In this laboratory, traditional (old) and new equipment will be set up to
(1) principles of recording
(2) limitations solved by more modern equipment
(1) With the smoked kymograph,
the frog nerve-muscle
preparation would use a stylus to scratch into paper that was covered with
soot using a wing-tipped bunsen burner
(2) the record was then dipped in shellac for preservation
For measuring Voltage, we have the Volt
meter. Try it.
displays voltage as a function of time
On the amplifier,
the following will be demonstrated:
(2) gain [mV/cm]
(3) time constant
Trace on physiograph
shows 1 mV calibration pulse:
(1) square wave [DC]
set mV/cm to 10 and multiplier to 10 (that comes to 1 mV/cm)
(2) low-pass [high-cut-off] filtering ["filtering Hz"]
(3) high-pass [low-cut-off] filtering ["time constant"]
For the sake of completion, the bottom of this
sheet shows the circuitry and math for RC filtering, and a few of you may
be interested in this, though I certainly will not hold you responsible
I have set up some "electrodes" that can be gripped in each hand
to get an EKG (ECG = electrocardiogram).
(1) set mV/cm to 2, multiplier to 10, time constant to 0.3 and Filter Hz
(2) doesn't look as good as the computer
EKG, does it?
(3) vary the settings to see why I suggested these starting values
Several students in the Fall, 2004 lab got pretty nice EKG's from the polygraph,
is a good one.
(1) graphs voltage as a function of time
(2) as a blue dot with a green afterimage
(3) here, the sweep is started by external input on the time base amplifier
(4) the gain is set on the differential amplifier [middle] (Volts/division)
Problems with the oscilloscope (solved by more modern computers)
(1) how to save image:
here the Grass (brand) camera
is shown, and a blue filter captures the blue dot
(2) how to get the response in the center of the screen
here the Grass (brand) stimulator
synch out (delay) and the delay feed the oscilloscope's time base
(3) I am showing you a tank
for developing 100 foot rolls of film with dark room chemicals
are spools for the film.
Microelectrodes (micropipettes) [Other researchers call these "needles"
and use them to inject DNA for transformations.)
We will make micropipettes
(1) We use a Narishige PD-5 (Tokyo) horizontal puller
with controls for an early
magnet, a heater, and a late (stronger
(2) The heater glows
red while the first magnet pulls gently.
(3) A microswitch
with a shim detects the melt and the early pull to kick in the harder pull.
(4) After the second pull, two electrodes
We will fill micropipettes
(1) Over the history of micropipettes, many tricks have been developed to
get the very narrow tip to fill. Currently, a capillary tube with an inner
filament has magic filling properties.
(2) First you back fill
the butt end a little with a spinal tap needle.
(3) The electrolyte is carried to the tip.
Then, you can finish back filling the elecrode with the syringe.
We will test micropipettes
(1) On the electrometer
(microelectrode amplifier), position, electrode check and capicity compensation
will be demonstrated.
(2) You will be provided with a micromanipulator
to insert electrode (in this case simply into a grounded block
(3) We will need to introduce some simple features of the Powerlab
and the T-chart program, selecting a channel, setting the range, and starting
and stopping a record. (Many more features will be demonstrated in later
(4) Then we can test the electrode resistance and demonstrate the "ringing"
(used by some single cell electrophysiologists to get the electrode into
cells) that comes from overcompensation of the capacity.
I work on Drosophila, recording extracellularly
in the eye using the ERG (electroretinogram), and here is a picture of a
fruit fly with an electrode in its eye.
Here is another example of electrophysiology used in my research. Here is
an outline of my work on
rescue from blindness in a mouse model of Batten's disease using stem cells,
and these data indicate
that the stem cell injected eye has a better ERG than the sham injected
Most of the apparatus discussed below will be demonstrated, but only the
most fundamental is on the web hyperlinks (above). After an initial demonstration
the groups of lab partners will rotate to get their hands on these most
fundamental pieces of apparatus in several separate work-stations. While
trying to be clear and concise (below), I do not hesitate to introduce the
students to the jargon electrophysiologists babble.
One of the main properties of life defined in the introductory chapter of
any introductory biology book is "excitability." There is no better
example in biology than in nerve and muscle cells of animals. As the pedagogy
of texts gets more and more advanced, the "cartoons" (diagrams)
get further and further removed from the real world. Here, in this laboratory,
I want to show you how biologists measure electrical signals (and not to
re-hash what is known about electrophysiology).
Way back before electronics, work could be done on the frog nerve-muscle
preparation. The nerve could be jolted, and the muscle connected to a stylus.
A kymograph was fitted with a piece of paper that was coated with soot from
a yellow bunsen burner flame. The stylus scratched a record onto the smoked
kymograph drum with the speed of rotation providing time on the X-axis,
and the record was preserved with shellac.
A hobbyist or an electrician would measure Voltage with a Volt meter. Bioelectric
signals are smaller than the 1.5 V flashlightlight battery (but not all
that much smaller). So the "voltmeters" we use must be sensitive
enough to measure mV. This is done with amplifiers, and so, in many respects,
the input of the physiologist's voltmeter is a differential amplifier to
measure voltage difference (say, from inside a cell relative to the extracellular
Perhaps more important than the greater sensitivity for physiologists is
the need to measure Voltage (on the Y-axis) as a function of time to monitor
fast changes. The "polygraph" achieves this by drawing a line
and rolling the paper out under the line (at various speeds) to put time
on the X-axis. If the signal is recorded directly, that is called DC. Although
DC is usually interpreted to mean "direct current" (as opposed
to alternating current), here it means "direct coupled." If a
signal is fast but your ability to see the signal is obscured by slow signals,
then a filter composed of a circuit with capacitance and resistance (RC)
provides high-frequency-pass (low-cutoff). If fast noise is interfering
with recording, then a different type of electronic filter is high-cutoff
The oscilloscope sweeps a dot across the screen at various speeds controlled
by the time base amplifier to put time on the X-axis. A stimulator can be
used to feed pulses into the oscilloscope for demonstration. The rate of
stimulation and the voltage output can be varied. To keep the record on
the screen, the position knob and the gain (sensitivity) can be varied.
To put the signal in the middle of the screen horizontally, the delay (on
the stimulator) measures the time between starting the oscilloscope sweep
and the output pulse from the stimulator. The stimulator's "synch out"
feeding the time base on the oscilloscope controls this.
The oscilloscope screen needed to be photographed to produce a permanent
record. A camera runs film from a roll. I used a blue filter to capture
the dot on the oscilloscope (and not get a blurred photograph from the green
afterimage of the dot). But then I needed to develop film in a developing
tank with chemical developer, fix, rinse, etc.
Nowadays, the face of the computer is used as if it were the face of the
oscilloscope or the polygraph paper (and that is what we will use to test
electrodes, below). An interface (the Powerlab) is used on the input, and
all the functions like gain (sensitivity) and time base are on the computer
program. A position knob is provided as "input offset on an additional
bridge amplifier. Permanent records are achieved by saving files to disk,
Traces can be zoomed, and put into montages using PhotoShop. In short, given
enough computer savvy, lots of things can be done easily that used to be
We will pull fine tipped microelectrodes. These are so small they can be
inserted into individual cells. They are filled with a concentrateed salt
solution. It would take several paragraphs to say how this used to be done
and how hard that was. Eventually, it was found that an inner tube helped
in the filling, and, since nobody understood why, I called this "magic
glass." 25 years ago, I bought a kilo of magic glass from Germany and
that'll probably last me the rest of my life. We need to cutt off pieces
the right length, then make and fill electrodes.
Using a micromanipulator, we will put a filled electrode into a grounded
block of agar. The amplifier used for microelectrodes has that essential
position knob to bring the trace into range. The resistance is checked with
the electrode check and it might be10 megOhms. There is a knob for capacity
compensation. Because of the high capacitance of glass and the high resistance
of the electrode, the electrode itself makes a low pass filter, and the
aplifier gives the researcher the opportunity to fudge the signal by adding
just the right amount of high pass filtered signal to make a square wave
square. If this were not done, fast signals (for instance, an action potential
might be only 1 ms) would be filtered out by the high cut off inherent in
the electrode's properties.
Last year, there was a quiz, and here it is along with the answers:
Your lecture professor briefly mentioned that intracellular recording electrodes
were typically filled with potassium chloride. Why not sodium chloride?
A simple answer to this question is that KCl would more approximately match
the high KCl inside cells. Many people had this idea but got confused in
how they worded this.
You've filled a microelectrode and you're ready to get started. Typically,
you can't just hook a glass microelectrode up to an oscilloscope to record
an action potential. State one of the two properties of a microelectrode
that force you to use a special microelectrode amplifier.
The high resistance through the fine tip and the high capacitance of glass
makes the electrode a low pass filter and it would miss the very fast signals
you want to record. Many people said that small (mV range) signals required
amplification, and although that is not "one of the two properties
of a microelectrode that force you to use a special microelectrode amplifier,
I gave half credit."
Your lab professor is showing you a voltmeter, such as your lecture professor
showed you as Fig. 5-37. Why can't you use such a device to measure a neural
signal? (i.e. Why do you need a chart recorder, a polygraph, an oscilloscope,
or a computer instead?)
You need to record fast signals. Again, so many students indicated lack
of sufficient sensitivity that I gave half credit, in this case, reluctantly
Pretend the face of the oscilloscope recording an action potential is a
graph in an algebra class. What is on the X axis?
Time, typically in ms
What is on the Y axis?
Voltage typically ranging from at least ­p;70 mV to +55 mV
Suppose you were trying to record an ECG (EKG, electrocardiogram). For the
purposes of this question, you need to know: (1) there are 5 components,
called P, Q, R, S, and T, (2) all 5 occur during each heart beat, (3) resting
pulse is typically 72 beats per minute, and (4) from the beginning of the
P wave to the end of the T wave might be typically 3/4 of a second. The
amplifier of the polygraph is set to DC. Your subject cannot sit still,
and the trace keeps going off scale top and bottom every 5 or 10 seconds.
What should you do electronically to give a line that stays on scale?
To filter out slow "drift" and still pick up fast changes, use
a high pass filter. Some people wanted to change the scale (lower sensitivity)
and that would work, though the EKG would be small, so I gave credit.
How do you make fine tipped microelectrodes from glass tubing that is about
1 mm in diameter?
Basically, you melt the glass and pull it apart. I gave half credit for
answers pertaining to filling rather than making
This page was last updated 9/10/04
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