PATCHMASTER TUTORIAL

Heka Elektronik

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• Publié par: Chiwyog
• Nombre de lectures: 139
• Langue: English

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Contents
1 Fluorescence Ratio Measurements 1
1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Configuring Patchmaster for Fluorescence Measurements 2
1.2.1 Turning on the Photometry Extension . . . . . . . 3
1.3 Editing the PulseGenerator Sequences . . . . . . . . . . . . 3
1.3.1 The Sequence ”TestFura” . . . . . . . . . . . . . . . 3
1.3.2 The ”RatioFura” . . . . . . . . . . . . . . 6
1.4 Editing the Online Analysis . . . . . . . . . . . . . . . . . . 6
1.4.1 The ”Background” Analysis Method . . . . . . . . . 7
1.4.2 The ”Ratio” Analysis Method . . . . . . . . . . . . . 8
1.5 The Experiment with Photometry Measurements . . . . . . 9
2 High-Speed Fluorescence Measurements 11
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.2 Editing the pgf-File. . . . . . . . . . . . . . . . . . . . . . . 11
2.2.1 The Photometry Stimulation . . . . . . . . . . . . . 13
2.2.2 Extraction of the F340 and F380 Signals . . . . . . . 15
2.2.3 Calculation of the Ratio Trace . . . . . . . . . . . . 19
2.3 Display of the Data in the Oscilloscope window . . . . . . . 19
2.4 Calculation of Compression Settings . . . . . . . . . . . . . 201. Fluorescence Ratio
Measurements
1.1 Introduction
Inthefollowingtutorialwewilldescribeawhole-cellexperimentwithload-
ing a ratiometric fluorescence dye (e.g. fura-2) through the patch pipette
into the cell.
Beforewestartthedetaileddescriptionhowtoset-upandconfigurePatch-
master, we will first outline the different phases of the experiment.
1. Approach of the cell with the patch pipette and formation of a seal.
2. During seal formation usually some fluorescence dye diffuses into the
bath solution. Therefore, after seal formation we will detect a de-
clining fluorescence signal since the dye molecules are diffusing in the
bath solution away from the detection area. Due to dilution effects
the fluorescence signal will decrease in the following tens of seconds.
In this phase of the experiment, we have to wait for a stable fluo-
rescence background, originating from auto fluorescence of the cell
and dye in the tip of the patch pipette. (To reduce the contribution
of fluorescence from dye molecules in the patch pipette you should
restrict the field of fluorescence excitation and detection to the area
of the cell.) When background fluorescence has been stabilized we
will record this fluorescence as background fluorescence for later cor-
rection of the fluorescence ratio.
3. Establishment of the whole-cell configuration (break through) and
recording of dye loading into the cell. During this phase of the exper-
imentwestartthefluorescenceratiomeasurement. Wecanfollowthe
dye loading level and get first measurements of a fluorescence ratio.2 Fluorescence Ratio Measurements
For the experiment described above we recommend a minimal set of con-
figurations. We will need:
Pulse Generator Sequences: Asequencewhichappliesatestpulse
and records one pair of fluorescence values. We will name this se-
quence ”TestFura”. When executing this sequence data will not be
stored. You will use this sequence e.g. during and short after seal
formation for measuring the decline of the background fluorescence.
Then we will use a sequence ”RatioFura” which is very similar to the
”TestFura” sequence but allows the storage of data and also calls a
different Online Analysis method. This sequence will be used when
recording a baseline, during resting times or e.g. during recovery
periods.
Online Analysis: We need two Online Analysis methods. The
method”Background”willbeusedtoanalyzethefluorescencevalues
and store them in a parameter value. The method ”Ratio” will be
used for the fluorescence ratio analysis during the experiment.
Protocols (optional): The Protocol Editor can be used to
link the different Pulse Generator Sequences and switch from one
phase of the experiment to the next. Depending on the design of
the whole experiment one can use only one protocol containing all
phases of the experiment. If more flexibility is needed one can cre-
ateindividualprotocolsorexecutethePulse Generator Sequences
manually.
1.2 Configuring Patchmaster for Fluores-
cence Measurements
In the following chapter we describe all tasks which should be done before
starting the final experiment. Our description outlines using photometry
setup which accepts an analog voltage for wavelength control and provides
an analog voltage (0 to 10V) proportional to the fluorescence signal. Usu-
ally, such system consists of a Monochrometer for fluorescence excitation
and a Photomultiplier or Photo Diode as fluorescence detector. Such a
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???1.3 Editing the PulseGenerator Sequences 3
system can be controlled by Patchmaster using the TILL Photometry
extension.
1.2.1 Turning on the Photometry Extension
After starting up the Patchmaster software open the Configuration
window(Windows→Configurations)andselecte.g. theTILLphotometry
extension (compare p. 109 Reference Manual) and follow the instructions
givenbytheprogram. Forcalibrationofthemonochrometerpleasereferto
the Reference Manual p. 114. Once the monochrometer is calibrated you
can specify the excitation wavelength in Patchmaster directly. Conver-
siontothecorrespondingcommandvoltagewillbedonebythephotometry
extension.
1.3 Editing the PulseGenerator Sequences
1.3.1 The Sequence ”TestFura”
Open the Pulse Generator (Windows→ Pulse Generator) and load the
defaultprotocolpool(DefPgf v9). Deleteallsequencesexceptthesequence
”TestSeries” and save the new Pulse Generator file under a new name,
e.g. ”Photometry”. We will then rename the sequence ”TestSeries” into
”TestFura”.
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Please compare the settings of the first channel with the screenshot shown
above. For stimulation you should use the ”Stim-DA” and the Stimulus
→ DA should be set to ”StimScale, Relative”. As AD channel please select
one of the current monitor (Imon) channels. The second channel we use
for control of the fluorescence excitation device and record the fluorescence
signalfromthedetectorofthephotometrysystem. AsDAchannelselectthe
channel that was specified in the Photometry configurations as wavelength
excitation channel (”Excit.”). Here e.g. DA-1. As Stimulus→ DA please
select ”use for Photometry”. As AD channel please select the channel that
has been specified as emission channel 1 (”E-mit 1”) in the photometry
configurations. Here AD-1. Optionally, one can use a data compression for
the photometry channel.
http://www.heka.com1.3 Editing the PulseGenerator Sequences 5
For the first channel we use four segments, each with 10 ms duration and
we apply a double pulse from the V-membrane relative +10 mV and then
step to -10 mV and finally back to V-membrane.
Forthesecondchannelweusealsofoursegments. Sinceweusethischannel
for Photometry, the amplitude of the stimulation can be entered in wave-
length units, nm. We start from a resting wavelength of about 260 nm,
then jump first to 340 nm and then to 380 nm and finally back to 260 nm.
Ontherightsideofthesegmentssectionwecanspecifyadditionalparame-
ters such as Filter Factor, Analysis Method and the Relative X- and
Y- segments. We specify the ”Background” which we
will define later and set segment number 2 as Relative segment.
http://www.heka.com6 Fluorescence Ratio Measurements
In the Timing section we enter a sample interval of e.g. 50.0 s and a large
number of Sweeps (e.g. 5000).
1.3.2 The Sequence ”RatioFura”
The ”RatioFura” sequence can be derived from the ”TestFura” sequence.
Please copy the ”TestFura” sequence and name it ”RatioFura”. We will do
two changes to this sequence:
First, we enable ”Store” of the sampled traces.
Second, we change the Online Analysis Method from ”Background”
to ”Ratio”.
1.4 Editing the Online Analysis
In the upper part of the Online Analysis window, we create new
Analysis Methods. We will start with the method ”Background”.
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??1.4 Editing the Online Analysis 7
1.4.1 The ”Background” Analysis Method
We select ”Background” and configure three Analysis Functions. The
firstfunctionweneedistheTimerTime. WenamethisfunctionjustTimer.
We will use this function to calculate the X-value for the online display.
For analysis of the fluorescence values we create two additional functions.
We click on the NEW button in the Analysis Function section, select the
function ”Mean” from the Y-Analysis functions, enter a name for the func-
tion, e.g. F340, and click Done.
In the Analysis Function section we have to set the Trace # to ”Trace
2”, since the fluorescence signal is recorded with the second channel (see
Pulse Generator). In addition, we will save the result in a Value that we
can subtract the background fluorescence on a later time. Hence, we select
”Store to Value-1”. And we set the cursor bounds from 40 to 90%. The
cursor bounds define the data range that is analyzed. The lower bound
of the cursor bound has to be set to a value large enough to allow the
fluorescence excitation source to settle at the specified wavelength. Note:
The photometry extension of Patchmaster does not use the ”dead time”
anymore.
Now we create another ”Mean” function with name ”F380”. In the
Analysis Function section of the function ”F380”, we set the Y-seg.
Offset