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Technical Notes

System
Specifications

System
Enhancements

Excitation Ratio is used to set up and run experiments for intracellular ion determinations using excitation-shifted probes such as Fura-2 for calcium and BCECF for pH. In this experiment, the excitation source must alternate between two different excitation wavelengths that are characteristic of the probe. The emission intensity at both excitation wavelengths is measured at a longer emission wavelength and the ratio of these intensities is calculated. The ratio is proportional to the concentration of the ion under investigation.

Excitation 1,2
Enter the excitation wavelengths in the text boxes. Your instrument will automatically alternate between excitation wavelength 1 and excitation wavelength 2. The rate of alternation is dependent upon the illuminator type. The patented PTI DeltaRam V can provide up to 250 ratios/sec while the DeltaScan X can produce ultra-fast switching allowing for 650 ratios/sec. A model 101M monochromator must move from one excitation wavelength to the other at the slewing speed set in the hardware configuration.

Emission
Enter the emission wavelength in the text box. If your instrument has two emission monochromators, FeliX32™ will ask for two emission wavelengths. The wavelengths you enter will be the wavelengths to which the monochromators will automatically move prior to data acquisition. If your system uses filters for wavelength selection, simply enter the peak wavelengths of the filters in these boxes.

Enable Single Point Screening
This is used to collect single data points into a spreadsheet display.

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Emission Ratio is used to set up and run experiments for intracellular ion determinations using emission-shifted probes such as Indo-1 for calcium and SNAFL for pH. In this experiment, a constant excitation wavelength is used and two emission wavelengths must be selected. This is normally done with two monochromators in a cuvette system, but one monochromator can be utilized. In a microscope-based system, the two emission wavelengths are selected using a dichroic assembly in the photometer. The emission intensity at both emission wavelengths is measured and the ratio of these intensities is calculated. The ratio is proportional to the concentration of the ion being determined.

Excitation
Enter the excitation wavelength in the text box. The wavelengths you enter will be the wavelengths to which the monochromators will automatically move prior to data acquisition. If your system uses filters for wavelength selection, simply enter the peak wavelengths of the filters in these boxes.

Emission 1,2
Enter the emission wavelengths in the text boxes. Your instrument will automatically alternate between wavelength 1 and wavelength 2. The rate of alternation is dependent upon the configuration. Dual emission systems provide up to 1000 ratios per second. Single monochromator emission systems slew between the wavelengths to provide up to 1 ratio per second. A model 101M monochromator must move from one excitation wavelength to the other at the slewing speed set in the hardware configuration.

Enable Single Point Screening
This is used to collect single data points into a spreadsheet display.

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In an Excitation Scan, the excitation monochromator is scanned between two wavelengths while the emission monochromator is fixed. The emission intensity is measured as a function of excitation wavelength.Due to the nature of fluorescence, the emission wavelength is set at a wavelength that is longer than the excitation wavelength range (red-shifted).

Start and Stop
Enter the initial excitation wavelength and the final excitation wavelength for the scan in these text boxes. Emission Enter the emission wavelength in the text box. If your instrument has two emission monochromators, FeliX32™ will ask for two emission wavelengths.

Length
This shows the length of the scan that will be run. If the starting wavelength and the length are entered, FeliX32™ will calculate the ending wavelength corresponding to these parameters.

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In an Emission Scan, the emission wavelength is scanned between two wavelengths while the excitation monochromator is fixed. The emission intensity is measured as a function of excitation wavelength. Due to the nature of fluorescence, the excitation wavelength is set at a shorter wavelength than the emission wavelength range.

Excitation
Enter the excitation wavelength in the text box. Start and End wavelength Enter the emission wavelength scanning range in the Emission 1 text boxes. If the system is equipped with two emission monochromators, FeliX32™ will request two wavelength ranges.

Length
This shows the length of the scan that will be run. If the starting wavelength and the length are entered, FeliX32™ will calculate the ending wavelength corresponding to these parameters. For dual emission systems, the length of the scan will be the identical for both emission channels. FeliX32™ will adjust the emission ranges to ensure they both have the same length.

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In a Synchronous Scan, the excitation and emission monochromators are scanned simultaneously at identical scan rates with a constant wavelength difference between them. A synchronous scan often results in the simplification of complex excitation or emission scans.

Excitation Range
Enter the excitation wavelength range in the text boxes.

Emission Range(s)
Enter the emission wavelength range in the text boxes. If the system is equipped with two emission monochromators, FeliX32™ will request two wavelength ranges.

Length
Enter the scan range in nanometers. This value will be calculated for you if a range is entered. If starting wavelengths and length is entered, FeliX32™ will calculate the ending wavelengths based upon these parameters. The length for all monochromators, excitation and emission, will be identical. FeliX32™ will adjust the range values to ensure that the length is the same.

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The Multiple Dyes function is used to set up and run experiments for intracellular ion determinations using several indicators in combination, such as Fura-2 for calcium and BCECF for pH. In this experiment, the excitation light source must alternate between four different excitation wavelengths that are characteristic of the two probes (e.g. 340, 380, 440, 490-nm). In addition the isosbestic wavelength for Fura-2 is frequently monitored at 361 nm to obtain a calcium-independent signal.

The emission intensity resulting from excitation at the above five wavelengths is measured at longer emission wavelengths (510 and 525 nm, respectively) and the ratio of these intensities is calculated. The ratio is proportional to the concentration of the ion under investigation. Any combination of up to 10 excitation and 10 emission wavelengths may be defined to accommodate the simultaneous measurement of both excitation- and emission-shifted dyes.

Use
Use the checkboxes to select the number of wavelength pairs for the experiment.

Ex
Enter the excitation wavelengths in these text boxes.

Emi. 1 and Emi. 2
Enter the emission wavelengths in these text boxes. If your system only has a single emission channel, the system will only display a single column for entering emission wavelengths.

Enable Single Point Screening
This is used to collect single data points into a spreadsheet display.

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In a Timebased experiment, the excitation and emission wavelengths remain fixed throughout the experiment. The emission intensity is measured as a function of time. Timebased experiments typically involve kinetic measurements.

Excitation
Enter the excitation wavelength in the text box.

Emission
Enter the emission wavelength in the text box. If your instrument has two emission channels, FeliX32™ will ask for two emission wavelengths. The wavelengths you enter will be the wavelengths to which the monochromators will automatically move prior to data acquisition. If your system uses filters for wavelength selection, simply enter the peak wavelengths of the filters in these boxes.

Enable Single Point Screening
This is used to collect single data points into a spreadsheet display.

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Polarization measurements can be done in several ways in FeliX32™. Some methods include using a timebased experiment or excitation/emission scans. One of the easier ways to perform polarization is to use the Timebased Polarization acquisition, which is useful for performing the polarization measurements under complete FeliX32™ automation, including measuring the G-factor. Anisotropy and polarization will be calculated in real time and displayed in the workspace. This experiment is mainly intended for systems with a single emission channel using motorized polarizers, although manual polarizers may be used as well. The various parameters have much in common with the other experiments.

Excitation
Enter the excitation wavelength in the text box.

Emission
Set the emission wavelength for the experiment in the text box.

Enable Single Point Screening
This is used to collect single data points into a spreadsheet display. It is useful for measuring the polarization of a number of samples. The two intensities, polarization, and anisotropy for the sample will be determined.

Background/G-Factor
The system can perform background subtractions and G-Factor calculations automatically. The parameters for these measurements may be set to different values than the above section, which contains the information to run the polarization experiment on the sample. The measurement of G-Factor and background may be controlled using either the Calculate G-Factor or Acquire Background checkbox.

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Toggling this control determines if the G-Factor is calculated during the experiment, or whether a previously acquired G-Factor is used. If a pre-determined G-Factor is used, it is set in the Configure menu found beside this checkbox. Filter based systems do not require the calculation of a G-Factor. To cancel the acquisition of a G-Factor, enter the Configure menu and set the G-Factor to a value of 1.

Configure G-Factor
Entering this menu allows the selection of a pre-determined G-Factor. There are two options that may be followed. You can enter a known G-Factor into the G-Factor text box or you may use a G-Factor lookup table. Please refer to Chapter 10 for more information on lookup tables.

Note. The value of the G-Factor is wavelength dependent, therefore remember to use an appropriate G-Factor for the experiment.

Point by Point Polarization
When selected, this feature forces FeliX32™ to rotate the emission polarizer between vertical and horizontal at a rate determined by the points/second. The polarization and/or anisotropy will be determined one point at a time. If toggled off, one full measurement for the set duration will be acquired for each polarizer orientation.

Display
The Trace Setup menu for Timebased Polarizations is different than that for the other acquisitions. The Curve Set in the Raw data window represents all the curves that will be generated during the experiment including G-Factor measurements, raw data, and anisotropy and polarization traces. If a four cuvette turret is used, it is possible to toggle the visibility of individual curve sets by selecting Hide Trace for each sample that you wish to hide. The rest of the Trace Setup features remain identical in nature to the other acquisitions.

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Int Time
This is the time in microseconds for which the integration window is open for each lamp pulse. Since, in this case, the observation window is defined by the integration time, increasing the integration time will increase the signal at the expense of lifetime resolution while decreasing the integration time will increase the lifetime resolution at the expense of signal strength. In particular, when the instrument is used to separate fluorescence spectra from phosphorescence spectra, care must be used in selecting the integration time. Since fluorescence is essentially over in the first 5 to 10 µs after the excitation pulse, the delay should be set to the excitation peak and the integration time to 5 to 10 µs. Longer integration times will contaminate the fluorescence with phosphorescence. When collecting phosphorescence, the delay should be set 5 to 10 µs after the excitation pulse and the integration time chosen to be larger to maximize sensitivity.

Shots
Enter the number of lamp pulses to be collected and averaged at each delay for each scan. Extra shots will improve the signal to noise ratio at the expense of additional acquisition time.

Frequency
The lamp frequency can be set up to 100 Hz. For very long-lived samples, the phosphorescence from one pulse may not have completely decayed before the next pulse arrives. At least ten sample lifetimes should be allowed between each lamp pulse. Thus a lamp frequency of 100 Hz may be used for samples whose lifetimes are shorter than 1000 µs.

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