Instrument Manual
Contents
1. Data Export Data can be exported from either the Acquisition or Analysis panels by pressing Export or right clicking the graph and selecting Export Data presented in each respective graph will be captured and displayed on the export panel as both an image and collected data arrays The units and filtering of original data are preserved in the exported data For example if the loaded data is presented in umole the exported data will be of umole Each data array s name is shown to its left To save the image and data select the image format and press Save All numeric data is saved to a csv excel file with the file path indicated in the field at To export only unprocessed unfiltered voltage data make sure that in the Analysis window that Lowpass Filter is set to gt 50 smoothing is set to 0 and that Voltage Only is marked in the Units Select section Then select the Concentration tab and ensure the desired data sets are selected in the legend Select Export and in the export panel press Save This will save raw voltage data acquired from the probes without baseline scaling or processing Troubleshooting Despite our best effort to design a robust user friendly system Clark electrode based measurements of biological gas exchange are still an art We have done our best to design a system which controls many of the confounding variables which may cause experi2. Vol 9 No 8 1251 1261 Patents Karns D Meuser JE Posewitz MC Dempsey E 2011 Hydrogen and Oxygen Sensing Clark Electrode System for Hydrogen Producing Algae Characterization Provisional patent 61 334 997 filed May 14th 2010 Non provisional Utility patent filed May 14th 2011 25
3. 2012 Photo catalytic conversion of carbon dioxide to organic acids by recombinant cyanobacterium incapable of glycogen storage Energy and Environmental Science Vol 5 11 9457 94561 Meuser JE Baxter BK Spear JR Peters JW Posewitz MC Boyd ES 2013 Contrasting Patterns of Community Assembly in the Stratified Water Column of Great Salt Lake Utah Microb Ecol 1 13 Meuser JE D Adamo S Jinkerson RE Mus F Yang F Yang W Ghirardi ML Seiber M Grossman AR Posewitz MC 2011 Genetic disruption of both Chlamydomonas reinhardtii FeFe hydrogenases Insight into the role of HYDA2 in H2 Production Biochem Biophys Res Commun 417 2 704 9 Meuser JE Boyd ES Ananyev G Karns D Radakovits R Murthy UMN Ghirardi ML Dismukes GC Peters JW Posewitz MC 2011 Evolutionary significance of an algal gene encoding an FeFe hydrogenase with F domain homology and hydrogenase activity in Chlorella variabilis NC64A Planta in press DOI 10 1007 s00425 011 1431 y Wecker MSA Meuser JE Posewitz MC Ghirardi ML 2011 Design of a new biosensor for algal H2 production based on the H2 sensing system for R capsulatus Int J Hyd Energ in press Work V H Radakovits R Jinkerson R E Meuser J E Elliot L G Vinyard D L Lauren M L L Dismukes G D Posewitz M C 2010 Increased Lipid Accumulation in the Chlamydomonas reinhardtii sta7 10 Starchless Isoamylase Mutant and Increased Carbohydrate Synthesis in Complemented Strains Eukaryotic Cell
4. Scheme Design button can be entered where specific light intensities are defined during the course of an experiment When the system is running the pump must be turned off using the software based pump switch Once the water pump has been switched off the glass cell may be removed by depressing the metal quick connects With the cell removed the LI COR or equivalent light meter sensor can be placed on the stir plate cross hairs representing the location of the sample cell and the calibration can be performed as described below Note External light sensor not included is required to perform additional lighting calibrations Please use the included default lighting calibration file Lighting Calibration Procedure itr 7 o Ic Save I Load I Delete Turn the pump off and remove the glass sample cell by disconnecting the quick connects at either end of the black mid m rg N an N Light Units w Ni T sideValue tntensty Panel Position your light sensor approximately above the blue 5 fas Gp I dotted cross hairs on the stir plate Enter the light meter 6 50 mp Ta 5 PP 10 C nst measurements in the Intensity field at a series of slide values 3 5 8 ranging from 0 10 at 0 5 increments of the Slide Value field 9 90 6 Slide values may be set using either the slide or the numeric 5 3 field Click Inser
5. calculate the O concentration in solution which corresponds to the voltage signal produced by the electrode polarized for O measurement so be sure to have the appropriate salinity values input before taking baselines Hydrogen Calibration H calibration can be performed using a 5 recommended H Ar gas mixture Unlike other Clark electrode systems the ALGI DGA LPT has built in amplification and filtering for the H measuring circuit that does not require the preparation of platinum black by alternating polarization overnight Instead a polished platinum YSI 5331 electrode response to a 5 H gas mixture when polarized at 0 6 V on the H responsive circuit should correspond to approximately 2 3 V The exact of H used for calibration should be entered in under the Probe Baselines tab To calibrate infuse a solution of 1 mL water or appropriate biological buffer in the sample cell by bubbling with the gas mixture wait for signal stability and record the High calibration point Insert the percentage gas mixture into the H Gas Cal field under the Probe Baselines tab e g 5 H gas mixture 5 It is important when doing the H calibration that the gas line is first completely purged with the standard gas or proper signal will not be reached To determine the electrode baseline simply purge with Argon or Nitrogen wait for signal stability and record the Low calibration point H Gas Cal I
6. plug that can removed by hand The provided external hose can be pressed into this fitting temporarily for filling The second provided hose attached to a male metal quick connect fitting can be pressed into the drain on the back right hand corner of the system Once the two hoses are connected a deionized water source can used to flow water through the entire system The user may need to hold the system at various angles to help allow the escape of entrapped air as the water flows through the circuit Once it seems that all the air has been removed both hoses can be removed and two drops of the provided Bio guard solution preventing growth within the water system can be added to the water before re applying the orange plug and reaffixing the lid and retaining screws Instrument Start up 1 To begin communication of the system to the computer plug in the provided USB cable connecting the internal National Instruments DAQ 6009 to the computer The green indicator light on the instrument blinks when communication has been established 2 Open the ALGI_app software On the main window the left most pane should indicate the name of the USB 60090EM data acquisition card DAQ in communication with the computer Click Open ACQ PCI 6225 Al G i 1201f4 Monitoring w Open ACQ Open Analysis 3 Once the software has been started and communication to the DAQ has been established the primary power
7. 6 C 3 13778E 6 PID Gains r r f S f Experimental Parameters r Chlorophyll 0 ug r Cell Count 0 xik r Salinity 0 ppt Volume 1 mL Cell counts Concentration of cells can be determined by hemocytometer or Coulter principle based particle counters like the Z Series COULTER COUNTER Cell and Particle Counter In the Acquisition panel under Experiment Data cell count data x 10 can be entered prior to an experimental acquisition and this data will be saved for downstream analysis Cell count data can also be used for data analysis after an experimental run but entering the appropriate data in the Analysis panel and hitting Reprocess A J l J i i 16 Chlorophyll Total chlorophyll Chl concentrations from algae or cyanobacteria cultures is typically determined by solvent extraction ethanol or methanol cell debris removal by centrifugation and spectrophotometric determination The experimentally determined ug Chl equivalents in the algae sample assayed can be entered into the Experiment Data section prior to analysis and will be stored as meta data for downstream analysis Similar to cell count data Chl data may also be entered in manually during data analysis with data appropriately recalculated using the Reprocess button in the data analysis section Salinity For measurements done in solutions which have
8. Algae Light and Gas Instruments ALGI Dissolved Gas Analyzer 1 0 DGA LPT 1 0 User Manual Revision 3 0 August 2013 ALGI LLC 6606 South Chase Court Littleton Colorado 80123 contact ALGInstruments com Hardware ed alg I com Software devin alg l com Experimental Design Biology jon alg l com Preface Software Installation and Instrument Set up Software Installation Other required hardware calibration gas purging systems Filling the fluid temperature control system Instrument Start up Probe Preparation Probe Polarization and Calibration Oxygen Calibration Hydrogen Calibration Probe storage and maintenance Lighting Preparing for lighting calibration Lighting Calibration Procedure Lighting Scheme Design LED temperature control Sample Temperature Control Experimental Parameters Acquisition Procedure Experimental data file naming and storage Starting an Acquisition Experimental Parameters Data Analysis Panel Data Export Troubleshooting References and Patents Preface Software Installation and Instrument Set up Software Installation The required software is provided on a USB flashdrive Updates can be provided from ALGI LLC by mail on a DVD disc Contact us through our website for a direct link to download the software directly from the web Our software requires additional drivers which are installed using the provided installer Simply transfer the provided folder to the appropriate place for program
9. NS ESS b b Yeh bh eke we KB 9 10 10 00 00 00 00 0010 00 00 20 00 00 30 00 00 40 00 00 50 00 01 00 00 0110 00 01 20 00 01 30 00 01 40 h To view acquired data click on the Analysis tab at the top of the window In the upper left most field click the folder icon to open the acquisition folder Navigate to the desired file and click OK The experiment and sample information will be loaded into the fields Cell Count Chlorophyll H2 Gas Cal and Salinity and the acquired data will be displayed in the bottom graph The experiment and sample information can be changed and will automatically update on the graph as will changing any field on the analysis panel but the new value will not be saved to disk until the file is right clicked and Save is selected 22 Similar to the acquisition panel the units of the loaded data can be changed by selecting the desired units format through the top buttons in the Units Select section The selected units will appear as a label on the left hand y axis while the lighting units will appear on the right hand y axis Selecting Voltage will disable any other unit selection and report unscaled voltage Acquisition data can be filtered and smoothed using both the low pass filter frequency and the smoothing slide On each graph are cursors that report y values of data sets By default these cursors are associated with the first data set Probe 1 23
10. al at the designed constriction points Care must be taken that Teflon membrane is not excessively folded or bunched as to cause pressure on the walls of the cell or breakage could occur Once the two probes are installed the provided 3 x 3 mm stir bar Wilmad Labglass LG 9566T 108 desired 1mL liquid solution and capillary air lock can be added to complete the initial set up Probe Polarization and Calibration Probe 1 Mode 02 Probe 1 Polarization 0 V Probe 2 Mode 02 Probe 2 Polarization 0V ad After finalizing the initial set up of the electrodes and glass cell set the appropriate polarization voltage based on the type of measurements desired The software should automatically set the appropropriate polarization voltage based on the mode you select The system is designed so either electrode may be used for O or H measurement in the event the polarization voltages are not correct these are the suggested settings for polarization O polarization 0 8 V H polarization 0 6 V low O baseline Probe Baseline Panel Probel Probe 2 Delete Load Delete J Delete _ Load _ Delete High ov High Jov Low ffov Low fov a i i H2 Calibration Gas 0 A rP U Save Probe ID names can be manually entered so that calibration values can be stored This is helpful for two reasons First in case of software failure the prob
11. cannot be assured Moreover if the LED is driven at high intensities for periods longer than constant temperature is maintained the LED lifetime and consistency of photonic output cannot be assured Thus we recommend lighting intensities and schedules that keep the indicator light highlighted Lighting Spectrum of Photosynthetic Active Radiation PAR at 400 700nm Atilon LED Visible Spectrum 14 Sample Temperature Control Temperature can affect the response of the electrodes the molarity of dissolved calibration gases and the behavior of a biological sample For these reasons it is important to define a constant temperature setting for each experiment To set the temperature desired for an experiment the user can refer to the top of the main Acquisition panel The actual temperature is displayed both as a red line on the graph and as a number in the Temp C window The set point can be easily adjusted by the user by either dragging the thermometer display to the desired temperature or simply inputting the precise temp C in the window above the the thermometer display Temperature Calibration 24186492 2013 05 11 TC the 8 Delete Load Save U 4 Cal Point 1 CalTemp1 OC Calibrate 0 4 J Cal Point 2 Cal Temp 2 0C Calibrate 0 t 25 1 1 Cal Point 3 Cal Temp 3 0C Calibrate 0 ed a Recalculate Coefficients A 0 00492012 B 0 000435
12. e calibration can be reloaded with ease Second saving calibration data allows the user to track the sensitivity of the electrodes over time to determine when new electrodes should be purchased due to an unreasonable decline in sensitivity Important note on calibration accuracy Probe sensitivity changes over time as the Ag anode becomes coated with tarnish The decline in sensitivity is very great in the first 30min to 1h then the slope of decline is reasonably low for many hours as the response become more stable However for greatest accuracy a new calibration can be performed prior to each experimental measurement by bubbling with calibration gases Oxygen Calibration Setting the probe polarization to O or unplugging the probes is our recommended method to determine the low baseline Other users prefer purging with inert gas or addition of sodium dithionite i e sodium hydrosulfite O calibration High baseline can be performed simply using the same solution which will be used in the subsequent assays in equilibrium with atmospheric gas concentration For high baseline add 1 mL of the same air saturated solution ensure that the micro stir bar is freely stirring in solution wait for signal stability and temp equilibration and click the High button under the appropriate probe menu to record the high calibration point On board barometric pressure sensor and user defined salinity values input into the software are used to
13. f performing H measurements it is required that the mixture of the calibration 9 gas in composition be entered into this window of the Probe Baseline tab The provider of the calibration gas should provide a spec sheet with this data attached to the provided gas tank This value is used by the software to determine the appropriate molar concentration represented by the voltage difference recorded during high and low calibration data acquisitions Probe storage and maintenance The probes are a consumable component of the ALGI DGA LPT system but their lifetime can be extended by proper care Electrodes should only be polarized when active measurements are being performed to extend their lifetime If the probes are left polarized for extended periods their sensitivity will be markedly reduced Sensitivity can be restored by cleaning tarnish from the silver anode however after multiple cleanings the silver itself will be removed and the probe will no longer function When not in use the probes should be stored dry covered with a membrane unpolarized and unplugged 10 Lighting Preparing for lighting calibration Using a common light sensor such as a LI COR LI 250A the output of light at the sample site can be calibrated to umol photon m s Photosynthetic Active Radiation PAR or any other desired measure of irradiance lumens ect Once this calibration has been performed and saved an experimental scheme design Lighting
14. from the silver anode but care must be taken to not remove excessive silver or the probe will be destroyed over time Also care should be taken not to clean the center platinum electrode too often since it is typically the silver side that becomes oxidized 3 An o ring should be applied to the provided plastic applicator in preparation for installing the membrane If this is a newly purchased system a membrane installation kit should be included that makes installing membranes a little easier There should be an included manual that explains how this kit should be utilized 4 A drop of saturated KCL solution should be applied to the probe head 5 A Teflon membrane should be stretched across the probe head to entrap the KCL droplet and held affixed with thumb and index fingers of the left hand The o ring can subsequently be applied with the right hand using the plastic applicator It is recommended for the majority of measurements to use a high sensitivity membrane If you find your experiments are saturating the probe circuits you can try using the standard sensitivity membranes both should be included 6 Each probe should be plugged in to the phono plug receptacles located on the rear left of the instrument and polarized for a minimum of 30 minutes such that probe baselines and responses stabilize prior to calibration using standard gas concentrations 7 The probes can now be gently pressed into the glass cell such that the o rings se
15. g Ctrl or Shift left click can be copied 13 or deleted using the controls at the bottom of the segment list H As well schemes can be cleared by clicking the Clear button Schemes can be saved and loaded for editing later by typing a name in the upper right most field and clicking Save or by selecting a scheme in the pull down menu and clicking Load G LED temperature control In the early stages of our research and development we found that the intensity of the LED output would drop off significantly as the LED heated up during an experiment Thus without maintaining a constant temperature we could not calibrate and define a specific photonic flux with confidence The current system features an LED illumination system kept at 30 C by a digitally controlled peltier thermoelectric temperature management system This system is capable of maintaining the LED at constant temperature well above the equivalent of full solar incidence 2500 umol photon m st PAR However the lighting can be driven up to approximately 8000 umol photon m s PAR which is above the point that the peltier system can maintain constant temperature for long periods Thus the ALGI software incorporates an indicator light which provides a visual reference that constant temperature of the LED is maintained When the LED Temp indicator is not highlighted the user is informed that proper temp and thus maintenance of calibrated LED output
16. ikely Also if the primary power supply is turned on when there is no communication to the ALGI software the LED light may turn on full brightness without any temperature control and damage to the LED by overheating could occur To maintain the longevity of the LED it is important to establish communication to the software prior to turning on the main power supply switch Probe Preparation The ALGI DGA LPT 1 0 Dissolved Gas Analyzer with Light Pressure and Temp control system utilizes YSI http www ysireagents com category php categoryld 328 5331 Clark type electrodes and the associated YSI 5775 Standard O measurement and YSI 5776 High sensitivity H measurment membranes The YSI 5331 probe consists of a platinum cathode silver anode and saturated KCL solution contained by a disposable Teflon membrane and held in place by an O ring Preparing the electrodes for operation video of electrode preparation 1 If a new kit is being used the KCL crystals provided in the dropper bottle should be dissolved with enough distilled water to fill the provided bottle to the top 2 If the electrode has been previously used tarnish which develops on the silver anode may need to be cleaned off One method to clean the probe involves using a cotton tipped swab dipped in 3 NH OH to wipe the silver anode followed by a DI water rinse Also a fine 4000 8000 grit emory cloth ALGI Electrode Cleaning Kit may be used to remove tarnish
17. lines are acquired appropriately defines that differences in voltage as equivalent to the appropriate molar quantity of gas in solution 21 Salinity For measurements done in solutions which have low salinity this value can be left set to zero However if measurements are done in solutions of high salinity ocean water or greater then it is appropriate to provide a salinity value ppt so that the differences in dissolved calibration gases compared to fresh water can be accounted for Also despite providing a salinity value for appropriate calculation of dissolved gases care also must be taken in consideration of the greater sensitivity of the electrodes in solutions of greater salinity because of the higher fugacity of the dissolved gases In other words the dissolved gases will be driven into the KCL electrolyte to a higher degree when the sample solution is high in solutes To properly account for the salinity affect on the electrode sensitivity simply be sure to do all gas calibrations in a buffer of the same ionic composition as the sample to be assayed Data Analysis Panel Voltage Enabled Hide Y Units Concentration Integral Rate Refit ean lense w opp o a ae O ENA ay 6eF NH kA wo Amplitude Zapnyjduny Chlorophyll 0ug Smoothing conn Salinity 0 ppt LP Frequency 1 Hz H2 Cal Gas 0 Volume 0 mL oud hk we KH Oe Yew eH Aas we w oOo i e ad om na e e a r I E
18. low salinity this value can be left set to zero However if measurements are done in solutions of high salinity ocean water or greater then it is appropriate to provide a salinity value ppt so that the differences in dissolved calibration gases compared to fresh water can be accounted for Also despite providing a salinity value for appropriate calculation of dissolved gases care also must be taken in consideration of the greater sensitivity of the electrodes in solutions of greater salinity because of the higher fugacity of the dissolved gases In other words the dissolved gases will be driven into the KCL electrolyte to a higher degree when the sample solution is high in solutes To properly account for the salinity affect on the electrode sensitivity simply be sure to do all gas calibrations in a buffer of the same ionic composition as the sample to be assayed Volume This parameter should not need to be changed The sample cell volume accommodates 1 mL of solution Barometric pressure Atmospheric pressure atm is recorded by an on board integrated circuit pressure sensor The value being acquired from this circuit is displayed in the software Acquisition panel adjacent to the temperature data This value is used to determine the precise molar concentration of calibration gases based on well defined values at varying partial pressures Simply put the molar concentration of dissolved oxygen in solution is directly proportional t
19. mental inaccuracies However problems with the probe chemistry teflon membrane are the most common cause of system malfunction For instance if sample solution gets around the o ring and makes contact with the Ag anode or a membrane is damaged by a needle during gas purging or sample introduction confounding effects on electrode response have been known to occur If an electrode stops responding normally it should be removed from the sample cell cleaned and fresh electrolyte and a new membrane provided Unfortunately the normal period of electrode stabilization is required after each membrane electrolyte change and calibration must be repeated This can be a source of great frustration when biological time courses require the instrument to perform properly at a defined timepoint To best avoid the possibility of this frustration two systems can be set up in parallel Also when measuring only O or H our two probe system allows for parallel measurements for experimental redundancy in case one electrode loses activity or stability YSI 5331 probes come with a one year manufacturer s warranty However they are the disposable component of the ALGI DGA LPT system and because they do lose activity over time or fail after significant use it is a good idea to keep new electrode s on hand in case the current electrode s in use fail unexpectedly 24 References and Patents References Carrieri D Paddock T Maness P Seibert M Yu J
20. o the atm So a solution saturated with atmospheric oxygen in Golden CO at approximately 0 83 atm will contain 83 of the moles of oxygen of the same solution at sea level The software takes this into account and when high and low calibration baselines are acquired appropriately defines that differences in voltage as equivalent to the appropriate molar quantity of gas in solution 17 18 Acquisition Procedure Experimental data file naming and storage User Name Data Set Acquisition Name J File Path i Comments Prior to running an experiment meta data which will control how the data is stored must be entered in under the File Info tab of the Acquisition panel Data folders are stored in a folder labelled Data within the ALGI software folder The User Name field will define the name of the folder which your data will be stored under in addition to a subfolder based on the date of acquisition Similarly the Acquisition Name field will determine the name of the file which is being acquired This field is completely flexible as to the format which the scientist is most comfortable with storing their data If subfolders are desired for storing data for instance in cases where the same user would like to do experiments for different projects on the same day we have provided the Data Set Name field which will define subfolders for storing data The comments field al
21. s on your computer s harddrive and click on the installer setup file within the folder Other required hardware calibration gas purging systems For H measurements a calibration gas cylinder 5 recommended appropriate regulators a purging station and purging needles capable of bubbling gases into the sample cell are required Similarly H baseline data can be acquired by purging the measured gas completely out of solution by bubbling with an inert gas Argon or Nitrogen The O consuming reducing agent sodium dithionite i e sodium hydrosulfite is also sometimes used to for the baseline measurement for O measurements however this reagent does not typically have a long shelf life and is prone to partial oxidation over time Therefore for greatest reproducibility we recommending taking Low O baseline measurements with the probes polarization set to 0 Filling the fluid temperature control system On Newer systems that include an ALGi logo on the front panel please use the following youtube video link to see demonstration videos of how to correctly fill the system http www youtube com user alginstruments feature results_main If your system arrives dry the water jacket system will need to be filled To do this remove the lid using a phillips head screwdriver to remove the four retaining screws Inside an orange T fitting can be located This fitting has a quick connect containing an orange
22. so allows the scientist to store additional notes as desired within the stored data file as meta data Starting an Acquisition r Start Acquisiti Execute Selected alas Scheme on Start Ws Stop Acquisition 00 00 00 After Scheme Completion Auto Analyze After Scheme List Acquisition a PhotosyntheticActivityAssay lts SaturatingProduction lts test assay lts tester lts gt Start Scheme To start an acquisition the user simply clicks the Start Acquisition button visible on the main panel When this button is depressed the current probe baselines experimental data file info temp set point and lighting scheme design information is all referenced to inform the experiment as designed by the experimentalist The blue indicator bar adjacent to the Start ACQ button can allow the progress of the experiment to be visualized 20 Experimental Parameters Chlorophyll 0 ug Cell Count 0 dk Salinity 0 ppt Volume 1 mL ad Cell counts Concentration of cells can be determined by hemocytometer or Coulter principle based particle counters like the Z Series COULTER COUNTER Cell and Particle Counter In the Acquisition panel under Sample Data cell count data x 10 can be entered prior to an experimental acquisition and this data will be saved for downstream analysis Cell count data can also be used for data analysis af
23. supply switch may be turned on All primary power except for probe polarization including stirring lighting amp sample temperature control are powered by the power supply box The power supply should be plugged into a grounded 100 264V AC power outlet The power supply has a single plug used to connect to the main instrument When this plug is connected to the main instrument care must be taken to line up the plug properly and it can then be turned clockwise to firmly connect all sources of power The provided stir plate also has an independent on off switch When the stir plate is turned on a green indicator LED on the surface of the stir plate can be seen illuminated 4 Important note There is a switch on the stir plate that initiates alternating stirring Care must be taken that this switch is always off so that stirring is consistent E r g pory ae ppano mr r File Info J Lighting Scheme Design SetPoint Water Bath Temp Pressure Probe1 Probe 2 Dismount ir irf Ig rf LED Temp v f f Ps EJ Device 7 7 5 jasc jf Nanc lj Pump On 0 440 atm s317 6 795 Export Refit PREN 1 Oxygen A 08 Oxygen 4 Important notes When the main power switch is turned on the user must confirm the cooling fans on the white power supply box are running and not blocked or power supply failure due to overheating is l
24. t to add the calibration point To save this EREE calibration enter a name and lighting units and click save You A e A can load previous calibration files by selecting them in the Intensity pull down ring and clicking load 11 Lighting Scheme Design 10 x Close 8 g 6 4 g 2 3 04 amp 2 4 6 8 10 i 1 i i i i 1 1 1 1 1 00 00 00 00 00 10 00 00 20 00 00 30 00 00 40 00 00 50 00 01 00 00 01 10 00 01 20 00 01 30 00 01 40 Initial Intensity fo o Duration 00 00 00 Instances 1 ej Add Scheme Name l New AA Save i Alf Square Wave i 2sec flash lt al I J T Final Intensity Wo I Smooth quare Wave I mls Scheme Files sec flash Its hl Load J x Delete Jl Copy f Remove j Lighting schemes are used to define illumination schedules at specific intervals and dictate acquisition lengths That is an acquisition will run for a maximum of the designated length of a saved lighting scheme To prepare a lighting scheme click the Lighting Scheme Design button Schemes are composed of a number of segments defined by the parameters at the top of the Scheme Design tab and all active segments are displayed both as a graph and textually in the right most field To design a segment Define initial and final intensities in units specified in the loaded calibration Define the duration of the segment in hh mm ss ss format Define the number of ins
25. tances of this segment that will be added to the scheme Specify whether or not the segment will have a frequency component and if so indicate its frequency and type Determine whether the segment will be added before or after the selected segment in the right most field Click Add 12 2 00 00 15 00 00 00 30 00 Flash 3 Hz Square Wave Ramp 0 100 3 00 00 30 00 00 00 45 00 Flash 3 Hz Square Wave Ramp 0 100 A 15 second 0 100 ramped 3 Hz square wave with segment multiplier of 3 1 00 00 00 00 00 00 30 0 Smooth Ramp 0 100 2 00 00 30 00 00 01 00 00 Smooth 100 4 00 01 45 00 00 02 45 00 Flash 1 Hz Square Wave Ramp 50 0 00 00 00 00 00 15 00 00 30 00 00 45 00 01 00 00 01 15 00 01 30 00 01 45 00 0200 00 02 15 00 0230 00 02 45 00 03 00 ratte 50 Oration 0 00 tance 2_ se aoa oj 2S oe oo Eee Oe ee A composite lighting scheme The segment selected in the right field is bounded by the red cursors in the graph Prior to inserting a segment into a scheme a single instance of the segment is previewed in the small upper graph F The segment will be by default inserted after whichever segment is selected in the list By selecting a segment in the segment list the new segment may be inserted either before or after the selected segment by toggling the Before After switch Segments or combinations of segments selected usin
26. ter an experimental run but entering the appropriate data in the Analysis panel and hitting Reprocess Chlorophyll Total chlorophyll Chl concentrations from algae or cyanobacteria cultures is typically determined by solvent extraction ethanol or methanol cell debris removal by centrifugation and spectrophotometric determination The experimentally determined ug Chl equivalents in the algae sample assayed can be entered into the Experiment Data section prior to analysis and will be stored as meta data for downstream analysis Similar to cell count data Chl data may also be entered in manually during data analysis with data appropriately recalculated using the Reprocess button in the data analysis section Barometric pressure Atmospheric pressure atm is recorded by an on board integrated circuit pressure sensor The value being acquired from this circuit is displayed in the software Acquisition panel adjacent to the temperature data This value is used to determine the precise molar concentration of calibration gases based on well defined values at varying partial pressures Simply put the molar concentration of dissolved oxygen in solution is directly proportional to the atm So a solution saturated with atmospheric oxygen in Golden CO at approximately 0 83 atm will contain 83 of the moles of oxygen of the same solution at sea level The software takes this into account and when high and low calibration base
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