Sloper manual Copyright 2007 Unisense A/S Version Unisense A/S

Transcription

1 Sloper manual Copyright 2007 Unisense A/S Version 2007 Sloper manual Unisense A/S

2 TABLE OF CONTENTS System Requirements...6 Features of Sloper....7 Installation of Software...8 The Light-Dark Shift Method....9 Measuring Setup...12 Starting of Sloper and Settings...13 Overview of the Main Window Motor Control...17 File Handling The Chart Recorder...19 Calibration Calibrating the sensors 20 Light sensor calibration 21 Photosynthesis Rate Measurements Slope masurement window 22 The Profile Windows References

3 System Requirements Features of Sloper Windows 2000/XP (XP recommended) Pentium-class processor 100 MB free hard disk space UNISENSE Data Acquisition Module ADC-216- USB (requires USB port) UNISENSE Light Sensor UNISENSE Motor Controller (requires serial port RS232) Sloper is a program for photosynthesis rate measurements. More exactly, it enables the measurement of the production or consumption rate of chemicals due to the light reaction during photosynthesis. Thus, the oxygen production rate is measured, if an oxygen microsensor is utilized. The measurement is based on the light-dark shift method described in the literature. A short description of this method is given below. The data from the microsensor are read by the computer via the UNISENSE Data Acquisition Module ADC-216USB. Additionally, the UNISENSE Light Sensor is necessary for the light-dark shift method. It is also connected to the data acquisition box. If desired, the readings of the microsensor can be calibrated with the calibration feature. In addition, Sloper can control a motorized micromanipulator via a UNISENSE Motor Controller, which is connected to the serial port. Basic features of the program are: Strip chart indicators for the indication of actual microsensor readings Microsensor calibration option Manual motor control Manual data acquisition Logging at defined time intervals Fast profiling Standard profiling Inspection of old data files 6 7

4 Installation of Software The Light-Dark Shift Method Install the driver software for the UNISENSE Data Acquisition Module ADC-216USB The voltage data from the microsensor and the light sensor are acquired by the use of a UNISENSE Data Acquisition Module (e.g. ADC-216USB). The user should first read the separate installation guide for this device and install the driver software (a copy of the installation guide can be found on the Sloper installation CD, e.g. Installation ADC- 216USB.pdf ). Uninstall older versions of Sloper Follow the instructions of your operation system in order to uninstall any older versions of Sloper. Install the new version of Sloper. Start the installation program setup.exe in the folder Installer Sloper 3.xy and follow the instructions given by the installation shield. During the installation process a new program group Unisense Sloper will be created. In this group the program Sloper itself and a copy of this manual can be found. This paragraph gives a short description of the theory on which the light-dark shift method is based. The tip of an oxygen microsensor is placed at a certain position r in a photosynthetic system. It measures at time t the oxygen concentration C(r,t). The time dependency (i.e. the derivative in time) of the oxygen concentration can be described by δc δt = - T - R + P, where P is the volumetric gross photosynthesis rate of oxygen (mol m -3 s -1 ), R volumetric oxygen respiration (mol m -3 s -1 ), respectively. The volumetric transport rate T represents the changes in oxygen concentration due to transport phenomena (mol m -3 s -1 ). The type of transport is dependent on the specific sample under investigation. It can be due to water current, convection, or diffusion. The light-dark shift method works for all types of transport. For the light-dark shift technique, it is important that the oxygen concentration acquires a measurable steady state value under constant illumination. At steady state the above equation must be equal zero: δc δt = 0 => - T - R + P = 0 If now the illumination is switched off the steady state is disturbed. The photosynthesis rate P is now zero, whereas transport and respiration rate are assumed unchanged within the next short time period. Thus, the time dependency of the oxygen concentration C immediately after the onset of darkness is given by. δc = - T - R δt 8 9

5 The latter two equations can be subtracted from each other, which yields: P = - Thus, the volumetric gross photosynthesis rate P can be determined by the slope of the oxygen concentration C immediately after the light-dark shift. This is indicated in the following figure. For a more detailed discussion please refer to the references given at the end of this manual. δc δt darkening. The user has to provide the program with the start and end time of the fitting interval relative to the switching event. Typical values for the fitting interval are and seconds for the start and end times, respectively. As Sloper has to know exactly when the switching took place, a light sensor is placed within the light field near the measuring site. Its signal is used by the computer as a trigger for the switching event. In order to realize this measurement the following setup is used. The oxygen microsensor tip is moved to the measuring site with the help of a micromanipulator. The sample is illuminated with C steady state dc dt light photosynthetically actinic light of constant intensity. Steady state is achieved when the microsensor readings do not change any more as a function of time. At this point the illumination is switched of. The switching event has to be fast (<<100 ms). So, just switching off the power supply to the light source will usually not work, as most types of light sources exhibit an afterglow. Mostly, shading the sample will be the best choice. This can be either done by blocking the light path manually (e.g. using a piece of cardboard) or with an electronic shutter. The computer samples the voltage readings of the microsensor. Typical sample frequencies are Hz, typical sampling intervals start 1-3 seconds before and stops 2-6 seconds after the switching event. The interval before the switching is used to calculate an average value of the steady state oxygen concentration. The volumetric gross photosynthesis rate is obtained by fitting a slope to the data sampled in the fitting interval immediately after dark t 10 11

6 Measuring Setup Starting of Sloper and Settings The UNISENSE Data Acquisition Module ADC-216USB is connected to a USB-port of the computer. The voltage signal of the microsensor is connected to channel A, while the UNISENSE Light Sensor is connected to channel B. The microsensor tip is positioned with help of a micromanipulator at the measuring site in the illuminated sample. The UNISENSE Light Sensor is placed with a lab stand such that it is placed within the light field, but without disturbing the light to the measuring site. Thus, the signal of the light sensor acts as a marker for determining when the darkening of the sample took place. The micromanipulator can be optionally motorized by a UNISENSE Motor Controller. Read the instructions provided separately for this controller. The controller is connected to the computer via the serial port (RS 232). After starting Sloper the user is asked whether the last calibration should be retrieved. Press No if you are running the program the first time. The option to retrieve the last calibration will be discussed later. Now the settings in the following dialog box have to be adjusted: The settings for the data acquisition are on the left side. Select the appropriate data acquisition module under Da t a Acquisition. Ra n g e A and Ra n g e B define the expected voltage range of the sensor signals and the light sensor signal. Different ranges can be chosen between ±1V and ±10V. The range should not be set too high, because this will cause a loss in resolution. A software Fi l te r is implemented in order to diminish any influence of the power line frequency on the measurements. Select either 50 Hz or 60 Hz depending on the region (e.g. Europe 50 Hz, USA 60 Hz). Du m m y DAQ can be checked for testing reasons, if no data acquisition module is connected to the computer. A sinusoidal signal will be simulated for both channels of the data acquisition module. On the right side, the settings for the motorized micromanipulator can be found. First select the appropriate m o t o r c o n t r o l l e r m o d e l

7 Se r i a l Po r t refers to the port number of the serial interface (RS 232) that the UNISENSE Motor Controller is connected to. This value is normally 1 if your computer provides only a single serial port. An g l e is the angle in degrees between the microsensor and the surface normal of the sample under investigation. If the microsensor penetrates the surface perpendicularly this value is 0. All depths used by Sloper are real depths inside the sample measured perpendicularly towards the surface. The actual distances the motor has to move are calculated by correcting the real depths with the value of Angle. For example, if the microsensor penetrates the sample with an angle of 45 and the user wants to move the microsensors 100 µm in depth, the motor actually moves the sensor 141 µm along its longitudinal axis. Du m m y Mo t o r can be checked if no motor controller is connected to the computer. In this case, Sloper simulates the behavior of a motorized micromanipulator. Sa v e can be used for saving the current settings in a file chosen by the user. The saved settings can be retrieved later by clicking Lo a d. Overview of the Main Window The following figure shows all elements in the main window When Sloper is terminated, the program saves the last settings. These settings are automatically retrieved at the next startup. After pressing OK in the settings dialog box, a data file name has to be selected. Finally the main window of Sloper shows up. The settings can be readjusted anytime by pressing the Se t t i n g s button in this main window. The area with the blue buttons is used for controlling the motorized micromanipulator. All operations for the saving of the data in a file are performed in the area with the grey buttons. In the area with the green Measure button, the parameters for the actual photosynthesis measurement are set. The large area to the right is used for monitoring the sampled data. The indicator Light Status shows information, whether the sample is illuminated or not. Next to this is the Channel A chart recorder. Here the readings of the microsensor connected to channel A are shown in real time. This recorder is especially used during the measurements in order to determine whether the sample under investigation has reached steady state. The readings shown in the chart recorder are not saved to a file

8 The two graphs Steady State and Slope show the results of photosynthesis measurements. The steady state value and the value of the fitted slope (x-axis) are plotted against the depth position (yaxis). All data points shown in these two graphs are always saved to the data file. Motor Control The box to the right shows the elements for controlling of the motorized micromanipulator. All values for depth indicated in this box represent the real depth in the sample (see paragraph Starting of Sloper and Settings under Angle). The depth unit is always micrometers. Ac t u a l De p t h indicates the current depth position of the microsensor tip. Pressing Go t o moves the microsensor to the new depth set in New De p t h. If you press either Up (or <Page up> key) or Do w n (or <Page Down> key) the microsensor will be moved one step up or down, respectively. The step size can be set in Ste p. While the motor is moving, the background of the Actual Depth indicator turns red and a red STOP Mo t o r b u t t o n appears. The motor can be stopped immediately by pressing this button (or by the <ESC> key). The velocity of the motor is set in Ve l o c i t y. The minimum and maximum values are dependent on the type of motor controller you are using. Refer to the provided manuals of the motor controller. The reference depth can be chosen by entering a value into the field next to Set Ac t u a l De p t h. After pressing this button, the Actual Depth indicator will be set to the entered value. An example will be used to clarify this: a convenient way to establish a reference point is to move the microsensor tip to the surface of the sample using the Up and Down button with relevant step sizes. When the sensor tip is touching the surface, type 0 next to the Set Actual Depth button and click this button. The Actual Depth indicator will be set to zero. Assuming also that the correct value for Angle was entered in the settings, all other depth values in the program now refer to the actual depth in the sample. Positive values indicate positions inside the sample, while negative values indicate positions above the surface. Important Under certain circumstances the motor cannot move after it has been commanded to move (e.g. due to a mechanical block). In this case STOP Motor appears, but there is no change in depth indicated in Actual Depth. Press the STOP Motor button and fix the problem with the motor

9 File Handling The Chart Recorder All data points acquired by Sloper are always saved into a text file. This box contains all the elements for the file handling. This file can be read by common spreadsheet programs. As separator characters tab and return are used. The current file name is indicated in File. You can choose a new file by pressing New File and selecting a file name in the file dialog box. The size in kb of the actual file is indicated in Size, while the space left on the volume (e.g. hard disk C:) in MB is indicated in Free. The data points saved in a file are separated in successive data sets. A header with date, time, data set number and current parameter settings of Sloper is written before each data set. The actual data set is indicated in Actual Data Set. You can generate a new data set by pressing New Da t a Set. By pressing Ch e c k File a window is opened wherein the current data file can be viewed as it would appear in a common spreadsheet program. Maximally the last 200 lines of the data file are shown. The content of the window will be updated each time Check File is pressed again. On the upper right side of the window you can find a chart recorder for the microsensor signal at channel A. It shows the actual value two times per second. The x-axis gives the time in seconds. Additionally the actual reading is shown above the chart. There are several possibilities to c h a n g e t h e r a n g e of the chart. You can click directly with the mouse onto the upper or lower limit of the x- or y-axis, and type in a new value followed by <Return>. The buttons or provide autoscaling for the x- or y-axis, respectively. This feature can also be permanently activated by clicking on the switches. The buttons and can be used for changing the format, precision, or the mapping mode (linear, logarithmic). The button offers several zoom options. After clicking the button, the user has the possibility to click onto the chart and move the whole area while keeping the mouse button pressed. By pressing with the right mouse button on the black part of the chart recorder, a pop-up menu appears. Clear Ch a r t removes all old data shown in the chart recorder. Under Up d a t e Mo d e it is possible to select three different modes for the graphics updating, when the visible part of the chart recorder is filled up. In the first mode the visible part is continuously scrolled. The second mode clears the chart recorder and starts again at the beginning, whereas the third mode also starts at the beginning but overwrites the old data. The actual position is indicated by a vertical red line. The latter mode is also the default mode. The items Au t osc a l e X and Au t osc a l e Y function in exactly the same manner as the auto scaling switches in the tool panel described above. Important Under certain circumstances the motor cannot move after it has been commanded to move (e.g. due to a mechanical block). In this case STOP Motor appears, but there is no change in depth indicated in Actual Depth. Press the STOP Motor button and fix the problem with the motor

10 Calibration Before the first measurement the light sensor at channel B has to be calibrated. The microsensor at channel A can also be calibrated (optional). If the microsensor is not calibrated all data are given as voltage data. calibration of a sensor, e.g. 100 % and 0 % air saturation for oxygen sensors. These two values are entered into the controls for Po i n t 1 and Po i n t 2. Now the sensor is inserted into a standard solution for each of the two points. It can be seen on the chart recorder when the signal achieves a steady state. By pressing Set Po i n t 1 or Set Po i n t 2, respectively, these signal values are read into the Ac t u a l Calibration indicator next to the corresponding button. The average time for reading this value can be set in the Av e r a g e Ti m e control. When this procedure has been done for both points, the square indicator to the left turns red and the sensor is now calibrated. All values in the main window now denote calibrated units. If you want to return to viewing the uncalibrated voltage data, press the Un c a l. button. Light sensor calibration If the microsensor in channel A is calibrated all values seen in the strip chart recorder and in the profile window are calibrated values, which are calculated by linearly mapping them onto the scale defined by the two calibration values. This is indicated by the red color of the round indicator. Furthermore, the unit label on top of the chart recorder switches from [V] to [cal.]. The actual calibration procedure is performed by pressing the Calibration button. Calibrating the sensors We start with the calibration of the microsensor at channel A. The current voltage readings are shown in the chart recorder to the upper right. The handling of this type of recorder was described in the previous section. Two points must be chosen for the The light sensor has to be positioned exactly as for the actual measurements. The current voltage readings are shown in the chart recorder to the lower right. Press Li g h t On during illumination. Now switch the illumination off and press Li g h t Off. The actual voltage readings of the light sensor are shown in the boxes next to the buttons. When the calibration is done, the squared indicator turns red. The measured light data (see below) are always given in units of %. 100% refer to the light intensity during Light On calibration, whereas 0% refer to the Light Off calibration. A calibration can be saved in a file by pressing Sa v e and later on retrieved by using the Lo a d button. When Sloper is terminated, the calibration is automatically saved on the hard disk. At the next startup Sloper will ask you whether it should retrieve the last calibration. This could be useful if the setup was not changed since the last measurement. After the calibration is finished click OK. You should check now whether the light calibration was successful. Switch the illumination on and off and observe whether the light status indicator in the main window responds correctly. If this is not the case, repeat the calibration procedure. Be sure to make the dark periods during the calibration and calibration check as short as possible, if measurements are to be performed shortly after. This is because long dark periods require a long period to attain a new steady state

11 Photosynthesis Rate Measurements Before the actual measurement can be started, some parameters have to be adjusted in the main window. These were already described in the paragraph The Light-Dark Shift Method. The sampling rate (or sampling frequency) is set in Sa m p l e s p e r sec. The total sampling duration is set in Be f o r e s w i t c h and Af te r s w i t c h. They define the recorded time interval in seconds before the switching event and after it, respectively. The time interval used for the slope fitting is defined in St a r t and En d. The values are given in seconds relative to the switching event. The fitting interval can be interactively changed after the measurement (see below). Now the actual measurement can be started. The microsensor tip is positioned at the measuring site and the sample is illuminated. The sensor readings in the chart recorder have to be observed until they reach a stable value indicating that the system is in steady state. Now click the Measure button (or the <F1> key) and wait at least as many seconds as set under Before Switch before you darken the sample. This is important because otherwise the trigger signal of the light sensor cannot be detected. Keep the sample in the dark until the Slope Measurement window is opened. Slope masurement window The upper chart shows the sensor data together with the fitted slope. The x-axis is the time in seconds relative to the switching event. The y-axis of the upper chart gives the microsensor readings either in volts or in calibrated units. The Steady State indicator shows the average value of all data points before the switching event (i.e. time < 0s). StDev gives the standard deviation for the average value. The fitted slope is shown as a thick red line. The fitting interval can be adjusted with the St a r t and En d fields. The fitted slope will be constantly updated. The slope value is shown in Slope, the r2 of the fitting in R-Square. The latter is a value between 0 and 1. The closer it is to 1, the better the fit. The lower chart shows the sampled light sensor data in units of %. The scale of its x-axis is identical to the one of the upper chart. This chart allows the user to check whether the darkening of the sample was sufficient. The user is additionally notified by a dialog box if the maximum light change was less than 90%. Comments to the actual measurement can be noted in Co m m e n t. These comments will be saved together with the measured data, if the measuring window is exited by clicking Sa v e Sl o p e. The slope value, r2, steady state, its standard deviation are saved to the actual data file. But the time series (i.e. all data points from the sensor and the light sensor) are not saved automatically. If the whole time series should be saved press Sa v e Ti m e Ser ies. Sloper creates a separate file with the name format datafile_x_y. txt, where datafile stands for the name of the actual normal data file, x for the actual data set number, and y for the actual depth in micrometer. If this file name already exists, the new data are appended to it. If the actual slope measurement should not be saved at all, press Ca n c e l and return to the main window. This might also be necessary, if Sloper did not detect the trigger signal (e.g. if you did not wait long enough with the darkening after clicking Measure, or if the light sensor is not correctly calibrated (see above)). Important Remember that the whole data series is only saved by pressing SAVE TIME SERIES 22 23

12 The Profile Windows There are two profile windows in the main window. The calculated slope and the steady state value are plotted against the depth in micrometer. The data of the current data set are indicated as white circles by default. Furthermore, the profiles of the last six data sets are indicated by different colors. The legend defines the plot mode of each data set, where the uppermost entry refers to the actual data set. The data points of the last recent 7 data sets are displayed. The y-axis refers to the depth position (µm), where the data points have been acquired. The x- axis refers either to the voltage value or to the calibrated value (in case the sensor was calibrated). The legend next to the profile graph defines the plot mode of each data set, where the uppermost entry refers to the actual data set. By clicking on an element in the legend a pop-up menu appears. The items Co m m o n Pl o t s, Co l o r, Li n e Wi d t h, Li n e St y l e, Po i n t s t y l e, In t e r p o l a t i o n can be used to change the appearance of the plotted data points (The items Bar Plot, Fill BaseLine, and Y-Scale are not appropriate to this application). If the graph becomes too crowded, the points of the oldest data set can be removed by clicking Cl e a r Oldest Co l o r. By repeatedly pressing this button all data sets except the current one can be removed. This operation does not effect the data file. The scaling of the profile graph can be modified by the user as described for the chart recorder (see section Chart Recorder ). Additionally, a yellow cursor is available inside the profile graph for reading precise values of data points. The actual position of the cursor can be read as x (voltage or calibrated units) and y (depth position in micrometer) in the cursor control panel below the profile graph (see illustration). In order to move the cursor, click onto the cursor button in the tool panel. Now you can click onto the center of the cursor and drag it to a new position. By clicking the cursor mode button a pop-up menu appears. The first three items Cu r s o r s t y l e, Po i n t s t y l e, and Co l o r can be used to alter the appearance of the cursor. The last two items of the pop-up menu are useful if the cursor is not within the visible part of the profile graph. If you click Br i n g to center the cursor will be moved to the center of this window. Choosing Go to c u r s o r will change the ranges of the two axes of the profile graph, so that the cursor appears in the center. The pop-up menu from the lock button gives several options for determining the operation mode of the cursor. The first three items define the degree of freedom of the cursor. If Fr e e is chosen, it can be moved to any position. Sn a p to p o i n t makes the cursor jump to the nearest data point when it is dragged. Lo c k to p l o t works similarly but with the additional restriction that the cursor only jumps to data points belonging to a specific data set which can be chosen from the last seven items of the pop-up menu. An additional possibility for moving the cursor is the diamond shaped button. It allows precise single step movements of the cursor in all four directions by clicking its four edges

13 References Glud, R. N., N. B. Ramsing & N. P. Revsbech Photosynthesis and photosynthesiscoupled respiration in natural biofilms measured by use of oxygen microsensors. J. Phycol. 28:51-16 Kühl, M., R. N. Glud, H. Ploug & N. B. Ramsing Microenvironmental control of photosynthesis and photosynthesis-coupled respiration in an epilithic cyanobacterial biofilm. J. Phycol. 32: