BIO340 Laboratory Guide #1
ELECTROPHYSIOLOGICAL
INSTRUMENTATION
Physiology is the study of how living systems work. Physiological measurements obtained from living systems are dynamic, that is they change with time. The most effective way to record many physiological measures is via electrical signals, specifically, by monitoring how an electrical voltage changes over time. Techniques and methods for this kind of measurement are collectively termed electrophysiology, and this laboratory will serve to familiarize you with some basic electrophysiological equipment. You will be using this equipment extensively in future labs.
It is important that each of you understands how these instruments work and becomes proficient in their use. Take turns setting the controls so that all members of your lab group get comfortable with these instruments. Now is NOT the time for a "division of labor" approach.
There are several reasons for devoting a lab to learning about instrumentation:
The first reason, as always is your SAFETY. Some electronic instruments, have very high voltages associated with internal devices. The stimulators, if set incorrectly, can produce 100 volts of direct current across the output jacks. All of this equipment operates on 110V AC line current. So: DO BE CAREFUL, DO PAY ATTENTION TO WHAT YOU ARE DOING AND WHAT YOU ARE TOUCHING, AND DON'T GET ANY OF THIS EQUIPMENT WET.
The second reason is that this is expensive equipment, which is difficult and time-consuming to replace or repair. It is quite possible to severely damage this equipment by using it carelessly. ALWAYS DOUBLE CHECK SETTINGS AND CONNECTIONS BEFORE TURNING THE EQUIPMENT ON.
The third reason is that for each subsequent lab you will need to be able to concentrate on the theoretical aspects of the lab and not have to devote time and attention to figuring out the instruments. Electrophysiological instruments are tools, and should be as comfortable to use as a hammer. KNOW WHAT YOU ARE DOING. DON'T EVER JUST TWIDDLE KNOBS AND HOPE FOR THE BEST. IF YOU HAVE QUESTIONS, ASK.
The fourth reason is courtesy. You will be working with one or more lab partners throughout this course. If you don’t bother to become proficient with the laboratory instrumentation, then you will be relying on your lab parner(s) to carry you through each lab. That would be just plain rude.
You will be learning to use a "state-of-the-art" system - the PowerLab system for digital signal acquisition, display, processing, and storage on the PC. This system has many profound advantages over the more traditional oscilloscopes and chart recorders used at some other schools. However, digital systems also have some limitations, and you need to learn to work effectively within those limitations. Because the PowerLab system is designed to emulate either a chart recorder or an oscilloscope, we will start with a brief consideration of these devices.
Finally:
INSTRUCTIONS WHICH APPEAR IN UNDERLINED BOLD CAPITALS CONTAIN IMPORTANT RULES TO FOLLOW IN ORDER TO AVOID INJURING YOURSELF OR DAMAGING THE EQUIPMENT. PLEASE PAY CLOSE ATTENTION TO THEM. THESE WARNINGS ARE INTENDED TO ATTRACT YOUR ATTENTION BY OFFENDING YOUR AESTHETIC SENSIBILITIES.
I. KYMOGRAPH, PHYSIOGRAPH, AND OSCILLOSCOPE
One of the early devices for monitoring physiological activity was the kymograph. A kymograph consists of a rotating drum with its axis oriented vertically and a stylus or pen which contacts the surface of the drum as it rotates. Vertical deflections of the stylus are either mechanically or electrically driven and reflect changes in the physiological measure being taken. The drum rotates at a constant speed, so time is represented around its circumference. Kymographs are marginally useful for tracking repetitive or episodic processes over multiple cyclic periods, such as days. We are fortunate not to have any kymographs in our inventory.
In the chart recorder or physiograph the drum has essentially been replaced by a continuous sheet of paper, which feeds off of a roll and passes under one or more pens at a constant rate. In the record or plot that this produces, the long horizontal axis is time and vertical deflections of each pen reflect changes in some physiological measure, such as muscle tension or pulse volume. In chart recorders each pen is driven by a galvanometer - essentially an electromagnet which tracks the voltage of the signal that is fed into it. Because the galvanometer and pen have an appreciable amount of inertia, they generally can't accurately track oscillatory signals which change faster than about 20 Hz (cycles/second). Chart recorders are, therefore, most useful for producing permanent, continuous records of processes or signals which don't change very rapidly. You can see a circular variant of a chart recorder on the outside of the environmental chamber outside MSC 109.
The oscilloscope is basically a cathode ray tube (CRT) that produces an image by projecting a focused beam of electrons at a phosphorescent screen. Two pairs of charged plates deflect the beam in the vertical and horizontal directions. In general, the horizontal axis of the display represents time. Repeated fixed rate deflections or "sweeps" along this axis are provided by the time base, which supplies a "ramp" voltage to the horizontal deflection plates. The vertical axis of the display directly reflects the voltage of the input signal. Because electrons have essentially zero inertia, oscilloscopes can be used to track signals which change very rapidly, on a time scale of milliseconds, or even microseconds. A major advantage of the oscilloscope is that it is fairly universal in design and operation. The oscilloscope is very versatile and fairly rugged (but not indestructible). The display can be rapidly and conveniently adjusted "on the fly", i.e. while it is being continuously updated. Furthermore, the oscilloscope is a precision instrument whose calibration and accuracy can be generally trusted. Input impedance is very high, so the oscilloscope draws very little current off of circuits that it is monitoring. These features make the oscilloscope valuable for troubleshooting electrophysiological experiments and experimental equipment, a process that unfortunately occupies a substantial amount of research time. The major disadvantage of the oscilloscope is that it is primarily a display device rather than a storage or analytical device. Information displayed on the oscilloscope screen generally cannot be conveniently converted into a useful permanent record for subsequent storage and analysis (there are, however, several decidedly inconvenient ways to save oscilloscope traces).
To save time and aggravation, we will deal with both the physiograph (chart recorder) and oscilloscope as short classroom demos or descriptions. Make sure that you get a feel for how both devices work, and what the advantages and limitations of each are. Chart and Scope are actually software digital emulations of a chart recorder and an oscilloscope, respectively. If you are having trouble understanding exactly what PowerLab displays are showing you, looking at the original devices may help.
II. ELECTRONIC STIMULATOR
The Grass SD9 stimulator is designed to deliver square wave voltage pulses for stimulating biological preparations. Pulses are applied across the + (red) and - (black) output jacks and are isolated from chassis ground. Stimulation may be delivered as single pulses, twin pulses, or as a train of pulses with a specified frequency. The stimulator can be internally triggered, triggered from an external electrical signal, or triggered manually. It also produces a separate synchronization pulse. Each individual stimulator output pulse is defined by its amplitude (in volts), its duration (in milliseconds), its polarity (normal or reversed), and its delay relative to a preceding pulse or to the synchronization pulse. The main advantage of this stimulator is that it is versatile and convenient to use. The only disadvantage is that the synchronization signal which it produces is too short to reliably trigger the PowerLab. For this reason, in future labs we will generally use the PowerLab stimulator (see below) to trigger both the electronic stimulator and the PowerLab Scope recording sweep.
TO AVOID DAMAGING EQUIPMENT OR INJURING YOURSELF ALWAYS FOLLOW THESE TWO RULES WHEN USING THE STIMULATOR:
1) NEVER , EVER TURN THE VOLTS MULTIPLIER TO 10
2) ALWAYS TURN THE MODE SWITCH TO OFF BEFORE YOU TURN THE POWER SWITCH TO ON.
To save time, you will be learning to use the stimulator in concert with the PowerLab Chart and Scope tutorials below. If there is some aspect of stimulator function which you don't understand, feel free to check it out with an oscilloscope at some later time. For now, set up the stimulator with the following settings, then go on to the next section:
FREQUENCY 1.0Hz (10PPS x .1)
DELAY 0.1msec (10ms x .01)
DURATION 100msec (10ms x 10)
VOLTS (amplitude) 1.0volts (1volt x 1)
STIMULUS SELECTOR REGULAR
MODE OFF
POLARITY NORMAL
OUTPUT MONO
DOUBLE-CHECK ALL SETTINGS BEFORE PROCEEDING.
Disconnect any cables between the stimulator and the PowerLab. Now turn the stimulator ON and leave it that way. The green power light should come on and stay on. If the red monitor light is blinking, then set MODE to OFF and PAY MUCH CLOSER ATTENTION TO WHAT YOU ARE DOING FROM NOW ON.
III. POWERLAB SYSTEM
For most of the experiments in this laboratory and for many of your independent projects you will be using the PowerLab data acquisition system. The hardware portion of the PowerLab system consists of an amplifier box and a cable that connects to a SCSI adapter card in the PC. PowerLab supports up to four channels of input, through BNC connectors on the front of the box. Each channel can function as a standard amplifier, which follows voltage at the positive (central +) lead relative to ground. Alternatively, each channel can function as a differential amplifier which follows voltage at the positive (central +) lead relative to the negative (central -) lead, where both leads are live and independent of ground.
The software portion of the PowerLab system has multiple functions. 1) It controls the internal settings of the amplifiers. During its operation you will often hear clicks from inside the box as switches are reset. 2) It functions as an analog-to-digital converter (ADC). The ADC samples the input voltage at discrete time intervals, and converts the continuous analog voltage into a discrete numerical value for each sample. The computer then represents the input signal as a sequence of pairs of numbers, each pair consisting of a time value and a voltage value at that time. Some information is lost in this process, but if the sampling frequency and the voltage digitizing range are set appropriately, these discrete points will reasonably accurately represent the input signal. 3) It displays the input signal on the computer screen in a manner that emulates an oscilloscope, a chart recorder, or a simple digital voltmeter. 4) It allows the input signal to be accumulated and annotated over blocks of time, and stored for future display and analysis. Storage may either be temporary in random access memory (RAM) or more permanent on the computer hard disk. 5) It emulates an electronic stimulator, controlling output through the TRIGGER and OUTPUT BNC jacks on the amplifier box. 6) It provides a user-friendly interface for general experimental control.
The principal advantage of the PowerLab over a conventional oscilloscope or physiograph is that it provides an effective permanent mode of storage of collected data. This data can be retrieved at a later time for printout or numerical analysis. For this reason, we will be using the PowerLab system for primary electrophysiological data collection. One principal disadvantage is that adjusting settings is comparatively slow, and temporarily blocks data acquisition. Furthermore, because sampling occurs at discrete time intervals, the sampled signal may not accurately reflect the actual input waveform. Absolute calibration of the PowerLab should not be regarded as being quite as accurate as an analog oscilloscope. Finally, PowerLab is only one of several computer data acquisition systems. Learning PowerLab will help you understand other systems you may encounter in other laboratories, but the procedures are neither standard nor universal.
Step through the following tutorial to get comfortable with the use of both PowerLab and the PC itself. Keep notes about both confusing procedures and any shortcuts that you discover. REMEMBER THAT "USER-FRIENDLY" IS NOT NECESSARILY SYNONYMOUS WITH "IDIOT-PROOF", SO PAY ATTENTION TO WHAT YOU ARE DOING AND MAKE SURE THAT YOU UNDERSTAND EACH STEP.
This tutorial is only an introduction to the most basic features of Chart and Scope. At any point in the course feel free to either ask questions of the instructor or RTFM (read the friendly manual).
A. Using Chart
The Chart application emulates an 8-channel pen and paper chart recorder. The digitized signals are "drawn" onto each channel at the right edge of the screen, then scroll across the screen from right to left, mimicking the passage of chart recorder paper. Ordinarily each of the first four digital traces is assigned to one of the four input channels. Each of these first four digital "traces" is thus an evolving plot of voltage as a function of time. The other four traces are available for displaying "derived" measures, such as event rates, integrated data, rectified data, etc., on a real-time, "on-the-fly" basis.
Chart is most useful for continuously monitoring physiological activity over relatively long stretches of time. The highest sampling rate that Chart can achieve is 1000 samples/second, or 1 msec/sample. Thus Chart shares the advantages (continuous recording) and disadvantages (inability to record very rapid signals) of a conventional chart recorder.
Turn on the PC (if necessary) and login as PhysioStudent. Turn on the PowerLab box with the switch on the back panel at the right. The two status lights on the left front of the box should show a continuous blue and green colors. From the desktop open LabChart7 from its icon. Don't be too alarmed by any clicking sounds emanating from the PowerLab box.
Chart should display a blank sheet with eight channels delineated. If a large setup window opens instead, simply close this window to start Chart itself.
Speed settings Chart allows you to control the rate at which the input signal is sampled (digitized), as well as the rate of scrolling (emulated chart paper speed) via the speed menu. This is an unlabeled pull-down menu located at the upper right of the display area. Open this speed menu and select 400 samples/second. The speed display should read "400/s" when the cursor is over any of the control boxes at the right edge of the display, indicating that the actual rate of digital sampling is now 400 times per second. The horizontal display scale can be further adjusted using the set of small boxes at the lower right, featuring two small "mountain range" buttons with a horizontal scale compression ratio in between). For now, set this horizontal compression ratio to 2:1, using the little mountain range buttons.
Individual channel settings Associated with each channel are two pull-down menus which can be accessed by clicking on the appropriate down arrow buttons. The Channel menu allows you turn the channel on or off, set-up the input amplifier, or convert the units on the vertical scale from volts to some other measure. You can also chose to display the raw data, a digitally smoothed version of the data, or any one of a number of digital “transforms” of the data. The smaller pull-down menu to the upper left of each channel bar lets you directly set the vertical gain (amplification and default display range) for each channel.
For now, turn on channel 1 and turn off channels 2-8. On Ch1 select Input Amplifier ... to access its dialog box. Notice that this box gives you a continuously updated widow of the incoming signal. Set the Range to 5V. Select only the Single-ended box, which sets up channel 1 as a simple non-differential amplifier. Click on OK to close the dialog box.
Initiating and terminating a sampling session Click the Start button at the lower right of the screen. The line being drawn across the screen is the digitized representation of the signal on channel 1. The vertical scale voltage calibration appears at the far left of each channel's display area. Since there is currently no input to the PowerLab box, the trace should read at 0 volts. Notice that the Start button in the lower right has been replaced with a Stop button. After you have collected 5 or more seconds of data, click on the Stop button to terminate sampling. Start and stop sampling again several times. Notice that each sample is separated by a heavy vertical line.
Changing the display area for individual channels You can expand or shrink the portion of the display area devoted to each channel. To expand the display area for channel 1, position the cursor over the line between channels 1 and 2. The cursor will now be a double-ended arrow. Using the mouse button, click and drag the line all the way down to the bottom of the display area. Channels 2-8 have now been compressed to the point where they are no longer visible, but they can be restored by dragging the lower border of the channel 1 area back up towards the top of the screen.
Clearing the display from active memory All of the data which has accumulated during sampling sessions so far has been stored in RAM. To clear this out select New on the File menu, select both Settings from Document “Document#” and Close Document# after creating new document in the first dialog box, and answer No to the Save question in the second dialog box.
Setting up to record stimulator pulses Make sure that the stimulator mode switch is set to OFF. Connect the stimulator output cable to the CH1 + input cable (white-banded BNC), using a double banana-to-BNC adapter. The strange "kludged" appearance of the stimulator cables is due to some misguided efforts at "child-proofing" the stimulator by Grass Inc. MAKE , VERY, VERY, VERY SURE THAT THE GROUND SIDES OF THE TWO DOUBLE BANANA PLUGS ARE CONNECTED TOGETHER AND THAT THE EXPOSED BANANA PLUGS AT THE JUNCTION ARE NOT TOUCHING ANY METAL. FAILURE TO DO THIS COULD SHORT OUT THE STIMULATOR, WHICH WOULD BE VERY BAD FOR IT. CHECK THIS CONNECTION WITH THE INSTRUCTOR BEFORE PROCEEDING.
Set the stimulator mode to REPEAT. Start sampling on Chart. At these settings Chart should produce a fairly crisp square wave trace. Stop your sampling after 5-10 seconds.
Scrolling along the horizontal axis As you've probably noticed, the simulated "chart paper" steadily disappears off the left side of the screen during sampling. However, you can use the scroll bar at the bottom of the screen to retrieve earlier parts of the record. Experiment with several horizontal scale compression scales using the "mountain range" buttons to the right of the window title bar compress or expand the horizontal axis. The box to the left the mountains and the Start button toggles the screen monitoring on/off, so that you can temporarily freeze the trace on the screen, while continuing to record data.. Notice that when you toggle the screen display back on that the time index along the x axis has continued to advance. This time index resets itself to zero every time you stop and restart active recording. Notice also that these adjustments of the horizontal display affect only the display; they do not affect anything about the data actually being collected and stored in the computer.
Adjusting the vertical axis The vertical voltage scale at the left end of the channel record can be either stretched or shifted. To stretch the axis, move the cursor into the vertical scale area and position it directly over a number. The cursor will now look like a tiny double arrowhead. Click and drag on the number to stretch or contract the vertical scale. To keep the scale constant, but shift the y axis, position the cursor between two numbers in the scale area. The cursor will now look like a double-headed arrow. Practice adjusting the scale using these cursors. You can also adjust the scale by clicking on the small + or – magnifiers at the lower left of the scale area. As a final, and much less frustrating alternative to all of this, just use the pull-down menu under the arrowhead button at the upper left of the scale area to set the scale.
When you are finished testing out these alternatives, set the vertical axis with 0 near the bottom and 2 near the top of the display area.
Making time and voltage measurements You can make a rough estimate of pulse amplitude and duration by simply "eyeballing" the record. To get a more precise measurement use the waveform cursor and the marker. When the display is stopped and the mouse cursor is inside the display area for one of the channels, it appears as a "+" cross. A second "X" cross (waveform cursor) appears above or below the mouse cursor, and is superimposed on that channel's waveform trace. Slide the mouse cursor from right to left and notice how the waveform cursor tracks the recorded waveform trace. The time and voltage scales in the upper right corner of the display window now reflect the current x and y coordinates of the waveform cursor, in seconds and volts, respectively.
The M in the lower left corner of the screen functions as a marker. Click on the M, drag it to some point along the waveform trace, and release it. Now the time and voltage scales in the upper right corner reflect the position of the waveform cursor, relative to this marker, as delta values. Double clicking on the marker, dragging it out of the channel display area, or clicking on its "home" box will reset the marker.
Use the marker and waveform cursor to measure the apparent duration, amplitude, period (the time from the start of one pulse to the start of the next), and frequency of the recorded pulses during the last sampling interval, and record your measurements below. (The frequency is the inverse of the period from the start of one pulse to the start of the next.)
Duration in seconds
Amplitude in seconds
Period in seconds/cycle
Frequency in cycles/second
Selecting, zooming, and calculating waveform statistics Individual segments of the recorded data can be selected and enlarged to facilitate measurement. Select a segment of the Ch1 record by clicking and dragging the mouse over that part of the record. The selected segment will appear "highlighted", that is as an inverse-colored trace on a black background. Now enlarge (Zoom) the selected region by selecting Zoom View from the Windows pull-down menu, or clicking on the little magnifying glass icon in the horizontal tool bar above the display area. Notice that the waveform cursor coordinates appear across the top of the Zoom Window. Use the marker and waveform cursor to again measure the duration, amplitude, amplitude, period, and frequency of the signal pulses and record your measurements here. Note: The Zoom Window has its own marker in its own little home box.
Duration
Amplitude
Period
Frequency
If you drag and click to highlight a portion of the trace in the zoom window, the trace automatically rezooms to display just this area. Close the zoom window.
Attaching comments to data records You can make notes about an experiment and attach them as "comments" to any part of your chart record. These notes can remind you of manipulations that you have made, or serve as event markers in the record.
To attach a comment while recording, click in the Comment box at the top of the window, then just start typing on the keyboard. When you hit the enter key, your comments will be saved and a labeled vertical dotted line will mark the location. When you reexamine the record, you will see a small box with a comment number under the time axis, at a location corresponding to the time at which you hit the enter key. To see a numerical list of the comments made during the experiment, select Comments from the Windows menu. The small box to the right of the text entry widow shows the comment number and lets you select which channel your comment will appear on.
To attach a comment to your data record after recording, use the mouse cursor to click anywhere in the display screen. Then select Add Comment... from the Commands menu, type in your comment, select the channels to wich to add the comment, then hit return.
Printing The PowerLab stations share a networked printer at the side of the room. Chart has its own Print function available from the icon at the top of the screen, but it frankly sucks. A much more versatile option is to use the Windows Snipping Tool to select exactly what part of the display you would like to print, to save the "snip" to a .jpg file, and then to use the Windows photo printing utility to print one or more "snipped" images on a page. The instructor will demonstrate how to do this at the end of the lab session.
Saving files To save data records in a more permanent form on the hard disk choose Save As... from the File menu, type a file name in the highlighted bow, and click on the Save button. The Save Selection... option obviously lets you save only a selected part of the data. Try to practice good disk hygiene by keeping all of your data files in a single, data folder, clearly labeled with your group logo.
The realities of digital sampling Record an additional ~5 second segment under the current chart speed setting of 400 samples/second and stimulator settings of 1 volt x 100msec pulses delivered at 1 per second. Stop the recording and zoom in on a single “square” pulse. Is the trace really square, or are the sides tapered? Reduce the sampling rate to 40 samples/second, record a short sample, then zoom in on a single square pulse. Is the “tapering” effect better or worse?
Q1: Why does a perfectly square pulse produced by an analog stimulator result in a “rounded-off” trace when digitally sampled at too slow a rate? Hint: think about what digital sampling means – namely that the signal voltage is only sampled at discrete, regular time intervals (hence the expression “samples/second”).
Now adjust the Chart speed settings to 4 samples per second and produce a recording of at least 30 seconds.
Q2: Why doesn't Chart record every pulse at these settings?
Setting the Chart speed settings a higher number of samples/division increases temporal resolution, but also increases data storage space requirements. This tradeoff always has to be considered for digital acquisition of physiological data.
Finally, set the Chart speed up to 20K samples/second and ste the horizontal display compression ratio to 50:1. Record a few pulses then stop the recording. Zoom in on a single pulse and accurately measure its height and width.
Q3: Do these values correspond precisely to the stimulator settings? Why or why not?
When you are finished with Chart quit the application by choosing Exit from the File menu. Do not save the changes which you have made.
REMEMBER TO RETURN THE STIMULATOR MODE TO OFF.
B. Using Scope
The Scope application emulates a 2 channel storage oscilloscope, by sampling, holding, and displaying the input signal(s) in discrete pages (sweeps) of a predetermined duration.
Launch Scope for Windows by double clicking on its icon or on its aliased icon in the apple menu. Notice that most of the screen is occupied by a display area with a dot grid which serves the same function as the oscilloscope screen and reticule.
Setting up the input amplifiers Controls for the two input channels, designated Input A and Input B, are located to the right of the display area. Each input can be assigned to any of the four PowerLab channels, or turned off by using the Ch # pull-down menu. The input gain (vertical scale) can be quickly set using the Range menu. The Input Amplifier... control box lets you set up the input amplifier with the same basic set of controls that Chart used. The Time Base portion of the window lets you choose a sampling frequency by choosing a total number of samples (Samples:) and a horizontal time scale (Time:). To determine the digital sampling rate, divide samples by time, or simply look at the Hz number in the Time Base window.
For now, assign Input A to Ch1, set the range at 5V and select only the Single-ended box under Input Amplifier... . Turn off Input B. Set the Time Base to 256 samples and 20ms. Note that the sampling frequency of 10kHz (10,000 samples/second) is displayed in the Time Base window. This means that Scope will sample the input and record a numerical amplitude value 10,000 time per second, in other words at 0.1 msec intervals. A signal event which lasts 1 msec will be represented by only 10 recorded points. The computer display will play "connect-the-dots" to provide a serviceable but not very aesthetically pleasing representation of the waveform shape of the event. On the other hand, an event which lasts 10 msec will be represented by 100 points and will be displayed fairly accurately.
You can reduce the amount of the screen display used by the unconnected Input B by dragging and clicking the lower display boundary line, much as you did with Chart. Alternatively, you can dedicate the entire display area to Channel A by selecting Computed Functions . . . under the pull-down Display menu, then setting the Display: box to Ch A only.
This Computed Functions . . . dialog box also allows you to digitally filter your displayed trace, using the smoothing option. We will use this in later labs to “clean up” records by eliminating unwanted high frequency fluctuations or “noise”.
Set the electronic stimulator to 1volt x 1msec pulses at 100Hz, and set the stimulator MODE to REPEAT. Because of the high frequency of stimulus pulses, the MONITOR light will glow a steady red.
Initiating and terminating a sampling session Changes in the sampling controls are made by choosing Sampling... under the Set-Up... menu. In this control window Mode: and Source: in the Sweep box correspond to the comparable controls on the oscilloscope which determine under what conditions a sampling sweep occurs.
To start with, set the Scope sweep Mode: to Repetitive, Source: to User, and Delay: to 0 seconds then click on OK. Initiate sampling by clicking on the Start button at the lower right of the screen. you will see a set of 2-3 pulses moving back and forth across the screen. In this sampling mode, the display is a continuing series of 26msec digital "snapshots" of the input. When you click on the Stop button , the most recent snapshot is held on the screen. Note that at this sampling rate the square waves coming from the stimulator are rounded off in the display. Increase the sampling rate on the time base to 2560 (100kHz) to partially solve this problem.
Using the marker and waveform cursor to measure the duration, amplitude, and period of the pulses, and calculate their frequency. Record your measurements below.
Duration
Amplitude
Period
Frequency
Practice changing the stimulus duration and voltage on the stimulator, and adjusting Scope to produce effective displays. DO NOT EXCEED 10 VOLTS OR 100 MSEC ON THE STIMULATOR SETTINGS. These are the kinds of changes that you are going to have to do quickly and efficiently in the upcoming experiments.
Paging and overlaying By now you have probably noticed that each time you start and then stop the display, Scope saves the final sweep and goes to a new page. Page numbers are listed across the bottom left of the display area. To see a previous page, just click on the appropriate page number. Alternatively, you can flip through the pages using the page "corners" at the bottom right of the display area.
Saving every trace To save a consecutive series of traces as distinct pages you will have to change the sampling mode. Choose Sampling... under the Set-Up... menu. In this control window, set Mode: to Multiple, Source: to User, and Delay: to 0 seconds. Set Number of Samples to 8, then click on OK. Clicking on the Start button at the lower right of the screen. Notice that Scope produces 8 sweeps, assigning each trace to a new page, then stops automatically. You can also use the Stop button to abort the series of sweeps at any time.
Displaying multiple traces You can superimpose the traces from two or more pages by choosing Show Overlay under the Display menu. Notice that the traces are now all superimposed with the trace from the selected page represented as a solid line, and the other traces as dotted lines. Restore the single page display by choosing Hide Overlay under the Display menu.
Adding page comments The simplest way to label a given page or to view a previously assigned label is to select that page, then click on the small notepad icon at the lower left of the display area. This opens a comment window, and the comment that you enter is associated with that page.
Additional features The following features of Scope work in much the same way as the comparable features of Chart:
stretching and shifting the vertical axis
selecting and zooming
saving files.
You can clear the current display from active memory at any time by choosing New under the File menu. Of course, this results in the loss of all screen data which has not been expressly saved to a disk file.
C. Using the PowerLab Stimulator
PowerLab also contains a built-in stimulator, which can be directly controlled by the Scope or Chart software. The PowerLab stimulator is a little harder to use than the electronic stimulator, but is more versatile in the stimulation pattern that it can produce.
To set up the PowerLab stimulator, first set the external Grass SD9 stimulator Mode to OFF, then disconnect all of the existing connections between the electronic stimulator and the PowerLab. Connect the OUTPUT+ cable (white+blue banded BNC) on the PowerLab to the CH1+ input cable (white banded BNC) cable of the PowerLab using a BNC T connector.
Set the Scope Time Base at 2560 samples and 20ms. Choose Stimulator... from the Set-Up menu. In the Stimulator window set Mode: to Pulse. This will produce square wave pulses comparable to those produced by the electronic stimulator. The pulse parameters can now be set any one of four ways:
1) By sliding the Delay, Duration, and Amplitude scroll bars in the Stimulator window. Delay here specifies a delay between the start of each Scope display trace and the start of the stimulus pulse.
2) By clicking on the A box above each scroll bar in the Stimulator window and typing in a value.
3) By clicking and dragging the dark dots on the sample trace in the Stimulator window to the desired locations.
4) With the Stimulator window closed, by directly adjusting the stimulus Parameters using the arrows in the Stim box at the upper right of the main display window. Holding down the Control key while clicking one of these arrows allows you to adjust the stimulus duration increment.
Practice changing the pulse parameters with each of these four methods.
Set the pulse to 1msec delay, 1ms duration, and 1V amplitude. Choose Sampling... from the Set-Up menu and set the sweep Mode: to Single and Source: to User. Select Show Overlay under the Display menu. Clear out the previously collected data by choosing New in the File menu. Generate 3 or 4 different pulses by clicking Start once for each pulse, and resetting pulse parameters between pulses. Notice that each pulse record has automatically been saved to a new page. Adjust the Input A gain and Time Base as necessary. Enter a comment for the currently displayed page.
If you choose Overlay Stimulator . . . under the Display menu, you can add a trace or marker lines which indicate only the onset and offset timing of the PowerLab stimulus pulse. This is handy when the PowerLab stimulator is used to trigger the electronic stimulator and synchronize stimulus pulses to Scope samples. This will be the most common configuration for future labs.
The built-in PowerLab Stimulator can also be used in conjunction with Chart to produce a continuous train of stimulus pulses. If you have time, you can explore this use of the stimulator on your own.
IV. SHUT-DOWN PROCEDURE
When you have finished, please go through the following procedure:
1) Make sure that the electronic stimulator mode is set to Off, then turn
off the stimulator.
2) Exit from Scope by choosing Quit from the File menu.
3) Turn off the PowerLab box.
4) Turn off all the powerstips except the one powering the PC cart.
5) Bye.