USER MANUAL Chlorophyll Fluorescence Imaging
Contents
1. Fv gt Fv Where Fv is the variable fluorescence in the initial dark adapted state and Fv is the variable fluorescence at the point at which the value of a particular coefficient is being calculated A significant problem with this method is that any change in the effective rate constant for PSII photochemistry will decrease the value of the supposedly non photochemical quenching coefficient that is being calculated An example of where this might occur is when a healthy plant is subjected photoinhibitory conditions e g a combination of high light and low temperature Under photoinhibitory conditions the rate at which PSII centres are inactivated exceeds the rate at which they are replaced with a consequent decrease in the effective rate constant for PSII photochemistry qN One of two widely used quenching coefficients the other being qP the so called co efficient of non photochemical quenching is usually calculated as the change in variable fluorescence between the dark and light adapted states This parameter is not calculated by Fluor mager NPQ provides similar information qP So called co efficient of photochemical quenching Mathematically gP is equivalent to Fq Fv PSII photochemical factor It has frequently been used incorrectly to estimate the fraction of PSII centres in the open state R S Stern Volmer quenching A convenient way of describing the relationship between chlorophyll fluorescence2. selection window position of saturating E pulses Length 00 00 00 5 8 points Trace s gt pulse details Accessed through Trace s gt Pulse details this dialog allows for the camera filter name to be changed and comments that are specific to the pulse to be edited Version 2 2 0 26 Fluorlmager user manual for use with cf Imager Pulse details Control FvfFim values were significanti higher than treated sarnples Trace s gt transient details Accessed through Trace s gt Transient details This dialogue allows for the camera filter name to be changed and comments that are specific to the transient to be edited Transient details Eg Fis 4 Big differences seen between the rate of increase and treated plants Trace s gt PSI fluorescence The dialogue allows the input of the contribution of PSI fluorescence to the total fluorescence signal Expressed as a percentage of the total fluorescence signal at Fo If this is unknown Fluor mager will assume a value of zero It is not necessary to complete this box Version 2 2 0 27 Fluorlmager user manual for use with cf Imager Sub Menus Sub menus can be found by right clicking the mouse button in different regions of the screen Many of these commands can also be found under the main menus and are made available as popup menus for convenience So far we have covered the Modify image menu and the Trace windows menus above Thum
3. Fm Fo This term is equivalent to 1 Fo 1 Fm When normalised to the initial value it provides an estimate of the change in the maximum PSII photochemical efficiency that can be normally be attributed to photoinactivation of PSII centres Fq Fv A factor relating the PSII maximum _ efficiency and the PSII operating efficiency Mathematically Fq Fv is equivalent to qP G Version 2 2 0 33 Fluorlmager user manual for use with cf Imager Genty factor A term often used to describe the PSII operating efficiency after Genty Briantais and Baker 1989 H irradiance Refers to the energy flux density of light which has the units of J m s or more usually W m See also PAR PPFD and photon irradiance J K Kautsky curve L M N Non photochemical quenching Quenching of variable fluorescence that can be attributed to processes other than photochemistry Non radiative decay In the context of PSII photochemistry non radiative decay usually refers to the transition of an electron in the outermost shell of a chlorophyll a molecule from the first excited state to the ground state that results in an increase in the overall kinetic energy of the molecule rather than the emission of a photon as fluorescence or a charge separation event NPQ The term NPQ is widely used to represent non photochemical quenching It is most frequently associated with the fluorescence parameter Fm Fm _ 1 which i
4. about the current image It is a modeless dialogue which means that other parts of the program can be accessed while it is open It can be switched between its larger and smaller formats by selecting Show more and Show less respectively from the popup menu that is launched when the right mouse button is clicked The dialogue can be hidden by selecting Hide window from the same popup menu If this is done the dialogue will appear in its last location and size format when accessed again rather than the default condition Mn deficient wheat A2b 26 of 69 Eg FavFin OE 06 03 2002 J00 pmol mr 5 1 Mola Fis 4 6 RO a ee ry I x Doe 4 i ee a a 1042 76 mye rr J 15 E EP 10 43 cm U 263 0 004 to 0 574 Mn deficient wh 26 of 69 Eg FaF OE SU0 umol m4 s1 10 43 cm 0263 View gt Trace info Accessed through View gt Trace info or by selecting Trace info from the popup that is launched when the right mouse button is clicked within the trace window This dialogue provides information about the Fm or Fm pulse that is currently selected It is a modeless dialog which means that other parts of the program can be accessed while it is Open Version 2 2 0 12 Fluorlmager user manual for use with cf Imager UU 4s 300 umol rrr s1 2750 umol nm s1 TE z1 l 00 tr ms i 43 3 0 47 0 42 0 45 RC E g The dialogue can be hidden by selecting Hide window from the popup menu that is launched
5. been taken the Apply isolation command can be used as an automated method for isolating the active area s within the image If the isolation was unsuccessful if required pixels were deleted by the automated process the isolation can be reversed using the Undo isolation command A manual isolation can then be performed using the tools provided within the Modify image dialogue The Image isolation options command launches the Isolation options dialogue Image s gt Isolation options If Map image is selected pixels within new images are only active if their value is greater than zero within both the newly acquired data array and the map image If Auto low cut is selected the program increases the value of a low pass filter until the number of active pixels within the newly acquired data array is the same as in the map image Image isolation Isolation method for new images Oo hiap image amp Auto low cut Mone Cancel If None is selected no isolation method is applied The Map image option is best for samples that don t move during the period of imaging The Auto low cut option is useful in situations where some sample movement is likely Image s gt Auto region images Runs the automated region image function This creates a region image for each Isolated area within the map image This function can only be performed after a map image has been taken iew Settings Image s Trace s Window Help eee A Ee I
6. being open when Qa is in the ground state and closed when Qa is reduced Conversely a PSI centre is open when P700 is in the ground state and normally closed when P700 is oxidised P700 P PET Photosynthetic electron transfer Photon irradiance Photon irradiance is a term that is equivalent to PPFD and is preferred by some phycology journals See also PAR PPFD and irradiance Planck constant Formerly Planck s constant The energy of a single photon epsilon can be related to it s frequency v nu through the Planck constant h such that The value of his 6 626 x 10 Js See also Dirac constant PPFD photosynthetically active photon flux density PPFD is the term preferred by most plant journals The usual units are Omol m s Please note that PPFD is NOT interchangeable with irradiance or PAR Some phycology journals prefer the term photon irradiance PAR photosynthetically active radiation Although it is not incorrect to use PAR to refer to the photon content of light it should be noted that a PAR of 250 mol m s for example is incorrect because it doesn t include a reference to what is being measured The correct statement would be a PAR of 250 Limol photons m s See also PPFD irradiance and photon irradiance PSII maximum efficiency The efficiency with which light absorbed by the pigment matrix associated with PSII is used
7. can be set through the Data plot options dialogue Data plot options E E EB E g Accessed through View gt Data plot options or by selecting Data plot options from the popup that is launched when the right mouse button is clicked within the Data plots dialogue Changes to settings are made when the Save and exit button is pressed View gt Zoomed pixel data Zone lines Zone data Colony numbers and Colony data Each of these options can be displayed on the current image if selected The zoomed pixel data is only shown when the zoom has been applied to the current image View gt Thumbnail information Thumbnail links and Thumbnail options These options can be selected to on and off to allow information such as actinic light level filter and time to be displayed on each of the thumbnails The thumbnails used to build images can be displayed using the Thumbnail links option The types of thumbnails displayed can be selected in Thumbnail options View gt Region map trace All region traces and No region traces Version 2 2 0 14 Fluorlmager user manual for use with cf Imager Option which allows the user to define which traces are displayed If Region map trace is ticked and No region traces is selected only the map trace will be shown If Region map is not ticked no traces will be displayed All region traces displays all traces with and without the map image View gt Light values with trace Selection of this view op
8. g 5min Apply pulse 4000 5s Change actinic 300 5min Apply pulse 4000 5s Change actinic 400 3 5min Apply pulse 4000 Only save images on the last pulse 14 5s Change actinic 500 5 min Apply pulse 4000 Change actinic Apply pulse 7 te This number of times Details Pulse length ms 800 Take F amp Fm images with pulses 55 Change actinic 600 5min Apply pulse 4000 55 Change actinic 6800 5min Apply pulse 4000 5s Change actinic 1000 21 5min Apply pulse 4000 _ Update Add EZE pen i pei 3 40 z a Cut Copy Paste Step 23 of 23 Total time 01 06 00 Time remaining 00 00 00 s Save and exit Version 2 2 0 13 Fluorlmager user manual for use with cf Imager View gt Data plots and Data plot options Kin deficient wheat x 4 igr Data plots This dialogue plots up to six fluorescence parameters calculated from the fluorescence trace data It can be shown with or without a key on the right hand side The default location for this dialogue is directly above the fluorescence trace area of the main window This is a modeless dialogue which means it can remain open while other parts of the program are being accessed It is updated dynamically which means that parameter values are calculated and shown immediately after the application of a saturating pulse and are updated if the assumed level of PS fluorescence is changed see PS fluorescence dialog The parameters that are shown
9. held by friction to the lens to remove the filter raise the camera rack and pinion stage from its normal position both zeros opposite one another to the top of its travel and lock into position using the clamp at the rear of the stage Firmly pull down the narrower tube with a slight rocking motion and store the filter safely avoid touching it Place a non reflective black cloth on top of the sample stage and ensure the sample stage is at its normal height 140mm from base of instrument Press the Connect camera button on the toolbar bar Left click Settings on the top toolbar left click Calibration at the bottom of the menu left click Run camera calibration Before you accept yes ensure the instrument door is shut it must not be opened during the calibration An on screen message will tell you when calibration is complete Remove the black cloth and restore the sample stage to preferred height Lighting Alignment The 16 light bricks are factory set for even illumination over the sample stage If they have been accidentally disturbed or realigned for optimum illumination over irregular samples they will have to be realigned again With the continuous actinic light on ata PPFD of a few hundred umol m s a light meter located at the correct height as in the above section Lighting Calibration should be moved about over the sample stage and recorded light levels noted Version 2 2 0 43 Fluorlmager user manual for use with cf Ima
10. individual microphytobenthic cells using high resolution imaging of chlorophyll a fluorescence Limnology and Oceanography 45 1420 1425 Version 2 2 0 46
11. pasted into spreadsheet applications or to other data analysis applications Edit gt Copy to clipboard gt current image gt Zone data The data from each zone in an image can be copied and pasted into Excel or similar This includes the active area within each zone the mean fluorescence parameter value and with transient images 1 I P 1 O P 1 O I and the area between O and P lf Zones have not been identified within the current image mean values of area and the fluorescence parameter for the particular image will be copied Edit gt Copy to clipboard gt current image gt Zone Transients When a transient image is selected as the current image there is an option to copy the transient points from each zone for pasting into Excel and other spreadsheet based applications Edit gt Copy to clipboard gt current image gt Colony data Similar to zone data colony data from the currently selected image are copied to the clipboard These data include colony number x y position area and the specific fluorescence parameter for all identified colonies within an image If no colonies have been selected image mean values for each data category will be copied Edit gt Copy to clipboard gt current image gt Histogram values The values from the data and palette histograms are both copied using this command Edit gt Clipboard copy All images Image info Ctrl fone data Ctrl D Options Version 2 2 0
12. preferential excitement of PSI relative to PSII by the far red light There are at least two potential sources of error with this method Firstly it is very likely that non photochemical_ quenching will partly reverse during the far red light treatment Secondly and more importantly this method relies on the complete oxidation of Qa which in turn relies upon oxidation of the plastoquinone pool within a relatively short time typically 3 s in the presence of a ApH across the thylakoid membrane It is almost certainly the case that under these conditions the oxidation of plastoquinone at the Qo site of the cytochrome bef complex imposes a much greater limitation to non cyclic electron flow than does the rate of excitation of PSI Bendall 1982 Consequently it is unlikely that the preferential excitation of PS by far red light treatment will have the desired effect and that the level of Fo will be overestimated particularly at high PPFD The Fluor mager program employs an alternative method of Fo estimation Oxborough and Baker 1997 With this method a virtual Fo image is created during the construction of Fv Fm and Faq Fv images This image is created using the Fm selected for construction of the Fv Fm and Fq7 Fv images plus suitable Fo and Fm images If there are multiple Fo and Fm images within a file the user can select the one s that are to be used in the construction of the virtual Fo image E Fo Pam i Foy lt has
13. this is done the dialogue will appear in its last location and size format when accessed again rather than the default condition Image details Also found under Image s gt Details Launches the Image details dialogue from the current image The type of image can be changed using the wheel top corner of image type text box If copy is ticked the copy Version 2 2 0 29 Fluorlmager user manual for use with cf Imager location will be identified The camera filter if appropriate and user are also displayed A text box allows comments to be included and saved for that image type Image setting Also found under Image s gt Settings Launches the Image settings dialoque Allows user to input selection area and dimensions The most useful parameter is the data limits allowing the user to define the low and high limits e g a series of images can all be displayed using an identical defined colour scale Modify image Modify image opens for further information see How To Guide Edit overlay Also available through Image s gt Edit overlay Edit overlay allows manual numerical selection of the area of the image Moving x y co ordinates give the position of the curser within the image Allow Zoom on off Also found through Image s gt Zoom on off lf this is checked the selected area of the image is displayed at the highest magnification that fits within the image area Show zoomed pixel data show zone lines show zon
14. 87 92 Havaux M Strasser RJ and Greppin H 1991 A theoretical and experimental analysis of the qP and qN coefficients of chlorophyll fluorescence quenching and their relation to photochemical and non photochemical events Photosynth Res 27 41 55 Kautsky H Apel W and Amann H 1960 Chlorophyllfluorescenz und Kohlensaureassimilation Biochem Zeitschrift 322 277 292 Kautsky H and Zedlitz W 1941 Naturwissenschaften 29 101 102 Maxwell K and Johnson GN 2000 Chlorophyll fluorescence a practical guide J Exp Bot 51 659 668 McAllister ED and Myers J 1940 The time course of photosynthesis and fluorescence observed simultaneously Smithsonian Inst Misc Collections 99 1 37 Oxborough K and Baker NR 1997 Resolving chlorophyll a fluorescence images of photosynthetic efficiency into photochemical and non photochemical components calculation of qP and Fv Fm without measuring Fo Photosynth Res 54 135 142 Version 2 2 0 A5 Fluorlmager user manual for use with cf Imager References Research with Imaging System Lawson T amp Morison JIL 2006 Visualising patterns of COs diffusion in leaves New Phytologist 169 641 643 Morison JIL et al 2005 Chlorophyll fluorescence imaging demonstrates that lateral CO diffusion in photosynthesising leaves is limiting Plant Physiology 139 254 266 Lefebvre S et al 2005 Increased SBPase activity in transgenic tobacco plants stimulates photosynthesis and growth during early
15. 9 Fluorlmager user manual for use with cf Imager Edit gt Clipboard copy gt All images gt image info This command creates a table of data from all images in the current file which can be pasted into Excel and other spreadsheet based applications Edit gt Clipboard copy gt All images gt Zone data This command creates a table of zone data from all images in the current file which can be pasted into Excel and other spreadsheet based applications Edit gt Clipboard copy gt All images gt Options This launches the Images data clipboard options dialogue which provides options relevant to the copying of whole image data and zone data Edit gt Clipboard copy Trace s All trace points Selected trace points All pulseftransient data Selected pulsestransient data Edit gt Clipboard copy gt Trace s gt All trace points This command creates a table of data from all trace points in the current file which can be pasted into Excel and other spreadsheet based applications Edit gt Clipboard copy gt Trace s gt Selected trace points This command creates a table of data from selected trace points in the current file which can be pasted into Excel and other spreadsheet based applications Trace points can be selected by left mouse click and dragging the cursor over the trace area to be selected whilst holding down the shift and ctrl keys A blue boarder on the trace will highlight the area that has been select
16. Fluorlmager user manual for use with cf Imager technologica USER MANUAL Chlorophyll Fluorescence Imaging System Fluorlmager user manual for use with cf Imager User manual for Technologica Fluor mager software for use with Technlogica cf Imager hardware Copyright 2006 TECHNOLOGICA LIMITED All Rights Reserved Published in UK www technologica co uk Version 2 2 D Fluorlmager user manual for use with cf Imager Contents User Manual Page Before you start 4 Main Menus 8 Edit Main Menu Items 8 View Main Menu Items 11 Settings Main Menu Items 16 Image Main Menu Items 20 Trace Main Menu Items 25 Sub Menus 28 Thumbnail Right Click Popup Menu Items 28 Current Image Right Click Popup Menu Items 30 Fluorescence Terminology 31 Fluor mager Software Terminology 38 References 7 echnical 43 References Research with Imager 44 Version 2 2 3 Fluorlmager user manual for use with cf Imager Before you start The following information is for instrument use and assumes the user has installed the supplied hardware and software The instrument exterior is designed using Dutch folded panels to virtually exclude all external light thus preventing background noise influencing the sample or fluorescence measurements If the instrument is being used in a dark room all side and front panels can be removed by releasing the four corner screws of each panel using the 2mm driver supplied This feature can also be useful f
17. Reached through Settings Transients Alt T it allows for the setting up of six stock transient protocols A F Version 2 2 0 16 Fluorlmager user manual for use with cf Imager Transients Eg s000 all 1 047 4600 all 053 4000 wil 1 063 3400 alll 1 072 a000 all 1 087 2600 zll 1 041 4105 wmol mre s1 The user sets the incident PPFD during the transient The Fluor mager program calculates the duty cycle for the requested value which is given as a percentage of the maximum level max The user also sets the number of Points Time ms and the initial Interval ms between points The Fluor mager program calculates the Multiplier value that is required to satisfy these settings Illegal settings are corrected automatically For example if the user sets Points to 30 when Time is 1000 Time is increased to 1500 the minimum time that 30 points can be taken in If Time is then shortened back to 1000 Points is decreased to 20 the maximum number of points that can be taken in this time The O fluorescence level same as Fo is recorded before the light is applied while the l fluorescence level is taken as the first point during the transient The program automatically searches the remaining trace points to find the peak P If the Fluor mager program is not connected to the camera the left most dialogue above is launched when Settings Transients Alt T is selected This is a modal dialogue which must be closed bef
18. ager Fy Fm Fw Fm Fo If the current image is an Fm image the available options are Fq Fm Fy Fm amp Fg NPG FmFm 13 No options are available with other image types Appropriate build options also appear on the popup menu that is launched if the left mouse button is pressed when the cursor is within the current image area Image s gt Auto build options Launches the Auto image build options dialog Auto build options Checking a box allows the user to define the Automatically Guild images that are built with each saturating pulse The construction of certain images will depend upon particular parameter images being available E g it is impossible to build an image of NPQ without a dark adapted Fm Mm NP FrjFrni 13 image E FwiFin Fo Fyfe and Favry Image s gt Zoom on off Applies to the current image If this is checked the selected area of the image is displayed at the highest magnification that fits within the image area Trace Main Menu Items ow Take images with pulses l Addnote Open selection window Pulse details PSI Fluorescence Version 2 2 0 24 Fluorlmager user manual for use with cf Imager Trace s gt take images with pulses With this item selected images of selected parameters established in auto build option will be automatically built and placed as thumbnails with each saturating pulse Trace s gt add note edit note amp delete note This enables
19. ails dialogue launches from the thumbnail that is selected at that time The type of image can be changed using the wheel top corner of image type text box If copy is ticked the copy location will be Comments identified The camera filter if appropriate 7 and user are also displayed A text box allows comments to be included and saved for that image type Image type FyF rr Copied from Wnknowr Camera filter Logged on user John Save and exit Cancel Image s gt Settings Launches the Image settings dialogue TapE SENINES d Allows user to input selection area and Limits Selection Low igh dimensions The most useful parameter is Eel Data limits M034 JEE the data limits allowing the user to define Top i 5 Width Height the low and high limits e g a series of Fight Dimensions 76 57 images can all be displayed using an Bottom 7 identical defined colour scale Image s gt Modify Launches the Modify image dialogue see How To Guide for information Image s gt Edit overlay Launches the Edit overlay dialogue Edit overlay allows manual numerical selection of the area of the image Moving x y co ordinates give the position of the curser within the image Version 2 2 0 20 Fluorlmager user manual for use with cf Imager Image s gt Horizontal and vertical flip Flips map image Horizontal Vertical Image s gt Apply isolation amp Undo isolation After a map image has
20. alette settings Shift Alk c Paste palette settings Shift Alk y CopyePaste palette settings to all Shift 4lk 4 Copy data limits Chr Alk 0 Paste data limits Chr l alk CopyePaste data limits to all Chr 4lk 4 Paste zone lines CopyePaste zone lines to all Copy to Clipboard Edit gt cut Delete image Copy image and Paste image Cut Copy and Paste the current image Images can be copied and or moved from one location to another within the same file or from one file to another Edit gt copy selected images and paste selected images Multiple images can be copied and pasted to another location within the same file or to a different file Multiple images are selected from the thumbnail images by clicking the left mouse button while holding down the Ctrl key Images are pasted in the order that they originally occurred within the file they were copied from not the order that they were selected in Edit gt copy and paste active pixels and copy amp paste active pixels to all The active pixel locations can be copied from one image and pasted to another or to all images One instance where this can be useful is when a second file is started with the same sample This command allows the active pixel locations within the map image of the first file to be copied to the map image of the second file These commands can also be useful when region images are being used to divide a sample Edit gt Cut selection size Copy selection si
21. been suggested that this method should not be used to determine Fo in situations where plants are stressed and significant amounts of photoinhibition have occurred Maxwell and Johnson 2000 In reality the only requirements for the calculation of Fo from the above equation are that i all the PSII centres are open at the point at which Fo is measured ii there is no reversal of down regulation between the measurement of Fo and Fm and ili there is no reversal of photoinhibition between the measurement of Fm and Fm In most situations these requirements are very easily met Given the inherent difficulty of accurately measuring Fo it can be argued that the calculation of Fo gives a more reliable estimate of this fluorescence level G H Fo Version 2 2 0 30 Fluorlmager user manual for use with cf Imager TFA CS Map image A map image is normally taken at the start of every experiment This step is important for two reasons Firstly the maximum pixel values within this image are used to set the optimum camera gain for all subsequent images including the images that are used in the construction of the continuous fluorescence trace If the signal is too high when the map image is taken the user is informed and asked to decrease the amount of light reaching the CCD this is normally done by closing down the aperture although neutral density filters can also be used Secondly The map image defines all the active non z
22. bnail Right Click Popup Menu Items m m ee wf View information view links Thumbnail options Cut Delete image Copy image Paste image Copy selected images Paste selected images Copy all image info to the Clipboard View information View links and Thumbnail options These options are identical to those found under View gt Thumbnail information Thumbnail links and Thumbnail options View information can be selected to on and off to allow information such as actinic light level filter and time to be displayed on each of the thumbnails The thumbnails used to build images can be displayed using the View links The types of thumbnails displayed can be selected in Thumbnail options Cut Delete image Copy image and Paste image These options are identical to Edit gt cut Delete image Copy image and Paste image found under the main Edit menu The options allow the thumbnail images to be Cut Copied and Pasted from one location to another within the same file or from one file to another Copy selected images and paste selected images Identical to Edit gt copy selected images and paste selected images Multiple images can be copied and pasted to another location within the same file or to a different file Multiple images are selected from the thumbnail images by clicking the left mouse button while holding down the Ctrl key Images are pasted in the order that they Originally occurred within the file they were copi
23. development Plant Physiology 138 451 460 Von Caemmerer et al 2004 Stomatal conductance does not correlate with photosynthetic capacity in transgenic tobacco with reduced amounts of Rubisco Journal of Experimental Botany 55 1157 1166 Lawson T et al 2003 The response of guard cell photosynthesis to CO2 Os light and water stress in a range of species are similar Journal of Experimental Botany 54 1743 1752 Barbagallo RP et al 2003 Rapid Noninvasive Screening for Perturbations of Metabolism and Plant Growth Using Chlorophyll Fluorescence Imaging Plant Physiol 132 485 493 Fryer MJ et al 2003 Control of ascorbate peroxidase 2 expression by hydrogen peroxide and leaf water status during excess light stress reveals a functional organisation of Arabidopsis leaves The Plant Journal 33 691 705 Lawson et al 2002 Response of photosynthetic electron transport in stomatal guard cells and mesophyll cells in intact leaves to light CO2 and humidity Plant Physiol 128 1 11 Baker NR et al 2001 High resolution imaging of photosynthetic activities of tissues cells and chloroplasts in leaves J Exp Bot 52 615 621 Leipner J et al 2001 Primary sites of ozone induced perturbations of photosynthesis in leaves Identification and characterisation in Phaseolus vulgaris using high resolution chlorophyll fluorescence imaging J Exp Bot 52 1 8 Oxborough K et al 2000 In situ measurement of photosynthetic performance of
24. e data show colony numbers show colony data Also accessed through View gt Zoomed pixel data Zone lines Zone data Colony numbers and Colony data Each of these options can be displayed on the current image if selected The zoomed pixel data is only shown when the zoom has been applied to the current image Merge colonies Found through Image s gt Merge colonies Merge colonies can be used to merge two or more isolated colonies Copy Paste Internally This menu gives the user several options of copy items including active pixels selection size palette settings data limits and zone lines all of which can be copied and pasted between images or pasted to all images Selection size and zones lines can also be cut from the current image The difference between copy paste options and copy paste internally is that items copied internally are not placed on the clipboard and can therefore not be pasted outside Fluor mager Copy Paste Internally Active pixels Selection size ut selection size Shift Ctrl Palette settings b Copy selection size ShiFE Ctrl C Data limits Paste selection size Shi k Ctrl V zone lines CopyePaste selection size to all Shifk Ctri 4 Copy to Clipboard Version 2 2 0 30 Fluorlmager user manual for use with cf Imager Copy to the clipboard allows access to many of the main copy items that can be found under Edit gt Copy to clipboard There are an extensive range of clipboard copy funct
25. e is designed to be as user friendly as possible Several new features and modifications have been included in the new version of the software Version 2 2 Below we have outlined several new features that help users identify images and options 1 Thumbnail map and regions images are given a colour bar at the top of the image which corresponds to the colour traces 2 To assist identification of image types all thumbnail labels are in different colours according to the image type Map image white Regions grey Fo red Fm blue Fv Fm Cyan Fq Fm turquoise NPQ yellow Fv Fm dull yellow Fq Fv yellow green sa 70D O29T0D 3 When the mouse is moved over a menu bar key the button is highlighted The button will change colour if it has been selected and is a permanent selection Version 2 2 4 Fluorlmager user manual for use with cf Imager and depressed if it is a transient selection When the cursor is held over a button a display menu appears showing the name of the button for further details on the button bar below E FluorImager Imager9 File Edit View Settings Imagels Tracels Window Help ca Mad fic EN e ele eS 4 A status tool bar has been added to the bottom of the screen Using colour coding this bar indicates 1 whether the camera is connected 2 the identification of an I O card 3 whether the trace is running or not 4 the settings of actinic and measuring p
26. ed Edit gt Clipboard copy gt Trace s gt All pulse transient data This command creates a table of data from all trace points for all regions in the current file which can be pasted into Excel and other spreadsheet based applications With Include header info selected all experimental details and trace comments are copied along with raw fluorescence With Include header info not selected the data and comments are copied but without the experimental and user information This menu can also be accessed by right clicking the mouse on the main fluorescence trace which opens up a dialogue box in which the user can select various trace copy options Edit gt Clipboard copy gt Trace s gt selected pulse transient data Creates a table of data from all selected pulses transient data for all regions in the within the current file which can be pasted into Excel and other spreadsheet based applications Selections are made by either clicking on the pulse transient of interest which will be highlighted with a blue triangle for a pulse or yellow triangle for a transient Alternatively an area of a trace can be highlighted by hold ctrl shift and dragging the mouse over the area of the trace you wish to copy the area will be highlighted with blue boarder lines on the top and bottom of the trace window With Include header info selected all experimental details and trace comments are copied along with the fluorescence parameters With Include
27. ed from not the order that they were selected in Copy all image info to clipboard This command is identical to Edit gt Clipboard copy gt All images gt image info This option creates a table of data from all images in the current file which can be pasted into Excel and other spreadsheet based applications Version 2 2 0 98 Fluorlmager user manual for use with cf Imager Current Image Right Click Popup Menu Items Image info Image details Image settings Modify image Edit overlay wv Allow Zoom infouk Flashup Raw Images w Show zoomed pixel data Show zone lines Show zone data Show colony numbers Show colony data Merge colonies Copy Paste Internally Active pixels Selection size Palette settings Data limits FT F F F F zone lines Copy to Clipboard Include Header Info Image Pixels Bitmap Image with Overlays Bitmap Data values zone data Colony data Histogram values Histogram Bitmap Options Image Info Also found under View s gt Current image info This dialogue provides information about the current image It is a modeless dialogue which means that other parts of the program can be accessed while it is open It can be switched between its larger and smaller formats by selecting Show more and Show less respectively from the popup menu that is launched when the right mouse button is clicked The dialogue can be hidden by selecting Hide window from the same popup menu If
28. ee ion igr Auto region images Isolated area within the map image labels them and automatically creates regions for each area Each region is displayed as a thumbnail after the map image in the order they were created If run trace is selected a trace will be displayed for each region The areas are numbered according to their position in the current image from top left to bottom right Version 2 2 0 21 Fluorlmager user manual for use with cf Imager At present only 14 separate regions can be created if there are more than 14 regions the following warning will appear Regions will be created for the first 14 isolated areas and although the remaining area will be numbered and identified regions will not be created FluorImager i i xX Region images will only be constructed For colonies 1 14 Image s gt Boolean operators gt These commands can be used in the construction of region images Both the A and B images which can be the map image plus a region image or two region images are selected from the thumbnail region First the A image is selected Next the B image is selected while holding down the Ctrl key Finally the required operator is selected from the menu The resultant image is inserted after the B image Image s gt Isolate colonies amp Merge colonies The Isolate colonies command isolates up to 250 colonies within the map image The Merge colonies command can be used to merge two or more
29. era have been replaced Lighting calibration Lights Alt L Transients AIE T Intervals Alt S Prokocal A E P pel prokocol Files set settings files p Hardware Camera Filters Measuring pulses Calibration b The camera must be connected to perform this calibration Left click Settings on the top toolbar left click Calibration at the bottom of the menu left click Run Lights Calibration Using a calibrated light meter such as the Li Cor LI 250 positioned in the centre of the sample stage enter recorded values in the initially blue highlighted check boxes The sample stage must be lowered from its standard position 140mm above the instrument base by Version 2 2 0 42 Fluorlmager user manual for use with cf Imager the height of the light meter sensor In the case of the above meter this is 25mm When all nine check boxes have been carefully completed Save and exit Camera Calibration Lights Alt L Transients AIE T Intervals Alt S Prokocal A E P pel prokocol Files set settings Files p Hardware Camera Filters Measuring pulses Calibration b To perform this calibration which must be made if the lights calibration has been changed follow the on screen instructions The calibration takes several minutes and no settings should be changed whilst the calibration is in progress You will be instructed to remove the infra red filter in the telescopic lens tube This is
30. ero pixel locations for new images If a pixel location within the map image is zero the same pixel location within all new images will also be zero even if it is greater than zero in the new image Pixels with a value of zero are not included in the calculation of mean values Non zero pixels can be deleted made zero from the map image using the tools built into the Modify image dialog Modal and modeless dialogs Modal dialogs must be closed before the user can interact with the rest of the program Modeless dialogs can be left open while the user interacts with other parts of the program N O P Parameter images The parameter image types constructed within Fluor mager are Fv Fm Fv Fm Fo Fq Fm Fv Fm Fq Fv and NPQ Protocol Protocols represent the highest level of automation within the Fluor mager program They are constructed through the Protocol dialog Q R Version 2 2 0 40 Fluorlmager user manual for use with cf Imager Raw images This term is applied to the Fo Fm F and Fm images that are used in construction of the parameter images Region image A region image defines a sub set of the active pixels within the map image A separate fluorescence trace is recorded for each region image up to 14 region images can be created within a file in addition to the map image These traces can be shown hidden by selecting View region trace from a popup menu that is launched when the right mouse button is cl
31. est available value this will normally be within 1 umol m s of the requested value This is a modal dialogue which must be closed before the user can interact with the rest of the program Version 2 2 0 15 Fluorlmager user manual for use with cf Imager Light settings Actinic off 4000 56 5 S00 50 D A 4000 56 5 a00 100 1 5 4500 abe ba a00 150 2 Mh s000 fo 1 BU 200 2 g S000 Po 1 oni 400 5 8 s000 fit lea a00 BOO 0 a s000 h 1 agi ani 11 7 S500 o0 4 agi 1000 14 6 K000 Br roo a00 1200 17 5 B000 Or FOO a00 1400 20 5 Bogi Off a oni 2000 29 2 6500 45 0 S00 J000 dagr paga 100 0 S00 B843 pmol m s1 When the program is connected to the camera the Light dialogue below is launched This is a modeless version which can be left open while the user interacts with the rest of the program This version of the dialogue can be shrunk by clicking the right mouse button within the background of the dialogue and selecting Just show actinic PPFDs It can be expanded again by clicking the right mouse button and selecting Show pulse details When the Actinic on button is pressed it turns Red Orange until it is pressed again to switch the light off When the Apply pulse button is pressed the button turns Red Orange for the duration of the pulse Light lt 50 100 150 200 400 BOL 1000 1200 1400 oO e 300 2000 Settings gt Transients
32. ger As a starting point arrange the LED panels in the following way a The six central panels should be horizontal b The eight corners panels should be adjusted in pairs to give the maximum signal close to the adjacent corner c The remaining two panels and the six central panels should then be adjusted to even the light out Please note that e Even small movements of the central six panels can have quite large effects e tis normal to end up with one or more of the central bricks pointing slightly away from the centre Version 2 2 0 44 Fluorlmager user manual for use with cf Imager References Technical Baker NR and Dominy PJ 1980 Cation regulation of factors limiting primary photochemistry in spinach chloroplasts Photobiochem Photobiophys 1 155 160 Bendall D 1982 Biochim Biophys Acta 683 119 Demmig B and Winter K 1988 Light response of COs assimilation reduction state of Q and radiationless energy dissipation in intact leaves Aust J Plant Phys 15 151 162 Duysens LNM and Sweers HE 1963 Mechanism of two photochemical reactions in algae as studied by means of fluorescence In H Tamiya ed Studies on Microalgae and Photosynthetic Bacteria pp 353 372 Japanese Society of Plant Physiology University of Tokyo Press Tokyo Genty B Briantais J M and Baker NR 1989 The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence Biochim Biophys Acta 990
33. header info not selected the data and comments are copied but without the experimental and user information Version 2 2 0 10 View Main Menu Items Experiment details Current image info Trace info Protocol Data plots Data plot options zoomed pixel data oy Zone lines fone data Colony numbers Colony data Thumbnail information Thumbnail links Thumbnail options m re 11 All region traces Mo region traces Light values with trace Trace selection window ow Toolbar Status Bar Refresh FS View gt Experimental details Fluorlmager user manual for use with cf Imager Opens up Experimental details dialogue where experimental information such as author project experiment and comments can be included This dialogue box allows input of current image and current trace pulse comments which can be copied with the data to a spreadsheet Experiment Details Tobacco x 12 colon X Author Kevin Oxborough Project Fluoarlmager test Experiment Tobacco in Petri dish Experiment comments Current image 026 comments Currenttrace pulse 004 comments View gt Current image info Version 2 2 0 11 Fluorlmager user manual for use with cf Imager Accessed through View gt Current image info or by selecting Image info from the popup that is launched when the right mouse button is clicked within the main image This dialogue provides information
34. hin the parameter image will be much greater than the true situation The Smoothing commands can be used to apply a smoothing algorithm to noisy raw images This algorithm has been written in such a way that it has minimal effect on the overall mean value of the image When a smoothing routine is applied to a non smoothed image a copy of the image is automatically made No copy is made if a smoothing routine is applied to an image that has already been smoothed at least once Image s gt Crop to selection boundaries amp Crop all to selection boundaries These commands crop the current image or all images within the file to the coordinates of the current selection Image s gt Restore map When a map image is taken a backup is automatically made This image is stored when the file is saved to disk If live pixels are subsequently deleted using the tools in the Modify image dialog The Restore map command can be used to revert to the original map image Image s gt Maximise image area The Maximise image area Command increases the size limits of the image to the maximum size possible see Image settings dialog and assigns a value of 128 to every pixel This option is no longer necessary for current software Image s gt Build gt The options that are available depend upon what type of image is current If the current image is an Fm image the available options are Version 2 2 0 93 Fluorlmager user manual for use with cf Im
35. icked while the cursor is over the thumbnail of the region image in question See Working with region images for further details S Saturation pulse A short pulse of light typically a PPFD of a few thousand Omol m s that is used to measure Fm and Fm Within Fluor mager individual PPFDs and saturation pulse lengths can be set at each of the stock incident PPFD levels through the Light settings dialog Settings files set When the program is started the default settings are loaded for the person who is logged on If different settings are loaded by opening a settings file these settings become the default and are loaded the next time the program is started by the same user Settings files can be loaded and saved through Settings gt set files gt T Thumbnail image Thumbnail versions of all the images within a file are shown in the thumbnail images area Transient In the context of Fluor mager a transient is the increase in fluorescence that results from the application of sub saturating incident light to dark adapted material the initial part of a Kautsky curve Fluorlmager has the facility for the setting up of 6 stock transients which can be applied at any time that a fluorescence trace is being recorded These transients can be set up through the Transient settings dialog A transient can also be applied as part of a protocol U V W Width and Skew of a colour palette The width and ske
36. ion window These are not Version 2 2 0 25 Fluorlmager user manual for use with cf Imager accessible when the selection window has been opened Several copies to clipboard options are also available within this popup menu Last 30 5 of trace Current selection ow Saturation pulse Open selection window Copy to Clipboard Include Header Info Selected trace points Selected pulse transient data Option 1 last 30 s of trace Displays the last 30 s of the current trace this will be updated as the trace is running Option 2 current selection Displays a user defined area The area can be selected holding the ctrl shift key down whilst dragging the mouse across the trace area of interest The selected area is shown on the trace by blue boarders Length 01 39 58 Length 00 03 28 s 214 points The selected area shown by the blue boards on the trace window is shown in the current selection box at the right of the main trace area Option 3 saturating pulse Displays the last saturating pulse This will be updated as the trace is running Particular saturating pulses can be user defined by clicking the mouse on the pulse of interest in the main trace window The selected pulse is shown by a downward arrow at the top of the trace The position of saturating pulses within the trace is illustrated by blue squares Length 01 39 58 s Pulse selected for display in the current Blue squares indicate
37. ions see Edit gt Copy to clipboard for more details Copy to Clipboard Include Header Info Image Pixels Bitriap Image with Overlays Bitmap Data values fone data Colony data Histogram values Histograrn Bitmap Options Version 2 2 0 31 Fluorlmager user manual for use with cf Imager Fluorescence Terminology ABCDEFGHIJKLMNOPQRSTUVWXYZ C D Dirac constant The Dirac constant h bar is equal to the Plank constant divided by 27 h 2z The value of h bar is 1 054 x 10 Us DeltaF AF This term refers to the difference in fluorescence signal at two defined points One of the many ways in which it has been used is to define the difference between F measured immediately before a saturating pulse and Fm measured at the peak of the same saturating pulse Within the Fluor mager program and these help files Fg is used in this context E ETR Electron transfer rate F F The fluorescence signal at any point between Fo and Fm Within the Fluorlmager program and these help files F is used in preference to Fs and Ft which have often been used in the same context Fm The maximum fluorescence signal when all PSII centres are in the closed state from dark adapted material Fm The maximum fluorescence signal when all PSII centres are in the closed state from light adapted material Fm Fm 1 A way of quantifying changes in the level of non photochemical quenching of ch
38. isolated colonies Map image before colony Colony isolation applied All Two colonies in yellow box have isolation separate colonies are numbered been merged Note now there is and labelled starting at the top left only one number and label for the two colonies Image s gt Auto space zone lines This command automatically distributes both horizontal and vertical zone lines evenly across the image Version 2 2 0 35 Fluorlmager user manual for use with cf Imager The left image shows uneven distribution of horizontal and vertical zone lines over a 96 well plate After the application of Auto space zone lines the horizontal and vertical zone lines are even distributed in both directions right image Image s gt Select as the current Fm lf the current image or trace saturation pulse was taken in the dark this command can be used to make it the current Fm image or pulse The current Fm image will be used to calculate all other fluorescence parameter based upon dark adapted Fm The position of the current Fm image on the trace is indicated by a red triangle Image s gt Smooth amp Smoothing of new Fo images gt lf the chlorophyll fluorescence signal from a sample is weak possibly due to a low concentration of chlorophyll within the sample the noise within the image can be significant This is particularly true with Fo images If noisy raw images are used in the construction of parameter images the spread of values wit
39. lorophyll fluorescence NPQ Version 2 2 0 39 Fluorlmager user manual for use with cf Imager Fo The minimum fluorescence signal when all PSII centres are in the open state from dark adapted material Fo The minimum fluorescence signal when all PSII centres are in the open siate from light adapted material Within Fluor mager virtual Fo images which are required for the construction of Fv Fm and Fq Fv images are constructed using the Fo calculation method Fes The contribution of PSI fluorescence to the total fluorescence signal Expressed as a percentage of the total fluorescence signal at Fo or Fo See PSI fluorescence dialog Fq The difference between F measured immediately before a saturating pulse and Fm measured at the peak of the same saturating pulse The non specific term DeltaF AF has often been used in the same context Fs In most situations this term is equivalent to F Ft In most situations this term is equivalent to F Fv Variable fluorescence difference between Fo and Fm from dark adapted material Fv Variable fluorescence difference between Fo and Fm from light adapted material Fv Fm Provides an estimate of the PSII maximum efficiency within dark adapted material Fv Fm Provides an estimate of the PSII maximum efficiency within light adapted material Fq Fm Provides an estimate of the PSII operating efficiency within light adapted material Fv
40. ng Settings gt set settings files gt load defaults Settings gt Hardware These are factor set for camera hardware and interface and should not be altered by the user Hardware settings Settings gt Camera filters Allows input of filter names Not applicable with current system Settings gt Measuring pulses Sets measuring pulse length interval and peak signal Version 2 2 0 18 Measuring pulse settings Fo MP length Fo MP interval ims Peak signal atF thigh PPFD Peak signal at F low PPFD Peak signal at Fo ero PPFD Values represent offull scale save and exit Settings gt Calibration 1200 ms Fluorlmager user manual for use with cf Imager Camera and light calibration This is factory set but should users wish to re calibrate the instrument see Appendix I Image Main Menu Items Details Settings Modify Edit overlay Horizontal Flip Vertical Flip Apply isolation Undo isolation Isolation options Boolean operators Isolate colonies Merge colonies Select as the current Fm Smooth Smoothing of new Fo images Crop to selection boundaries Crop all to selection boundaries Maximise image area Restore map image Build Auto Guild options zoom on off Version 2 2 0 19 Fluorlmager user manual for use with cf Imager Image s gt Details Launches the Image details dialogue Image details x orien The image det
41. nies Command ii split colonies iii re run the Image s gt Isolate colonies command iv merge colonies Current Fm All of the fluorescence parameters calculated within Fluor mager require a value for Fm or Fm with the exception of the parameters calculated from fluorescence transients some parameters that require a value for Fm also require a value for Fm and in some cases Fo Specifically Fv Fm Fo Fv Fm and Fq Fv all require values for Fm and Fo while NPQ requires a value for Fm The Current Fm is used in the calculation of these parameters See Select the current Fm for details Current image The large image in the middle of the Fluor mager program screen the currently selected image Current pulse This is the saturation pulse that is currently selected from within the main trace area Version 2 2 0 32 Fluorlmager user manual for use with cf Imager Current transient This is the transient that is currently selected from within the main trace area A saturation pulse or a transient can be selected from within the main trace area Consequently there can be a current pulse or a current transient but not both D E F Fo calculation method In order to calculate Fv Fm and Fq Fv the value of Fo must be known A widely used method for the estimation of Fo is to expose the sample to weak far red light in the absence of actinic light It is assumed that under these conditions Qa is maximally oxidized due to
42. of PSII centres in the open state tends to decrease with increasing incident PPFD this parameter becomes an increasingly inaccurate method of estimating the yield of non radiative decay processes at PSII Version 2 2 0 37 Fluorlmager user manual for use with cf Imager Fluor mager Software Terminology A Active pixels The active pixel locations within new images are defined using the map image If a pixel location contains a zero value within the map image the same location in the new image will also be zero When trace data points are constructed pixel locations are excluded from the pixel count if they had a zero value within the map image or the data point image Administrator of the Fluor mager system Setting up a system for data acquisition or the running of either calibration routine requires a password This password is known to the administrator the person responsible for the instrument B C Colonies Up to 250 colonies can be isolated within the map image using the Image s Isolate colonies command Mean values are calculated for each colony within raw fluorescence images and parameter images Colonies can be merged by isolating the required colonies from the rest of the image and then selecting Image s gt Merge colonies A single colony can be split into two or more by using the tools within the Modify image dialog box These procedures should be performed in the following order i Run the Image s Isolate colo
43. or examining leaves that are still attached The base of the instrument can be removed for in situ measurements The front user access door of the instrument is detachable by gently lifting upwards off its hinges This allows increased access which can be particularly useful when aligning samples Cams on the central sample stage enable a variety of well plates to be accurately located If required the sample stage can be removed from the base plate using the 3mm socket driver supplied Your instrument is factory set for light and camera calibrations that are unique to your instrument with the data held on the installation CD In the unlikely event that you need to recalibrate the camera or the lights refer to Appendix This may be necessary if the camera is replaced or the lights require readjustment It is also important to note that the factory default setting refers to a nominal sample stage height of 140 mm above the base of the instruments zero on the fixed scale aligned with 30 on the moving scale of the sample stage rack and pinion This can be locked into position using the small lever at the rear of the stage It is important to ensure that all measurements are conducted using this 140mm reference height as light intensity alignment and area measurements are all calibrated to this height Because measurements are referenced to this nominal height the sample stage must be lowered by the mean height of the sample The Fluor mager softwar
44. ore the user can interact with the rest of the program When the program is connected to the camera the middle dialogue is launched This is a modeless version which can be left open while the user interacts with the rest of the program This version of the dialogue can be shrunk by clicking the right mouse button within the background of the dialogue and selecting Just show PPFDs It can be expanded again by clicking the right mouse button and selecting Show details When the Apply transient button is pressed the button turns Red Orange for the duration of the transient Settings gt Intervals Accessed through Settings Intervals AlttS the values set here define the time intervals between saturation pulses in the dark and light adapted states and between transients These values are used when continuous pulses and or continuous transients are applied Version 2 2 0 17 Fluorlmager user manual for use with cf Imager Intervals seconds Eg 1200 BU Settings gt Protocol Opens the protocol dialogue window allowing users to define an experimental protocol The protocol may include changing actinic light level applying a saturating pulse applying a transient or auto pulse These options can all be set with precise timing and repeated in cycles Settings gt pcl protocol files and set settings files Users can save and reload protocols and settings files and the default settings file can be restored usi
45. s a rearrangement of the Stern Volmer equation Bilger and Bjorkman 1990 This equation simply states that the reciprocal of fluorescence yield is proportional to quencher concentration So for example an increase in Fm Fm 1 from 1 to 2 could be interpreted as a doubling of quencher concentration In reality there is little evidence that fluorescence yield is actually modulated by quencher concentration and it should be appreciated that a change in the rate constant of a quenching mechanism at any location within the PSII pigment bed would be indistinguishable from a change in quencher concentration Although Fm Fm 1 can clearly be a useful parameter it is important to recognize that it is simply monitoring the rate constant for non radiative decay normalized to the dark adapted level Consequently it is only valid to compare Version 2 2 0 34 Fluorlmager user manual for use with cf Imager values among samples if it is known that the samples started off in the same condition very similar initial values of FvV Em would be a good indication of this O Open and closed PSII and PSI centres Reaction centres are defined as being open if they are capable of stable charge separation photochemistry and closed if they are not In the case of PSII stable charge separation results in oxidation of P680 and reduction of Qa Since the oxidation of Qa is orders of magnitude slower than reduction of P680 a PSII centre is defined as
46. the user to add specific notes to the trace at any time points specified by the user in hours minute and seconds Enter the time into the appropriate boxes and comment in the text box below When save and exit is pressed the position of the comment is indicated by a green square located above the trace at the point of insertion Added comments are copied with the data into spreadsheets Trace note Trace time Comments Save and exit Trace comments can also be included directly on the trace using the mouse cursor Move the mouse cursor to the point at which a comment is to be added and click the right mouse button Select Add note This will launch the Trace note dialog box Trace info Pulse details PSI Fluorescence Add note Copy to Clipboard Include Header Info All trace points All pulseftransient data Le n 5 Se Added comment s can be edited or deleted using the main menu edit note delete note and the correct time being entered into the trace time box The same menu can also be accessed by a right mouse click on the trace menu at the position of the note to be changed or deleted Trace s gt Open selection window Opens the trace selection window box on right side of main trace The open window can be enlarged using the mouse with drag and drop Three options are available for data displayed in the selection window These options can be accessed using right mouse click in the closed select
47. tion add an additional light values trace to the trace display View gt Trace selection window Options within this menu allow either Last 30 s of a trace to be displayed in the selection window or the current section or the selected saturating pulse View gt Toolbar and Status Bar and Refresh Toggles between on and off for the display of the Toolbar top of the screen and Status Bar bottom of the screen New changes to the view display can be viewed using Refresh or F5 Settings Main Menu Items Lights Alt L Transients AIE T Intervals Alt S Prokocal A E P pel prokocol Files set settings Files p Hardware Camera Filters Measuring pulses Calibration b Settings gt Lights Allows manual setting of the light levels and manual control of light level changes If the Fluor mager programme is connected to the camera the light setting dialogue is launched when Settings gt Lights Alt L is selected In addition to zero light twelve different stock PPFDs can be set A to L which are used when the data is being entered in manual mode All the user has to do is to enter the desired PPFD in the PPFD column as an integer value between O and the maximum actinic PPFD The Fluor mager program calculates the duty cycle for the requested value which is given as a percentage of the maximum level max If the precise PPFD value cannot be provided the program updates the requested value with the near
48. to drive stable photochemistry when all PSII centres are in the open state An estimate of the PSII maximum efficiency is provided by Fv Fm in the dark adapted state and Fv Fm in the light adapted state PSII operating efficiency The efficiency with which light absorbed by the light harvesting system associated with PSII is used to drive stable photochemistry in the light adapted state Because a fraction Version 2 2 0 35 Fluorlmager user manual for use with cf Imager of PSII centres are closed in the light adapted state the PSII operating efficiency is lower than the PSII maximum efficiency It s value can be estimated through Fq Fm which is equivalent to the so called Genty factor PSII photochemical factor A factor relating the PSII maximum efficiency and PSII operating efficiency which provides an estimate of the fraction of the PSII maximum efficiency that is actually realised Q Quenching coefficients The system of quenching coefficients includes one term qP that is related to photochemical quenching All of the remaining quenching coefficients relate to non ohotochemical quenching processes qN is the coefficient of total non photochemical quenching gE the coefficient of energy dependent quenching qT the coefficient of non photochemical quenching associated with state transitional changes and gl the coefficient of non photochemical quenching associated with photoinhibition They are usually calculated as
49. ude Header Info With this box checked details including user name image information comments and experimental details are copied along with the data to the clipboard Edit gt Clipboard copy gt Current image gt All images gt and Trace s gt Leads to sub menus for copying the current image and related data data from all images and trace s and related data Edit gt Clipboard copy Histograms and Histogram options The histograms associated with the current image can be copied in various ways Edit gt Clipboard copy gt Calibration data The calibration data option is normally greyed out the calibration data are only meaningful to trained technical support Version 2 2 0 g Fluorlmager user manual for use with cf Imager Edit gt Clipboard copy gt Current image Include Header Info Current image Image Pixels Bitriap All images p Image with Overlays Bitmap Trace s Data values Ctrl U l zone data Chrl 2 Histogram Bitmap Ctr 3 peren PRONE Colony data Ctrl l Calibration data Ctrl B Histogram values CErl H Edit gt Copy to clipboard gt current image gt Image pixels Bitmap amp Image with overlays Bitmap The current image can be exported as a Windows bitmap to programs such as PowerPoint If an area of the image has been selected only this area is copied Edit gt Copy to clipboard gt current image gt Data values The image can be copied as an array of pixel data values which can be
50. ulse lights and 5 whether the machine needs calibrating a display will appear in the far right of the status bar Version 2 2 5 Fluorlmager user manual for use with cf Imager Connect disconnect previous to imager camera and Focus ioe Saturating Trace Run Fo amp Fm or Scroll Previous Last ofthis Last of Open hardware reflected g Trace pulse Transient protocol F amp Fm up image image type this type a i EER T ee p ASE IEE E E S E BE E a E e949 New Save Focus Camera Reflective Show new Continous Continous Fo or F Scroll First Next First of Next of fluorescence Filter image trace saturating trace down image image this type this type image pulse transient lt is worth noting that not all menu bar button options are available at any one but are determined by the user options For example Trace button options are not available if a trace is not running Version 2 2 6 Fluorlmager user manual for use with cf Imager Main Menus The main File Window and Help menus are standard windows menus and need no further explanation here Edit Main Menu Items Cut Delete image Chri Copy image Chrl 0 Paste image Ctrl Copy selected images Ctrl F Paste selected images Chrl H Copy active pixels Cbrl E Paste active pixels Ctrl I Copy amp Faste active pixels to all Ctrl Copy selection size Shift Chrl 2 Paste selection size ShiFk Ctrl CopyePaste selection size to all Shift Ctri 4 Copy p
51. w controls provide a high degree of control over the presentation of false colour images The width of a colour palette determines the dominance of the colours at either end of the palette range It s value can be set anywhere between 0 and Version 2 2 0 A Fluorlmager user manual for use with cf Imager 100 The skew control moves the central region of the palette to the left or right of centre It s value can be anywhere between 50 left and 50 right XYZ Zone line Zone lines can be used to divide an image into a maximum of 96 zones This method of dividing an image is particularly useful when working with well plates and other well ordered samples When working with randomly distributed samples the colonies or region images methods may be more suitable See Work with zone lines 96 well plate example for more information Zooming When an area is selected within the current image the selected area is magnified by the highest possible integer value while keeping the selection within the boundaries of the current image area of the program screen or Modify image dialog This magnification can be switched off and on by clicking the right mouse button within the selected area of the image and selecting Zoom on off from the popup menu or by selecting Image s gt Zoom on off from the menu bar Appendix 1 Lights and Camera Calibration The lighting and camera calibrations do not need altering unless the light units or cam
52. when the right button is clicked If this is done the dialogue will appear in its last location when accessed again rather than the default location The dialogue will be updated with the selection of a different pulse from the trace left click on trace pulse If the current level of PSI fluorescence FPSI is assumed to be greater than zero see the PSI fluorescence dialog the value of all parameters is calculated assuming both zero PSI fluorescence and the selected value of PSI fluorescence the latter value being shown in brackets View gt Protocol Opens current protocol dialogue window A protocols represent the highest level of automation within the Fluor mager program A protocol allows the user to set up an automated procedure that can be saved and retrieved A user can change light intensity apply pulses apply transients and auto pulses all of which can be timed exactly There is also an option to repeat various steps within a protocol using different cycle numbers Commands within a protocol can be altered updated copied and pasted SSS OEE EE a Protocol x After a delay of 5 nin S Q ap Dela y Actio n amp y cles P F did D um ol m7 E Perform the following action 5S Apply pulse 4000 4s Change actinic a 5min Apply pulse 4000 Apply transient Auto pulse 4 5s Change actinic 100 lt 5 min Apply pulse 4000 55 Change actinic 150 5min Apply pulse 4000 max 8 5s Change actinic 200 Pulse PPFD 4000 100 00
53. yield and downregulation is to treat downregulation as a Stern Volmer quenching process The Version 2 2 0 36 Fluorlmager user manual for use with cf Imager Stern Volmer equation simply states that the reciprocal of fluorescence yield is proportional to quencher concentration p k A k B Where F is the yield of chlorophyll fluorescence A and B are Stern Volmer quenchers and ka and kg are the rate constants for the quenching of the singlet excited state of chlorophyll by A and B respectively See NPQ for further information T U V Variable fluorescence The difference between Fo and Fm or Fo and Fm Termed Fv in the dark adapted state Fv in the light adapted state Variable fluorescence is modulated by both photochemical and non photochemical quenching processes XYZ 1 Fo 1 Fm Originally described by Havaux et al 1991 This parameter is equivalent mathematically and conceptually to FvFm Fo which was originally described by Baker and Dominy 1980 1 Fv Fm This parameter originates from a study by Demmig Adams et al 1986 in which it was used incorrectly to estimate the yield of non radiative decay processes at PSII in the light adapted state This parameter actually provides an estimate of what the combined yield of non radiative decay processes and chlorophyll fluorescence would be if all PSII centres were in the open state at the point of measurement Since the fraction
54. ze Paste selection size and Paste selection size to all A rectangular selection can be made from within an image by clicking the left mouse button at one corner of the required area and dragging to the opposite corner while Version 2 2 7 Fluorlmager user manual for use with cf Imager holding down the Shift and Ctrl keys The coordinates of this selection can be copied and pasted to other images in the same file or a different file Edit gt Copy and Paste palette settings and Copy amp paste palette settings to all The palette settings for one image can be copied from one image and pasted to another Edit gt Copy and paste data limits and copy amp paste data limits to all Allows users to copy palette settings from one image and paste them to another or all other images This is particularly important if selected images are being compared with each other using the same colour palette scale Edit gt Cut zone lines and copy zone lines amp paste zone lines and copy amp paste zone lines to all Zone lines can be copied from one image to another or pasted to all images Using the cut Command removes all zone lines from the selected image Edit gt Copy to clipboard The extensive range of clipboard copy functions are described below Edit gt Clipboard copy Include Header Info Current image All images Traces Histogram Bitmap Ctrl G Options Calibration data Ctrl B Edit gt Clipboard copy gt Incl
Download Pdf Manuals
Related Search