RF Application - Demonstration Guide For Super Ion Probe Software RF-5301

Shimadzu Scientific Instruments, 7102 Riverwood Drive, Columbia, MD 21046
Tel: 800.477.1227/410.381.1227; Fax: 410.381.1222; www.ssi.shimadzu.com
Demonstration Guide For Super Ion Probe Software
I. Introduction
This document describes the operation of the Super Ion Probe software for the
RF-5301PC spectrofluorometer. It consists of a brief description of the
application addressed by the software, a tutorial on the software using stored
data files, a description of the steps involved in gathering real time data using
a test sample, and an appendix containing additional details on the
calculations involved and sources for the reagents required for duplicating the
exercise on data acquisition.
The Super Ion Probe software allows the user to measure changes in the
intracellular concentrations of physiologically important ions. Cells, as
fundamental units of all tissue, continuously monitor and respond to changes
in their external environment. A scheme illustrating some of the features of
this process is shown in Figure 1. There are sensors in the outer membranes of
cells which monitor the conditions present in the external media. In Figure 1,
such a sensor is labeled 'Response Element'. The response elements can detect
changes in chemical, electrical, or mechanical stimuli. Usually, there are
specific response elements called 'Receptors" for a particular type of stimulus.
If a stimulus exceeds a threshold, a series of events is set into motion. The
response element or receptor receives the primary signal that a given threshold
has been exceeded. The receptor then triggers one or more processes which give
rise to secondary signals called 'Second Messengers'. These second messengers
are frequently simple chemical compounds or ions. The second messengers
work with some type of transducer which is capable of translating the change
in concentration of a simple compound into a more complex response and which
also typically amplifies the response. Transducers are often enzymes or
regulatory proteins. Transducers operate with 'Effectors' to produce an
appropriate response to the original stimulus. The response, too may be
chemical, electrical, or mechanical. An example might be a nerve cell in contact
with a muscle cell as shown in Figure 2. An electrical signal is transmitted
down the long part of the nerve cell called the axon. When it reaches the end of
the axon, the electrical signal triggers the release of a chemical called a
transmitter. The transmitter interacts with the receptor on the muscle cell. The
receptor initiates a sequence resulting in the mechanical response of muscle
contraction. In this example, there are changes in an intracellular ion, i.e. Ca+2
involved in the sequence of events in both cells. The final trigger causing
release of the transmitter in the nerve cell is an increase in calcium
concentration. Likewise, when the transmitter interacts with the receptor on
the muscle cell, one of the first things that happens is an increase in the
concentration of calcium within the cell.
There are a number of intracellular ions including K+ , Na+, Mg+2, and H+ with
important regulatory functions. However, the most common and critical ion
regulator is Ca+2.
Figure 1
Cellular Stimulus-Response Sequence
Figure 2
Interaction Between Nerve And Muscle Cell
Because calcium is a crucial participant in so many cellular functions, it has

earned the titles "Universal Regulator" and "King of the Second Messengers". It
is readily understandable, then, why there is such an enormous amount of
basic and applied research where measurement of intracellular calcium
concentration is required. For example,the cardiovascular pharmacologist
seeking more effective calcium channel blockers, and the neurobiologist
studying the mechanism of programmed cell death in degenerative disorders
are both likely to use intracellular calcium measurements in their research.
One of the most widely used techniques for measurement of intracellular ion
concentrations involves the use of ion-sensitive fluorescent dyes. Such dyes are
typically chelators containing carboxyl functional groups and are introduced
into the cell by incubating a cell monolayer or suspension with the ester form of
the dye. The dye-ester enters the cell via diffusion. Once inside the cell, the
ester is hydrolyzed and the acid form of the dye is released. An equilibrium is
established between the dye and a particular intracellular ion and the
fluorescent signal in such a sample is proportional to the ratio of free dye to
dye-ion complex. Quantification of intracellular ions with these types of
indicators generally requires that measurements be made at two different
excitation and/or emission wavelengths. The ratio of the signals is used to
calculate the concentration of the ion(See appendix for more details).
The fluorescent dye 'Fura-2' has been used extensively as a fluorescent

indicator for intracellular calcium. This dye is structurally similar to the
calcium chelator EGTA. The free dye, in the absence of calcium, has an
excitation maximum wavelength of 380nm. In excess calcium, so that all of the
dye is calcium-bound, the excitation maximum wavelength shifts to 340nm.
The emission maximum wavelength is approximately 510nm for both
conditions. Thus, the ratio of fluorescence at 510nm using excitation
wavelengths of 340 and 380nm is proportional to the ratio of calcium-bound to
calcium- free dye.
The next section of this document is a tutorial on the Super Ion Probe software
using data obtained from measurements on Fura-2.
II. Tutorial Using Stored Data
This tutorial does not require an instrument. The only requirement is

successful installation of RF-5301PC and Super Ion Probe Software.
Open the 'Shimadzu' program group. Double click on the 'Super Ion Probe' icon.
The display should resemble that shown in Figure 3:
Figure 3
Initial Display
Click on 'File' in the menu bar. Click on 'Open'. Select 'Supercap/data' as the
directory. Select 'Fura.exp' as the file and click on 'OK'. Click on 'View' in the
menu bar. Three types of graphs may be displayed simultaneously: Data

Graphs, Ratio Graphs, and Concentration Graphs. These graphs may be
displayed or hidden by clicking on them in this drop-down menu. Use the left
mouse button to hide the Ratio and Concentration Graphs and display the Data
Graph. Place the cursor near the center of the Data Graph (Any position on the
graph will work) and double-click to perform an autoscale of the data. The
display should resemble that shown in Figure 4:
Figure 4
Data Graph
Click on 'View' and 'Data Display'. The menu shown in Figure 5 will be
displayed. Highlight 'Wavelength Pair 1' and click on the 'Params...' button.
The parameters associated with wavelength pair 1 will be displayed as shown
in Figure6. Note that the data was obtained using excitation and emission
wavelengths of 340 and 510nm, respectively. The other pushbuttons in this
window are: 'Export' for exporting the data in ASCII or RF-5301PC format,
'Rename' for renaming data, 'Show/Hide' and 'Show All/Hide All' for control of
on-screen data display, and 'Delete' for deleting data from RAM. Highlight
'Wavelength Pair 2' and click on the 'Params...' button.
Figure 5
Data Display
Figure 6
Parameters associated with wavelength pair 1
The parameters associated with wavelength pair 2 are displayed as shown in

Figure 7.
Figure 7
Parameters associated with wavelength pair 2
In Figure 4, the upper tracing is wavelength pair 1, i.e. excitation at 340nm and
emission at 510nm. This tracing represents the signal from Ca+2 - bound dye.
Conversely, the lower tracing in Figure 4 is wavelength pair 2, i.e. excitation at
380nm and emission at 510nm. This tracing represents the signal from Ca+2
-free dye.
The data in Figure 4 is the first step in the process of calculating calcium
concentrations. Background data, if used, would be obtained separately, using
an aliquot of cells that did not contain dye, or an aliquot of dye-loaded cells that
received no additional treatment. With such a sample in the cell holder, the
'Bkg' button on the bottom of the screen would be pressed. The results would be
stored as a .bkg file and subtracted (under 'Manipulate', "Background

Subtract...' )from corresponding sample data before any further calculation.
Background data at 340nm excitation would be subtracted from sample data at
340nm excitation. A similar procedure would be used for background and
sample data at 380nm. For additional details, see section III, Data Collection
Demonstration.
Note that there are a couple of steps in the tracings in Figure 4. Each of these
steps corresponds to a specific addition made to the sample during the run
using the 'Pause' and 'Continue' keys. In Figure 8, the points labeled 1 and 2
represent additions of FMLP and Digitonin, respectively. FMLP is a peptide
which increases intracellular calcium in neutrophils. The magnitude of the
response shown in Figure 8 is typical of a variety of agents which induce
increases in intracellular calcium. Digitonin is a surface active agent which
solubilizes the cell, releasing entrapped intracellular dye into the external
medium. This treatment is one method of generating calcium-saturated dye
and achieving maximum signal (Rmax). The point in Figure 7, labeled 3,
represents addition of excess EGTA and base to the sample which generates
calcium-free dye and yields the minimum signal (Rmin).
Figure 8
Additions to sample at Events Marks
Note that under conditions where the concentration of calcium increases, there
is an increase in the signal using 340nm excitation and 510nm emission and a
decrease in the signal using 380nm excitation and 510nm emission. This is
characteristic of the dye Fura-2. A table containing information on additions
similar to that shown in Figure 9 can be viewed by clicking on 'View' and 'Event
Marks'.
Figure 9
Event Marks
The table of event marks can also be saved as a text file for incorporation in
plots and reports.
The ratio of signals using 340 and 380nm excitation are calculated and
displayed on the screen in real time (See Section II, Data Collection
Demonstration). In order to display the Ratio Data, click on 'View' and 'Ratio
Graph'. Place the cursor on the Ratio Graph and double click to autoscale the
data. Note that the screen is now split with the Data Graph on top and the
Ratio Graph on the bottom. Click on 'View' and 'Data Graph' to hide the
individual data files collected using 340 and 380nm excitation and display only
the ratio data. The graph should resemble that shown in Figure10.
Figure 10
Ratio Graph
The events at the points labeled one, two, and three are the same as those
described in Figure 8. As indicated in Figure 11, the plateau after the addition
of digitonin is the region of maximum signal Rmax while the portion of the curve
after the addition of EGTA and base is the region of minimum signal Rmin. In
order to calculate calcium concentration, these boundary conditions of
maximum and minimum signal must be generated. In a typical experiment,

each sample would be so treated as to yield the required boundary conditions.
In this way, each individual sample serves as it's own control.
Figure 11
Identification of boundary conditions Rmax and Rmin
The next step is to calculate calcium concentrations. Click on 'Manipulate' and

'Concentration'. A dialog box similar to that shown in Figure 12 will be
displayed. Configure the items in the dialog box as shown in Figure 12:
Figure 12
Concentration Dialog Box
Experiment=Fura, Source=Pair1/Pair2, Method= C=((R-Rmin)/(Rmax-R)) Sf2/Sb2 .

Enter 125 for Kd and 49 and 32 for Sf2 and Sb2 respectively. Click on 'Next'. A
dialog box similar to that shown in Figure 13 will be displayed.
Figure 13
Selection of Maximum and Minimum Values
Position the cursor over the upper horizontal index line in the figure labeled
'Max'. Note that the cursor becomes an up/down arrow. Hold the left mouse
button down and drag the line to the top of the tracing, as shown in Figure 13.
While you are moving the line, the number in the box on the right labeled 'Max'
also changes. A value for 'Max' may also be entered manually. Position the
index line or make an entry so that a value of 3.678 is in the box labeled 'Max'.
In a similar fashion, use the cursor to move the lower horizontal index line or
make an entry such that a value of 1.129 is in the box labeled 'Min'. This
procedure designates the values for Rmax and Rmin to be used in calculating
calcium concentrations.
An alternative procedure allows averaging data to obtain Rmax and Rmin. To use
this feature, check the box labeled 'Average max and min'. The dialog box
changes as indicated in Figure 14. Note that there are now four vertical index
lines on the graph. Two of these lines are labeled 'Max' at the bottom and two
are labeled 'Min'. These lines may be positioned to designate start and stop
points on a range of data on which an average will be calculated. The data
between the lines labeled 'Max' will be averaged to give Rmax. Similarly, the
data between the lines labeled 'Min' will be averaged to give Rmin. Position the
cursor over one of the lines. Note that the cursor becomes a left/right arrow.
Hold the left mouse button down and drag the line to the desired position.
While a line is being moved, the corresponding value in the box on the right
also changes.
Figure 14
Using Average Max and Min
Click on the box labeled 'Average max and min' to deselect this feature. Note
that the display returns to a figure with one horizontal line labeled 'Max' and
one horizontal line labeled 'Min'. As described earlier, position these lines or
make entries so that 3.678 and 1.129 are in the boxes labeled "Max' and 'Min'
respectively.
Press the 'Next' button. A screen similar to that shown in Figure 15 will be
displayed.
Figure 15
Execute Concentration
Note that in this screen, there is a box in the upper left labeled 'Points'. To the
right of this box is a scroll button. Pressing this button displays a menu
containing 'Discrete' and 'Range' as selections. If 'Discrete' is selected, calcium
concentrations will be calculated at designated points along the ratio data
curve. Select 'Discrete'. Note that there is a box to the left labeled 'Time'. In the
center of the screen is the ratio data curve and a single vertical index line. The
bottom of the screen contains a table labeled 'Selected Points' with columns
labeled 'Point No.' , 'Time' , and 'Ion Concentration'. Position the cursor over the
vertical index line. Note that the cursor becomes a left/right arrow. Hold the left
mouse button down and drag the vertical index line along the ratio data curve.
Stop at 25 seconds, as shown in Figure 16.
Figure 16
Execute Quantitation
Press the 'Execute' pushbutton. Note that an entry is made in the table at the
bottom of the screen. The 'Point No.' is 1 , the 'Time' is 25sec, and the 'Ion
Concentration', in this case, Ca+2, is calculated to be approximately 128. Now
move the vertical index line along the ratio data curve and stop at 65 seconds.
Press 'Execute'. A second entry is made in the table, i.e. point number two at 65
seconds, with a calculated concentration of approximately 276. The display
should resemble that shown in Figure 17. Note that no units for concentration
are displayed. This is because the units depend on the method used for
calculation. In the example here, the units are defined by the units for the K d .
If the Kd was entered with units of nM, then the results calculated and
displayed in the column labeled 'Ion Concentration' will also be nM.
Press the 'Add' pushbutton. A dialog box similar to that shown in Figure 18 will
be displayed.
Figure 17
Execute Quantitation
Figure 18
Add Data Set
Select 'Add to the existing exp.' . Pressing 'OK' will append the calculations to
the original data. Note that in this case, the name appearing in the box is the
name of the
original experiment. In this example, the original experiment was named 'Fura'
so "Fura' appears in the box. Since calculations are being added to an existing
experiment, it is not possible to edit the experiment name. Press 'OK'. A dialog
box similar to that shown in Figure 19 will be displayed.
Figure 19
Naming a Data Set
For 'Data Set Name', enter 2pt. For analyst, enter 'Your Name', and for
Comment enter 'Comment'. Click on the 'OK' button to save this set of data in
RAM. At this point, the experiment file named 'Fura' has appended to it a set of
data consisting of calculated calcium concentrations at two time points. This
set of data is named 2pt. To illustrate this, return to the main menu and click
on 'View' and 'Data Display'. The display should resemble that shown in Figure
20. In accordance with the commands just executed, the experiment named
'Fura' has two data files of raw data named 'Wavelength Pair 1' and
'Wavelength Pair 2', a ratio data file named 'Pair1/Pair2', and the appended file
containing the calculated calcium concentrations named '2pt'.
If desired, a separate experiment can be created from the calculated data and
given a different name. To illustrate this feature, return to the main menu and
select 'Manipulate' and 'Concentration'. Configure the 'Concentration' dialog

box as follows: Experiment=Fura, Source=Pair1/Pair2,
Method=C=Kd((R-Rmin)/(Rmax-R))Sf2/Sb2 , Kd=125, Sf2=49, Sb2=32. Click on
'Next'. Configure the 'Max and Min' dialog box as follows: Max=3.678,
Min=1.129. Click on 'Next'. Configure the 'Execute Quantitation' dialog box as
follows: Points=discrete, time=75. Click on 'Execute'. Click on 'Add'. Select
'Create exp'. Enter 'Onept' for 'Data Set(Experiment)Name and click 'OK'.
Figure 20
Data Display Showing Appended Data
Enter 'Your Name' for analyst, and 'Comment' for comment. Click on 'OK'.
Return to the main menu and select 'View' and 'Data Display'. Click on the
scroll-down arrow and observe that a new experiment named 'Onept' is in RAM.
Select 'Onept' and observe that this experiment has in it, a single data set
named '75'. The display should resemble Figure 21.
Figure 21
Creation Of A New Experiment From Appended Data
Note that at this point, neither data set '2pt' nor data set 'Onept' has been
saved to disk. In order to save any file to disk, the 'Save' command under 'File'
must be executed.
Calcium concentrations may also be calculated over an interval rather than at

discrete points. Return to the main menu. Select 'Manipulate' and
'Concentration'. Configure the 'Concentration' dialog box as follows:
Experiment=Fura, Source=Pair1/Pair2, Method=
C=Kd((R-Rmin)/(Rmax-R))Sf2/Sb2, Kd=125, Sf2=49, Sb2=32. Press 'Next'. Configure
the 'Max and Min' dialog box as follows: Max=3.678 and Min=1.129. Press
'Next'. Configure the 'Execute Quantitation' dialog box as follows:
Points=Range. Note that at this point, the display changes to that shown in
Figure 22.
Figure 22
Quantitation Over A Range
Note that there are two vertical index lines. Position the cursor over one of the
index lines. When the cursor becomes a left/right arrow, hold down the left
mouse button and move the index line. Note that while doing so, the value in
the corresponding box labeled 'Start' or 'End' also changes. The value in the box
labeled 'Start' corresponds to the position of the left vertical index line while
the value in the box labeled 'End' corresponds to the position of the right
vertical index line. Values for 'Start' and 'End' may also be entered manually.
Enter values for 'Start' and 'End' of 1 and 100, respectively or move the vertical
index lines until these values are displayed. Click on 'Execute'. Note that a new
graph, similar to that shown in Figure 23 has been displayed. The ordinate of
this graph is calcium concentration. Press 'Add', and select 'Add to the existing
expt.' Press 'OK'.
Figure 23
Execution of Quantitation Over A Range
Enter 'Your Name' for analyst, and 'Comment' for comment. Press 'OK'. Return
to the main menu. Press 'View' and check on 'Concentration Graph'. Note that a
third graph, i.e. concentration vs time has been added to the display. Place the
cursor on the concentration graph and double click to perform an autoscale.
The display should resemble that shown in Figure 24. Click on 'View'. Click on
'Data Graph' and 'Ratio Graph' to deselect these graphs and display only the
Concentration Graph.
When selecting the start and end points, it is important to exclude the
boundary conditions as shown in Figure 25. By definition, the boundary
conditions are limits which are used in calculating calcium concentrations in
the remainder of the range. Although the software will allow inclusion of the
maximum and minumum boundaries in the calculation range, it is
inappropriate to do so and the results will not be useful.
Data may be exported as ASCII or RF-5301 files. Click on 'View' and 'Data
Display'. Highlight 'Range1' and click on 'Export'. Note that a box is displayed
with header information and a list of data points. The display should resemble
that shown in Figure 26.
Figure 24
Display Of Data, Ratio, And Concentration Graphs
Figure 25
Exclusion Of Boundary Conditions
Figure 26
Data Export
Click on 'File'. A drop-down menu will be displayed similar to that shown in

Figure 27.
Figure 27
Selection Of Export File Format
The format of the file for export may be ASC II or 5301PC. These choices are
shown in Figure 27. If the file is lengthy, it is possible to edit it before export.
Click on 'Parameters'. A dialog box such as that shown in Figure 29 will be
displayed. A starting, ending, and skip value may be entered.
Figure 28
Editing Data Before Export
This concludes the tutorial for Super Ion Probe software. Features such as
background correction are described in the guide to data acquisition for Super
Ion Probe software.

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Shimadzu Scientific Instruments, 7102 Riverwood Drive, Columbia, MD 21046

Tel: 800.477.1227/410.381.1227; Fax: 410.381.1222; www.ssi.shimadzu.com

Demonstration Guide For Super Ion Probe Software

Because calcium is a crucial participant in so many cellular functions, it has

The fluorescent dye 'Fura-2' has been used extensively as a fluorescent

II. Tutorial Using Stored Data

This tutorial does not require an instrument. The only requirement is

The display should resemble that shown in Figure 3:

menu bar. Three types of graphs may be displayed simultaneously: Data

The parameters associated with wavelength pair 2 are displayed as shown in

stored as a .bkg file and subtracted (under 'Manipulate', "Background

Additions to sample at Events Marks

maximum and minimum signal must be generated. In a typical experiment,

The next step is to calculate calcium concentrations. Click on 'Manipulate' and

Experiment=Fura, Source=Pair1/Pair2, Method= C=((R-Rmin)/(Rmax-R)) Sf2/Sb2 .

select 'Manipulate' and 'Concentration'. Configure the 'Concentration' dialog

Calcium concentrations may also be calculated over an interval rather than at

expt.' Press 'OK'.

Display Of Data, Ratio, And Concentration Graphs

Click on 'File'. A drop-down menu will be displayed similar to that shown in

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