)/(Rmax-R)
Both Rmin and Rmax need to be ascertained from the measurement. At the conclusion of a measurement, calcium is used to saturate the intracellular dye using a Ca++ ionophore and saturating concentrations of calcium.
The AM form, which is insensitive to calcium, will not undergo a wavelength shift like the sensitive form. Figure 1 shows the effect of residual unhydrolyzed dye on the 340/380nm ratio.
Figure 1. Effect of remaining unhydrolyzed dye on the 340/380nm ratio
Avoiding Over- or Under-Estimation of [Ca++]1
Due to an increase in the level of the background signal, the Rmax ratio has a reduced value. This occurs often because various chemicals are added during the course of the measurement.
However, when detergents such as triton or digitonin are added to solubilize membranes, they can significantly lower the background because of the solubilization of light-scattering particles suspended in the medium.
In case background subtraction is performed in real-time and the background level changes, the data will be incorrect.
Monochromators Offer Continually Adjustable Bandpass
The excitation monochromators deliver adjustable bandpass from 0.25 to 25nm with the 1200 lines/mm grating. When bandwidth of the excitation is narrow, one can obtain better dynamic range of measurement.
In order to measure pH or intracellular [Ca++] using BCECF or Fura-2, respectively, 2 or 3nm bandpass is normally used on the monochromators of the PTI DeltaScan illuminator. Figure 2 shows the effect of increasing bandwidth of the excitation light on the 340/380nm fluorescence ratio.
Figure 2. Effect of increasing bandwidth of the excitation light on the 340/380nm fluorescence ratio
Excitation Wavelengths at Isobestic Point
In some experiments, it is advantageous to select one of the excitation wavelengths at the isobestic point of the dye. In Fura-2, the fluorescence intensity at the isobestic point does not vary with [Ca++]I and hence offers a measure of events irrespective of the [Ca++]I, such as dye leakage, light scatter or shape changes.
Depending on the intracellular environment, the wavelength of the isobestic point may be in the range 356-362nm and here accurate positioning is important. Even a small error of 1nm will result in a [Ca++]I-dependent contribution and make the measurement invalid.
Microscope objectives are advanced assemblies of optical elements having multiple optical surfaces. When the excitation spectrum of the dye is measured, the transmission properties of the objectives can be verified.
Based on the quality of the microscope objectives available for work with Fura-2, one may need to measure 352/380, 345/380 or 347/380 ratios instead of the 340/380 ratio to attain desirable signal-to-noise ratios in the measurements.
Measurement of Fluorescence Excitation Spectra of Sample
When working with multiple probes, it is important to assess the amount of spectral overlap between the probes and the resultant interference between them. The dyes’ fluorescence quantum yield may be considerably different depending on their relative concentrations, and hence the optimal wavelengths of excitation and observation may be different from those utilized with the individual dyes.
Moreover, the two parameters pH and [Ca++] must be measured independently, despite the spectral overlap between their emission spectra and fluorescence excitation.
Figure 3 shows the results of an inappropriate choice of measurement parameters used to attain a meaningful titration of two dyes. Figure 4 shows a successful concurrent double titration obtained using the same buffers with appropriately chosen measurement conditions.
Figure 3. Results of inadequate choice of measurement conditions to acquire meaningful titration of two dyes.
Picture 4. Results of successful double titration obtained using same buffers with properly chosen measurement parameters.
Scanning Enables Detection of Inner Filter
Scanning helps in detecting the inner filter effect that pinpoints the cells overloaded with indicator dye. A Fura-2-loaded cell may prevent buffering of intracellular calcium.
Figure 5 shows the fluorescence excitation spectra of two adjoining Fura-2-loaded cells acquired with the PTI ImageScan.
Figure 5. The fluorescence excitation spectra of two Fura-2-loaded cells achieved using PTI ImageScan.
Additionally, when electronic excitation energy transfer takes place between acceptor and donor, the contribution from the fluorescence excitation bands of the donor molecule can be identified in the fluorescence excitation spectrum of the acceptor. Furthermore, the spectral overlap integral between acceptor absorption and donor fluorescence helps in measuring intermolecular distances between acceptor and donor molecules.
Monochromators are excellent alternative to optical filters, which tend to degrade, crack, solarize, smudge and get lost over time. In contrast, monochromators do not degrade even with extended use.
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