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HelpSmith - WinFluor V3.7.6 - Spider

1. Select the F0 frame option to define the baseline fluorescence level as the average fluorescence between the range of frames defined in the adjacent range box or select the FO option and enter the value into the FO box Select the F F0 F0 option to plot the fractional difference relative to FO or F F0 to plot the fraction of FO or Select the Fluorescence ratio option to the display fluorescence ratio of two wavelengths only available for multi wavelength image files M Fluorescence Display Fluorescence ratio v Exc 340 ao gt ch ho a ratio 380 10 Exclusion so Threshold Y Rmax 1 Eqn Eqno gt Select the wavelengths to be ratioed from the Exe ch ratio wavelength selection lists Enter the lowest acceptable intensity level for the denominator wavelength in the Exclusion Threshold box Ratios with denominators below this level are set to zero To display the ratio as a fraction of a maximum ratio tick the Rmax option and enter the maximum in the Rmax box To display ion concentration computed from the fluorescence ratio tick the Eqn option and select an equation 6 Click the Add to New Plot button to create a new Y axis and to plot the time course graph of the ROI selected in the Source list OR click the Add all ROIs button to add all ROIs except that selected as the background subtraction ROI to the plot If a plot already exists you can ad
2. m Markers Add Marker gt A graph of the time course of pixel intensity from a selected region of interest ROI within the live image one for each image pane in multi wavelength sequence is displayed along with the images Analogue signals if they are being acquired are also displayed in this area qn Zoom option can be used to magnify or reduce the region of the image displayed within the image panel mages Zoom 100 2010 Zoom factors of 25 800 are currently supported The magnified region of the image displayed within the image panel can be panned horizontally and vertically within the image Recording gt Images Signals gt Image Capture The Image Capture group of settings define the size and location of the image to be captured and the time interval between frames Image Capture A Exposure Interval 50 ms Min 0 ms CCD Area 338 x 482 _ Full CCD area Binning 2 Use rectangle Gain Set CCD area 300 y V Split CCD image Exposure Interval Sets the required time interval in ms between successive image frames The shortest valid inter frame interval is indicated below the entry box This will vary depending upon capture region size and CCD binning factor CCD Area If the camera supports CCD sub region readout the CCD area controls allow you to acquire the image from a defined sub region within the CCD
3. 1 The printer selected for output is indicated in the Output to box and can be changed by clicking the Printer Setup button to open the Printer Setup dialog box 2 The size and position of the graph on the page can be adjusted by changing the Left Right Top and Bottom page margin settings 3 The typeface used to label the axes can be selected from the Typeface list and the size of the text set by the Size field The thickness of the lines on the graph and the size of data point marker is set by the Line width and Marker size fields Lines are printed in colour as on the display screen If the Use colour option is ticked 4 Clicking the OK button plots the graph on the printer Printing 8 Copying Graphs gt Copying a Graph as Data to the Clipboard The data points of graph s displayed in a plotting window can be copied to the Windows clipboard for pasting into other programs by clicking on the window to ensure that it the active window and selecting Edit Copy Graph as Data Printing 8 Copying Graphs gt Copy a Graph as an Image to the Clipboard An image of the graph s displayed in a plotting window can be copied to the Windows clipboard for pasting into other programs by clicking on the window to ensure that it the active window and selecting Edit Copy Graph Image to open the dialog box Copy Image xi Dutput to Window Clipboard Printer Setup Calibration Bars r Typeface Time 61 20000
4. Set the upper limit of the ratio display range in the Display Max box Enter the minimum signal level that each fluorescence signal channel must exceed to be included in the ratio into the Min Signal Level box The ratio is set to zero if either signal within the ratio falls below this level Enter the event detection criteria Detection Criteria Posttive going events Threshold 5 9786 mY Level Exceed for 0 01 s Baseline Fixed Baseline At 39 532 mv Rolling Average Period E s Dead Time 43 Polarity Set the polarity of the signals to be detected selecting Positive going events for signals which rise to a positive peak relative to the detection baseline Negative going events for signals which rise to a negative peak Select Positive or Negative for to detect both positive and negative going signals Threshold Set the signal baseline and detection threshold levels using the horizontal Baseline and Threshold display cursors a y Threshold 40 Baseline Scroll through the displayed detection channel signal to locate and display the smallest signal to be detected Place the Baseline cursor on the signal baseline before the event Place the Threshold cursor at a level which is crossed by the signal and which exceeds the signal background noise Note a pair of and thresholds are displayed when the Positive or Negative detection option is selected E
5. Stage conte 101 x Stage Position List Stage Position 1 X mm 1 10 000 20 000 2 90 000 60 000 Y mm X E al a E Delete Position Al y Move To h y Coarse Fine Time Lapse Action Ir Incr Stage Position Stage movement The XY stage position can be moved by using the left right and up down arrows or centred by clicking the H button The Coarse and Fine options select the size of the movement step Stage Position List The current XY stage positions can be recorded in the stage position list by clicking the Add Position button Clicking the Move To button moves the stage to the selected position in the list The complete list or individual positions selected in the drop down menu can be deleted from the list by clicking the Delete Position button Time Lapse Action The Incr Stage Position option allows the XY stage position to be controlled during during a time lapse recording sequence in the Record Images amp Signals window When the Incr Stage Position option is selected the XY stage position is incremented to the next position defined in the stage position list between time lapse exposures cycling through the list of positions for as long as recording continues The image data at each stage position is extracted and stored in a series of Winfluor IDR data files one for each position denoted by the suffixes XY 1 idr X Y 2 idr etc
6. Graph Data or as an image Edit Copy Graph as Image Table 1 Waveform Measurements Event No Time Interval Fre quency Sequence number of event in detected event list Time since start of recording that event was detected s Time between event and previous event s Event frequency reciprocal of Interval Cursor Peak T rise T 90 Tau decay Duration Baseline gt 5 Average of block of points around display cursor Peak amplitude within defined analysis region positive negative or level can be selected Integral of signal within analysis region Time taken to rise from 10 to 90 of signal peak amplitude Time taken to fall from peak to 10 of peak Exponential decay time constant Event duration level at start of display window Analysis gt Event Detection amp Analysis gt Averaging Events Series of detected events can be combined into a single ensemble average event by averaging corresponding fluorescence and analogue sample points lined up relative to the detection point To average a series of events 1 Select the Average Events page of the Event Detection amp Analysis window 2 Select the range of events to be averaged either by select All Events OR by selecting Range and entering the range of events Note Events marked as Rejected are not included in the average 3 Click the Average Events button Displayed traces can be printed File Print Graph or copied to the
7. Recording gt Images Signals gt Z Axis Position When a lens stage positioner is available the Z Axis Position group of settings defines the lens focal plane Z position TZ Axis Position a 48 5 um KI Bj wj Z Stack Y Enable Start At o um Step Size 5 um No Steps 10 The plane of focus can be shifted up our down using the Z Axis Position slider or moved to a specific position by entering the position in microns in the Z Axis Position box and pressing the return key Z Stack The Z Stack option can be used to record a set of images at a series of focal planes To record a Z stack enter the position of the initial image plane in the Start At box the number of planes to be acquired in the No Steps box and the distance between planes in the Step Size box Tick the Enable option to enable the Z stack recording Recording gt Images Signals gt Recording Image amp Signals To record a series of images and analogue signals 1 2 Create a new data file to hold the recording by selecting File New Data File and entering a file name in the New Data File dialog box Enter the duration of the recording into the Recording Period box Select the Recording mode Select the Continuous option if continuous recording at a rate defined by the camera Exposure Interval setting is required Recording Record stop Mode Continuous v Recording Period BOs Select t
8. 480 nm y Fluorescence dF F0 Select the Fluorescence dF F0 option to display the fluorescence signal as a fraction of a predefined baseline fluorescence level FO E Fluorescence Display Fluorescence dF FO v Excit ch 480 nm y FO points 48 58 C FO 1 0 C F FO FO FIFO Display Max 10 Select the F0 frame option to define the baseline fluorescence level as the average fluorescence between the range of frames defined in the adjacent range box or select the F0 option and enter the value into the FO box Select the F F0 FO option to plot the fractional difference relative to FO or F F0 to plot the fraction of FO Enter the upper limit of the display range into the Display Max box Fluorescence ratio Select the Fluorescence ratio option to the display fluorescence ratio of two wavelengths only available for multi wavelength image files Fluorescence Display Fluorescence ratio Exc 480 nm ch bos 480 nm Exclusion 30 Threshold Rmax E Eqn El Display Max 10 Select the wavelengths to be ratioed from the Exc ch ratio wavelength selection lists Enter the lowest acceptable intensity level for the denominator wavelength in the Exclusion Threshold box Ratios with denominators below this level are set to zero To display the ratio as a fraction of a maximum ratio tick the Rmax option and enter the maximum
9. Select the source of the waveform and copy it to the Windows clipboard Waveforms may be copied from a WinWCP signal record using the Edit Copy Data menu option or from a spreadsheet or similar program Drag a digitised analogue waveform icon from the toolbox and drop it into the protocol list Insert the waveform into the protocol by clicking the _Paste_ button The waveform appears in the waveform display and its data points appear in the parameters table The parameters table consists of Initial delay defines the delay period before the series of pulses begin A list of data points for the analogue waveform The waveform can be altered by modifying this list There are a number of limitations when using the digitised waveform element Only one digitised waveform element is permitted per protocol Digitised waveforms must consist of less than 1000 data points The sampling interval of the digitised waveform must be greater than 0 1 msec If digitised waveforms are created with a spreadsheet the data points must be formatted as a pair of columns containing time msecs in the first and amplitude mV in the second E g To Vo Ti Vi etc Stimulus Protocols gt Digital Stimulus Waveforms Digital pulse fixed duration This produced a digital pulse on the selected output line of fixed duration It is defined by 4 parameters Initial delay defines the delay before the start of the pulse Duration defines the dura
10. In the Images window place one or more ROIs over regions of interest on cells where the intensity spectra are to be computed and displayed Place an ROI on an image region not containing cells to provide a measure of background intensity Select the Spectrum page in the Spectral Analysis window Select a region of interest for display in the ROI list Select a background region to be subtracted in the subtraction ROI list optional OY ee IN Individual spectra throughout the image series file can selected using using Spectrum No slider bar The Frame number and time of the start of the spectrum within the file is also indicated The displayed graph can be printed File Print Graph or copied to the Windows clipboard as data Edit Copy Graph Data or as an image Edit Copy Graph as Image Analysis gt Spectral Analysis gt Plotting Fluorescence Time Course at Selected Wavelengths Spectral Analysis Oo x Spectrum Time Course ROI ROL1 y oL 2 y M wavelengths All Wavelengths C Single Wavelength 34011 0 nm Range All Spectra C Range s 1 204 ROI 1 ROI 2 340 10 nm 380 10 t 19 05 s 7 7 340 10 nm 1 i 350 10 nm 15 Set Axes 360 10 nm 75 320110 nr 123 380 10 nm 151 To display the time of intensity at selected regions of interest 1 Select the Time Course page in the Spectral Analysis window 2 Select
11. To use the full imaging area of the CCD click the Full CCD Area button To acquire the image from a CCD sub area defined on screen use the mouse to drag in the edges of the capture rectangle superimposed on the live displayed image until it encompasses the region to be acquired then click the Use rectangle button To define the CCD sub area in terms of its X and Y pixel coordinates within the CCD click the Set CCD Area button and enter the required X and Y pixel range into the Set CCD Area dialog box which pops up Set CCD Area o x CCD readout area x pixel range Y pixel range 0 519 ok Cancel Binning If the camera supports CCD pixel binning to bin together blocks of pixels to increase image intensity and or alow faster frame capture rates enter a value greater than one into the Pixel Binning box then press the lt Enter gt key A value of 2 bins together 2x2 blocks of pixels increasing image intensity by 4 Gain Sets the camera CCD readout amplifier gain With EMCCD cameras the gain is expressed as a percentage of the maximum electron multiplying gain Split CCD Image When selected splits the the upper and lower halves of the CCD image into two separate panels This option can be used with optical image splitters such as Cairn Optosplit Recording gt Images Signals gt Display Contrast The display colour mapping options determines how pixel intensities within images are displayed on s
12. series of pulse trains varying in frequency ramp user defined waveform Details of each waveform shape and its parameters are defined in Table 1 Digital stimulus waveforms To create a digital stimulus waveform drag a digital stimulus element from the Toolbox Dig Waveforms TTL Si mer L MUL mscr r and drop it into the selected digital output channel Digo 7 Dist 2 Dig2 C Diga gt Diga gt Digs gt Digo gt pig 1 Each digital output channel controls the on 5V off OV state of a digital output line The duration and inverted non inverted signal polarity for each protocol is defined in its parameters table which can be made to appear by clicking on the element in the protocol list Repeated and linked protocols Waveform protocols can be made to repeat by ticking the Repeated Stimulation option and entering a repetition time in the Stimulation Period box Multiple stimulus protocols can also be linked together by selecting the name of another protocol in the Next Protocol list When linked protocols are in use the time set in Stimulation Period determines the time interval between protocols Stimulation Period 11 s Y Repeated stimulation Next Protocol None Divide factors Most voltage and patch clamp amplifiers divide down their command voltage input signals by some factor Enter the scaling factor into the Divide Factor box This f
13. 6xs d only 50 Shading Correction I Correction Enabled 0 Regions of Interest _Delete_ all ROIs gt To display a line profile 1 Define a Line or multi segment line ell ROIs within the image displayed in the Images window 2 Frame Type If the file contains multi wavelength images select the wavelength to be displayed from the Frame Type list 3 Select the region of interest from the list of available ROIs displayed in the ROT list 4 Line Width Define the number of pixels at right angles to each pixel point on the line to be averaged to produce the line profile in the Line Width box 5 Axes Range Select the Auto Scale option to automatically scale the plot axes range or Manual to specify a fixed range define using Set Axes The displayed plot can be printed File Print Graph or copied to the Windows clipboard as data Edit Copy Graph Data or as an image Edit Copy Graph as Image Analysis gt Line Profile gt Tracking Edges The Edge Track facility within the Line Profile module can be used to track frame by frame changes in the position of the left or right edges or width of a line profile Frame No x 41 451 7 iva a a ml gt mi Line Profile Edge Track Time ai S gt 0 6 Display Fr 0 Right edge gt Grey scale 7 Plot Time Course Zoom 100 Plot Range All Frames 204 Display Contrast Sliders C Range E Full Range _ Best_ BE 22 255 X Axis 2 IV Auto
14. Photometrics PVCAM cameras currently support this option Em Filter Em Range The Em Filter column selects the index number of the emission filter 0 No Filters 1 to be used by the filter combination If an emission filter is not available this entry is ignored The passband of the emission filter is entered in to the Em Range column This is added to the image panel labels in the recording windows m Excitation emission wavelength table No Wavelength Bandwidth exposure Em Filter Em Range 0 480nm 10 nm 50 0 500 520 1 SSOnm S nm 100 1 600 650 2 0nm 0 nm 100 0 3 0 nm 100 0 Multi wavelength Sequence The multi wavelength sequence list defines a series of up to 9 wavelengths selected from the wavelength table which can be applied in a cyclic sequence at either of two rates when the multi wavelength excitation option is selected within the Record Images window Up to 10 sequences can be defined and each given a specific name Add Wavelength fo gt Divide factor 4 Clear Sequence To define a wavelength sequence click Clear Sequence to clear the wavelength sequence list Select the row number of a wavelength from the Excitation Wavelength table then click the Add Wavelength button The value in the Divide factor box for each wavelength determines the rate at which it is applied within the sequence When all wavelengths have a divide factor of 1 or the same divide factor a simpl
15. Sets the desired size in microns of the display calibration bar Calibration bar width Sets the thickness of the displayed calibration bar in pixels Split Image Labels Defines the labels of the frames containing the upper and lower halves of the camera image when Split Image mode has been selected in the Record Images amp Signals window Upper Defines the frame label associated with upper section of the camera image Lower Defines the frame label associated with lower section of the camera image Camera Dark Level Sets the upper and lower limits of the camera dark level range used by the time lapse mode camera acquisition failure detector When an acquired time lapse frame contains an image containing only pixels in the dark level acquisition is restarted A dark level range of 1 to 1 disables this function Getting Started gt Cameras gt Qlmaging Camera Trigger Inputs QImaging cameras can be externally triggered via a TTL digital or a 5V analog output signal applied to the 6 pin mini DIN external trigger port connector on the rear of the camera The specific pins to which the signal is applied depends upon whether the camera uses an optocoupled trigger input most Q Imaging cameras or not QI Click Rolera MGIFast Optocoupled trigger inputs The trigger signal must be connected to pin 1 signal and 2 ground Non Optocoupled trigger inputs The trigger signal must be connected to pin 2 signal and 6 ground Con
16. Waveform Save File As 100 me Vout OL Set Folder 100 m Toolbox 00 4 vout Waveforms gt m Protocol Vout 0 Dig 0 L Diga Vout Digam TIL m Diosa TIL VYout 2 Dig 4 L Dios Dige Dio gt Rectangular pulse fixed size Ende Stimulation Period T5 Amplitude 100 00 rn 7 Repeated stimulation Amplitude Duration 0 1000 s Next Protocol None Divide Factors 7 Vout 0 10 Vout 1 10 Vout 2 1 To begin click the New File button to create a blank protocol or Open File to load an existing protocol Voltage waveform stimuli Voltage waveforms are constructed by dragging Vout waveform elements from the Toolbox Vout Waveforms ne mm J VN and dropping them into the selected Vout 0 Vout 1 or Vout 2 voltage waveform sequence within the protocol Protocol Yout O Vout Vout 2 A voltage waveform can consist of a sequence of up 10 separate waveform elements The amplitude and duration for each element is defined in its parameters table which can be made to appear by clicking the element in the waveform sequence Rectangular pulse fixed size 0 0000 s 100 00 mY Duration 0 1000 s Seven different waveform elements are available rectangular pulse family of pulses incrementing in amplitude family of pulses incrementing in duration train of pulses
17. connected to the external trigger input of the camera Active High Low Sets the polarity of the trigger pulse required by the camera Active High for a 0V to 5V pulse or Active Low for a 5V to 0V pulse Trigger Delay Determines the delay ms between the change of excitation wavelength in multi wavelength imaging and the triggering of camera exposure Bulb Exposure Mode When this mode is selected camera exposure time is determined by the duration of the trigger pulse and can be varied from frame to frame This option must be enabled to support the variable exposure time in WinFluor s programmable excitation emission wavelength control Clear CCD before exposure Sets the camera to clear the CCD at the beginning of a exposure Note Not all cameras support this option Enabling this option may reduce maximum frame rate of the camera Post exposure CCD readout Forces CCD readout before the next exposure can take place Note This option reduces camera performance This option is automatically selected for cameras which only support post exposure readout Extra Readout Time In externally triggered exposure mode camera exposure time is normally set to 90 of the inter frame interval The Extra Readout Time value default 0 allocates extra time for CCD readout by reducing the exposure time of the camera Calibration Lens magnification Sets the magnification factor of the microscope objective lens Calibration bar
18. each channel and pressing the Enter key Note Stimulus voltages waveforms generated during a voltage protocol are added to the holding voltage To record to file a series of images with signals if analogue channels are defined Recording gt Images Signals gt Time Course Window The Time Course Plots Live window displays the time course of the fluorescence intensity at a selected region of interest in the image and analogue signals Time Course Plots Live loj x r V Fluorescence ROI ROI y a ROI size nxn 10 Ratio 488 25 y 560 30 y Display Max 2 Excl 1 Threshold P A pren 7 ESS AD Channels a aL RS B Display P20 R 488 25 1194 560 30 1220 A TT EEEE a 300 100 1218 activo Ym 546 3 mv Im 0 1221 pA Ch 22 0 9155 mV 7 FixedZei Secs Mins aj i25s p B06 H00H0BF Fluorescence Select the Fluorescence option to display the time course of the fluorescence intensity of a selected the region of interest ROI 1 2 etc ROIs consist of n x n pixel square regions with the centred on the marker on the image The size of the ROI square is defined in the ROI size nxn box Initially ROI 1 is defined and placed at the centre of the displayed image It can be relocated within the image by moving the mouse pointer to the new location and double clicking the mouse Up to 10 additional ROIs can be defined by clicking the Add ROI butt
19. in the Rmax box To display ion concentration computed from the fluorescence ratio tick the Eqn option and select an equation Analysis gt Event Detection amp Analysis gt Viewing Detected Events The set of events detected within a data file can be viewed by selecting the View Events page in the Event Detection amp Analysis window Event Detection amp Analysis oj xj Detect events View Events Average Events Plot Graphs m Event No ars fes E E E Rejected Region of Interest ROI Ron Fluorescence Display Fluorescence d Excit ch 480 nm y 8068 r Export Export A D Channels Export Fluorescence 33 309 m 0 5 Superimpose Events Enable al 258 gt Individual events can be displayed using the Event No slider bar or by entering the number of the event in the Event No box and pressing the return key The selected fluorescence signal is display in the top display panel and the analogue channels if any in the bottom panel The duration of each signal display panel can be adjusted separately using the Duration box at the right hand edge of each display Events can be marked as rejected by ticking the Rejected option Rejected records are excluded from averages export lists or waveform measurement plots Displayed events can be printed File Print Graph o
20. incremented between recording sweeps It has 5 parameters Initial delay the delay period before the pulse begins Start at Amplitude the amplitude of the first pulse in the protocol sequence Incre ment by the increment to be added to the pulse amplitude between records Number of increments the number of steps in the sequence Pulse duration the duration of the pulse This element is typically used to explore the voltage sensitivity of ionic conductances by generating records containing the whole cell membrane currents evoked in response to a series of voltage steps to different membrane potentials Family of rectangular voltage pulses varying in duration This is a rectangular voltage pulse whose duration can be automatically incremented between recording sweeps It has 5 parameters Initial delay the delay period before the pulse begins Amplitude the amplitude of the pulse Pulse duration the duration of the pulse Incre ment by the increment to be added to the pulse duration between records Number of incre ments the number of steps in the sequence This element is most commonly used as a variable duration preconditioning pulse in 2 or 3 step protocols for investigating inactivation kinetics of Hodgkin Huxley type conductances Train of rectangular voltage pulses This is a train of rectangular voltage pulses of fixed size It is defined by 5 parameters Initial delay the delay period before the series of pulses b
21. the right edge of each plot Recording gt Images Signals gt Excitation Light Control The application of fluorescence excitation light is handled by the group of controls on the Excitation Light page Light Stimulator A Stimuetor Photo stin Excitation Light C On Off Y Turn on when recording m Wavelength C Single fo 340 10 y Multi wavelength 340 380 X C Spectrum Set Laser Intensity On Off The On and Off buttons turn the fluorescence excitation light on and off Turn on when recording Select the Turn on when recording option if you wish excitation light to be turned on at the beginning and off at the end of recording Wavelength Select the Single option to apply a single wavelength selected from one of the 10 available wavelengths listed below The list of available wavelengths is defined in the excitation wavelengths table in the Light Source Setup dialog box Select the Multi wavelength option to acquire multiple 2 9 wavelength image series using the sequence of 2 9 wavelengths from in the excitation wavelengths table defined The wavelength sequence is defined in the excitation wavelengths table in the Light Source Setup dialog box Select Spectrum to acquire a spectral series of images with the excitation wavelength being stepped over a defined range of wavelengths The spectral range is defined in the excitation wavelengths table in t
22. 0762 OO OF mmm Size 10 pixels 380 nm 712 3 Times New Roman Line Width 2 pixels Show zero levels Y Use colour Iv Show labels Iv Image size Set to 10 range Width 600 pixels Height _Ok_ Cancel 400 pixels 1 The dimensions of the bit map which will hold the image can be set using the Width and Height image size boxes The more pixels used in the bit map the better the quality of the image 2 The typeface used to label the axes can be selected from the Typeface list and the size of the text set by the Size field The thickness of the lines on the graph and the size of data point marker is set by the Line width and Marker size fields 3 Clicking the OK button copies the image to the clipboard
23. Low Bulb exposure mode E Clear CCD before exposure I Post exposure CCD Readout J Trigger Delay 0 ms Extra Readout Time 9 ms m Calibration Lens magnification 4 x Pixel size i Calibration bar size 49 Calibration bar width 4 pixels Split Image Labels Upper a Lower E z Camera Dark Level Range Tempr Set Point 50 DEGC Lower 4 Upper Disable Exposure Time Checking or cas Camera Camera Selects the required camera support library for the camera attached to the system Note Before selecting a camera ensure that the camera software drivers and support library are installed and the camera is connected and switched on Readout Speed Select the camera CCD readout rate of multiple rates are supported usually the highest COM Port If the camera is controlled via a serial communications line selects the computer COM port Tempr Set Point The Tempr Set Point setting determines the target temperature for camera equipped with Peltier cooled CCDs Disable Exposure Time Checking Q Imaging cameras only Tick this option to disable the checking of the camera exposure interval against the CCD readout time allowing exposure intervals shorter than the reported readout time Exposure Trigger Output Exposure Trigger Output Selects the analog or digital output line to be used to trigger camera exposures or Internal camera triggering The output line is
24. Sequence 13 Onm 0 nm 100 0 E 14 Onm 0 nm 100 0 500 00 nm O a S 16 Onm 0 nm 100 0 Step Size 10 nm 17 Onm 0 nm 100 0 Bandwidth 49 nm 18 Onm 0 nm 100 0 Hra Ro 19 Onm 0 nm 100 0 Excitation Emission Wavelengths Table The excitation emission wavelengths table defines up to 20 excitation emission light wavelength combinations Wavelength Bandwidth For monochromators the Wavelength column defines the centre wavelength nm of the excitation light passband and the Bandwidth column the width nm of the passband Note The Bandwidth setting is ignore by monochromators without digitally controllable slit width For fixed wavelength light sources filter wheels LEDS filters are selected by row number and the centre wavelength and bandwidth of the filter LED associated with each row should be entered in the Wavelength and Bandwidth columns Exposure The Exposure column allows the camera exposure time expressed as a of the camera exposure interval selected in a recording window to be varied for different wavelength combinations allowing high and low level fluorescence images to be captured within a single sequence without overloading the camera Reducing Exposure from its default value of 100 reduces the fraction of time during the exposure interval when the camera is being exposed Note Bulb exposure mode must be enabled in the Camera System Setup page for exposure settings to take effect Only
25. Settings ee IN CTI e e es CCAA SST ECC O CSC ECC ace es CCAA SST CECI A CET Getting Started gt Light Sources gt Sutter Lambda 10 2 Filter Wheel The connections and settings for a Sutter Lambda 10 2 filter wheel attached to a PCI 6229 interface board Camera System Setup settings er ca CT co Pt eos ECT E CCT ECC CCT e enero Signal Connections PCI 6229 68 way connector left Pie oe 53 gnd a pete AAA e ECONO ECON ESAS COMO CTS ma e o p e E mm p e po o pe m p p po pe m p e sr poo fe fs CAT pow fe CO CTC paw p ES CO ECT po fe e ETT paw e ES O CINETECA Getting Started gt Stimulus Outputs WinFluor can generate analog or digital stimulus waveforms simultaneously with image and analogue signal recording If sufficient output channels are available up to 3 voltage stimulus waveform output channels and TTL digital pulse output channels can be supported Stimulus output channels are configured on the Stimulus Outputs setup page Camera Light Source Stimulus Outputs Analog Inputs amp Timing Z Stage Voltage D A Outputs Digital Outputs Range Yout 0 Hone Start hone Vout 1 hone y End one y Vout 2 Jone y M Photo Stimulus Galvos Attenuators Shutter Meter X Galvo Control Hone y Y Galvo Control None v Voltage D A Outputs To select an analogue output channel for voltage stimulus waveform 0 select a DAC chann
26. WinFluor V3 7 6 Copyright 2002 2015 John Dempster University of Strathclyde Table of Contents Introduction Welcom Ss els ita a adn 3 Main Features ESA AIR IA 4 Supported Ha War a AEEA ooi 5 License COMMONS e enea a A A a suena ae tew 6 Getting Started Installing WIR PIO tc aie iuhel et hel eh ecole ete etcetera E 7 Hardware CORI GUI eau oNire ce wrest ccc i E cd erneaes oy nec E A opted cones ty AEA 8 Cameras Cameras ier O 9 QImaging Camera Trigger INputs sisticiosniaja ati ac is 11 Analogue Digital Interface Uni Gvcit2558Sasustonssteelintaie ata Madchooiuclokningtnh Make 12 Analog Inputs amp Amplifier S ttingS ccccesssssssssssesessesesesscsesseseseeeceesseseseseesesesecseseeaeseeeeseseeaesenesaes 13 Patch Clamp Signal CONnMECtIONS 00000 A eee ae 16 Light Sources e 3 Siac coils coin Souris leet aetna Sak lide aaa uit Sole olor 18 Cairn Optoscan Monochromator erstes 20 Sutter DG 4 Filter Changer rica a a a a a aaa 21 TULO PTI MONO NOM alot An 22 Sutter Lambda 10 2 Filter Mica e ds ees 23 Sumulls CUP ie sane A E A E E AE gun nie ain ke 24 XY Z Stages stage Controller Set Dia 25 XY Stage Controller Setup LO 27 Recording Images Signals Recording Images ad cidos 29 Image Capt re nioma rn AP E RA EEE E ENEA ECET EE TOCE ER 31 Display Contrasti i a ea e e icon 32 Shading a A E ETON E Ta E ER O eer nee 33 St imu lato ni ds 34 Time Co rse WIN Wars ati ies 35 Excitation Light Gon olivia oia da 37 Ex
27. WinFluor recording windows This is typically chosen to be a wavelength blocked by the dichroic or barrier filters in the microscope epi cube Opening time Defines the time ms allowed for the shutter to open before each exposure or sequence of exposures when multi wavelength imaging in the time lapse recording mode Frame blanking period Defines the period of time default O ms for which illumination is turned off at the end of each camera acquisition frame Shutter Control Output If the light source requires a shutter control line select the analog or digital output line to be used to open close the light source shutter from the Shutter Control list and set the polarity of the trigger pulse required by the shutter Active High for a OV to 5V pulse or Active Low for a 5V to 0V pulse Emission Filter Control lines and settings for controlling a filter wheel placed in the emission light pathway allowing the selection of specific emission wavebands to be selected along with as excitation light wavelength Filter Control Outputs Selects the group of digital output lines used to control the emission filter Select the first output line in the group from the Start list and the last output line from the End list Output lines are connected to TTL filter selection inputs Change Time Specifies the time ms taken for the filter wheel to switch emission filters Filter changes take place at the end of each exposure interval and the camera ex
28. Windows clipboard as data Edit Copy Graph Data or as an image Edit Copy Graph as Image A plot is selected for printing copying by clicking on it Selected plot is outlined in red Event Detection amp Analysis 101 x Detect events View Events Average Events plot Graphs Average Events Range All Events C Range s 4g a Im gt a D ps O 40 E A a O B 39 776 mv Analysis gt Event Detection amp Analysis gt Exporting Detected Events Detected event fluorescence and analogue channel waveform records can be exported to WinWCP Axon PCLAMP Igor Binary Wave and CED CFS format files and in ASCII tabular form To export a series of detected records 1 Click the Export A D Channels button to export the analogue data channels of one or more event records or the Export Fluorescence button to export the currently displayed event fluorescence waveform Export Export A D Channels Export Fluorescence Select the format of the export file Axon for Axon Instruments ABF format WCP for Strathclyde WinWCP format Text for ASCII text files Igor Wave for IGOR Pro Igor Binary Wave IBW format or CFS for CED Filing System format Export Events x Output file F Amy DataWWinFluor Atrial myocyte D PaujiCell3_003 Im Ym abf Change Output Fle Range Channels Export format All Events IV Im Axon Range Y Vm Text 1 8 C WCP C Ig
29. Y position lower limit mm Note WinFluor requires the Thorlabs APT System Software to be installed on the system before MLS203 stage can be selected 28 Recording gt Images Signals gt Recording Images amp Signals To acquire images from the camera and analog signals and record these to file select Record Images amp Signals to display the Record Images amp Signals window Record Images amp Signals E ioj xj Recording Expt CF V Fluorescenc Record Stop ROI 55 4 M Images Mode Continuous Ed eN Recording Period Ss m Image Capture Exposure Interval 50 ms Min 33 ms ROI size nxn Ratio 340 10 340 10 Display Max Contrast 340 10 la Grey scale Excl 1 Threshold Display Contrast Sliders Full Range Best 1172 5389 M Auto adjust iv All image panels 6 xs d only M Display 340 10 4578 L M Shading Correction Correction Enabled Add ROI Delete ROIs Display calibration bar Vv Y Acquire Backg Image Light Stimulator IR Stimulator Photo Stim Excitation Light COn Off IV Turn on when recording Wavelength Single fo 340 10 gt C Multi wavelength sa0 3e0 C Spectrum Set Laser Intensity Z Axis Position afassun ld oF 2 Z Stack Y Enable tart At 0 um Step Size Sum No Steps 10
30. a region of interest for plotting in the ROI list and optional a background region to be subtracted in the list 3 Wavelengths Select the All Wavelengths option to plot the time course for all wavelengths in the spectrum or Single Wavelength and select a single wavelength from the list 4 Range Select the All Spectra option to include all spectral time points in the plot or select Range and enter a selected range of spectra within the file 5 Click the Plot Graphs button to plot the time course s 6 The displayed graph can be printed File Print Graph or copied to the Windows clipboard as data Edit Copy Graph Data or as an image Edit Copy Graph as Image Analysis gt Pixel Intensity Histogram gt Pixel Intensity Histogram The pixel intensity histogram window displays the distribution of pixel intensity values within the image or a region of interest within the image Intensity histograms can be plotted both from live images and those recorded on file The mean minimum maximum and standard deviation of the image intensity values is also displayed Select Analysis Pixel Intensity Histogram to open the Pixel Intensity Histogram display window y iS DOS IDH Frame Type 480 nm 300 Histogram Range Full Range Auto Scale Set Axes ts o ns a ho o ya Mean 266 511 8 D 153 1 mi es Min 114 Max 870 218 me Pixel Intensity Select the source of the images to b
31. actor is used to scale the stimulus voltage output to the voltage output channel to obtain the correct voltage at the cell NOTE The voltage divide factors for Vout O and Vout 1 set automatically when amplifiers supported by WinFluor have been selected as Amplifiers 1 and 2 M Divide Factors Vout 0 10 Vout 1 10 Vout 2 1 Saving a stimulus protocol On completion a stimulus protocol can be saved for use by clicking the Save File As button and providing a name for the protocol file Stimulus protocol folder Protocol files are stored in the folder c winfluor vprot the default location and appear in the stimulus protocols list in the live recording window To change the folder used to store protocol files click the Set Folder button to open the folder selection dialog box and create or select another folder Stimulus Protocols gt Voltage Stimulus Stimulus Waveforms Rectangular voltage pulse of fixed size This is a simple pulse which does not vary in amplitude and duration between records It has 3 parameters Initial Delay the delay period before the pulse begins Amplitude the pulse amplitude mV Duration the duration of the pulse This element can be used to provide series of stimuli of fixed size or in combination with other elements to provide fixed pre conditioning pulses Family of rectangular pulses varying in amplitude This is a rectangular voltage pulse whose amplitude is automatically
32. adjust Time s o 0 2 Y All image panels Frames re M 6xs d only Set Axes Shading Correction Correction Enabled A 0 0 Regions of Interest ROI F _Delete_ ai ROIs 3 To track the edge of line profile that has been set up on the Line Profile page 1 On the Line Profile page use the horizontal Threshold cursor to set the tracking point on the selected edge of the line profile 150 Zthreshold 100 m ta 2 Track Select the part of the profile to be tracked Left Edge or Right Edge for the right or left hand end edges of the profile or Width to track the distance between the left and right edges 3 4 Plot Range Select the range of frames within the data file to be tracked Select the All Frames option to track all image frames in the file or select Range and enter the sub range to be used 5 6 X Axis Select Time s to plot the edge vs time or Frames to plot vs frame number 7 8 Click Plot Time Course to plot the edge track graph The X and Y axis range and labels of the graphs can be modified by clicking the Set Axes button to open the Customise Graph dialog box The displayed plot can be printed File Print Graph or copied to the Windows clipboard as data Edit Copy Graph Data or as an image Edit Copy Graph as Image File Import Export gt Creating Movies To create an AVI format movie showing images and plots of fluorescence time course select File Cre
33. age panels of multi wavelength files simultaneously Unticked the contrast can be adjusted for individual panels by clicking on a panel to select it then clicking the Best or Full Range button Display contrast can also be adjusted manually by selected the Sliders tab and adjusting the Brightness and Contrast slider bars to achieve the desired image contrast Display Contrast Sliders Contrast Du EDE Brightness Dn E op Recording gt Image gt Shading Correction The shading correction function can be used to cancel out uneven illumination intensity across the image by subtracting a smoothed background image a Shading Correction Correction Enabled Acquire Backg Image Image block size 10 Images averaged Hormalise to Mean Exrndbrdiarm inl To apply shading correction 1 Set the of number pixels n x n in a smoothing block in the Image block size box default 10 Set the the number of images to be averaged in the Images Averaged box default 1 Select the grey level to which the corrected image is normalised from in the Normalise to list Select Mean Min or Max to normalise to the mean minimum or maximum grey level within the uncorrected image or Zero to normalise about zero default Mean Click the Acquire Backg Image button to acquire a background image Select the Correction Enabled option to apply the shading correction to live image
34. al pulses of fixed intervals and of fixed duration It is defined by 5 parameters Initial delay defines the delay before the start of the first pulse in the series Pulse duration defines the duration of the each pulse in the series Inter pulse interval defines the time interval between pulses in the series Number of pulses defines the number of pulses in the series Invert Signal defines whether the digital pulse is an OFF ON or an ON OFF pulse If set to No the digital line is initially OFF OV and switches to ON 5V during the pulse If set to Yes the digital line is initially ON 5V and switches to OFF OV during the pulse This element can be used to apply a rapid train of stimuli to a cell Viewing Measuring Recorded Images gt Regions of Interest Regions of interest ROIs are used to define specific areas within each image for measurement required for intensity time course plots pixel intensity histograms and image region export functions Up to 50 ROIs can be defined within an image Four shapes of ROI are available e Point A single pixel within the image e Line A line of pixels e Rectangle A rectangular block of pixels e Ellipse An elliptical block of pixels e User defined line e User defined region Adding a Region of Interest To add a new ROI to an image select the shape of ROI to be added from the ROI shape list ROls o Ea 5 Drag on to image Drag the ROI on to the image and adjust its pos
35. ate Movie to open the window MM Create Movie fluorescence ratio and or analogue signal Oo x Export File Name Change Name Famy DataWinFluor Atrial myocyte D PaujiCell3_003 A I Plot Channels vm Im Fluorescence Ratio Y 480nm Range All Frames C Range 1 1008 Skip frames 0 m AVI Playback Rate 20 0 fps Clear Plot Subtract ROI gt OK Cancel Calibration Bars Text Set Font Size Arial 9 pts PlotArea 50 50 To create a movie 1 Range Select All Frames to plot a movie using all images in the data file or Range and enter a sub range of image frames Set the number of frames within the data file to be skipped to reduce the size of the AVI movie file in the Skip frames box optional 2 Plot Channels Select the Fluorescence option to plot raw fluorescence time course from the region of interest selected in the ROI list If a background ROI has been defined and is to be subtracted from the fluorescence plots select it from the Subtract ROT list For files containing multi wavelength images select which wavelength is to be plotted Plot Channels Add To Plot ROI Fluorescence Ratio jv 480nm or select Ratio to plot the ROI fluorescence ratio from a selected pair of wavelengths in a multi wavelength data file Add To Plot C Fluorescen
36. ation bar Frame Op 0 PASTA 29 19 5 20 20 5 21 21 5 22 50 Ym D my 75 65 442 mi 0 8 Secs Mins Fix Zero Levels Dis play Calibration Bar Checking this option displays a calibration bar at the left edge of the line scan image Viewing Measuring Recorded Images gt lon Binding Equations The parameters for up to 10 ratiometric ion binding equations can be entered and stored using the Ion Binding Equations setup dialog box To enter edit an equation select Setup lon binding equations To display the setup box lox Equation Add New Equation fura hd Name fura lon ca Delete Equation RMax 0 5 R Min is Kerr oa Units um OK Cancel To add a new equation 1 Enter the name of the ion binding fluorophore in the Name box and click the Add New Equation button 2 Enter the name of the binding ion in the Ion box 3 Enter the maximum fluorescence intensity ratio achieved at high concentrations of the binding ion in the R Max box 4 Enter the minimum fluorescence intensity ratio achieved at high concentrations of the binding ion in the R Min box 5 Enter the effective binding coefficient for the ion in the K Eff box 6 Enter the units to be used to express ionic concentration e g nM uM mM M in the Units box 7 Click the OK button to save the equation Equations stored within the program can be displayed edited by s
37. can be toggled on and off as required Recording gt Images Signals gt Stimulator The application of voltage and or digital stimulus pulses is handled by the controls on the Stimulator page m Light Stimulator a Excitation Light Stimulator Photo Stim fisec stim y Start Stop IV Start on Record m Hold Default VOutO o my Set Stim Folder Stimulus Protocols To generate a stimulus waveform select a stimulus protocol from the Stimulator list and click the Start button Stimulus protocols are also started automaticaly when the Record button is pressed Clicking the Stop button terminates the stimulus and returns the output voltage to the holding level Click the Set Stim Folder to change the folder holding the stimulus protocol files Stimulus protocols can consist of a series of one or more pulses incremented in amplitude or duration to create a family of pulses Complex stimulus waveforms can be produced including series of rectangular steps ramps and digitised analogue signals Individual protocols can also be linked together to automatically apply a series of different protocols during an experiment Protocols are created using the Stimulus Protocol Editor and stored as protocol files vpr files See section 2 V Hold De fault The holding voltage applied to each voltage stimulus channel VOut 0 2 can be set by entering a new voltage into the appropriate box for
38. ce Ratio 480 nm 480 nm Excl 9 Thresh Select the wavelengths to be ratioed from the two wavelength selection lists Enter the lowest acceptable intensity level for the denominator wavelength in the Excl Thresh box Ratios with denominators below this level are set to zero Click the Add To Plot button to add this plot to the Movie Add To Plot ROI1 jim RoI Ym Fluorescence ROIs and analogue channels selected for plotting are indicated in the plot list The plot list can be cleared by clicking the Clear Plot button Repeat steps 1 4 for additional plots to be added to the movie a movie can contain up to 4 plots Calibration Bars Once all ROl analogue channels have been added define the upper and lower limits of each time course plot in the calibration bars table m Calibration Bars Set the percentage of the height of the movie allocated to the time course plots in to Plot AreaSize box optional Text Click the Set Font button to change the font size and typeface used to label the time course plots optional AVI Set the AVI movie playback rate by entering the desired rate frames per second in the Playback rate box optional Optional Click the Change Name button to change the name of the AVI file to be created Click the OK button to create the movie File Import Export gt Exporting Images To export the images stored in a WinFluor data file select Fil
39. ch supports the simultaneous collection of cell fluorescence images at multiple excitation wavelengths and patch clamp current and voltage or other signals Recorded images and electrophysiological signals can be displayed analysed and plotted together on the same screen The main features of the software include e High speed image acquisition 500 frames sec depending on camera e Multi spectral excitation imaging 2 9 wavelengths per file e 1 8 analogue input channels e Continuous image analogue recording to disk e Stimulus pulse generation e Time course analysis for up to 50 regions of interest e Detection and analysis of event waveforms e Display of of excitation spectra e Pixel intensity histograms e Line profile plots e Averaging of images e Creation of ratio and ion concentration images e AVI movie creation e Import Export of images to PIC TIF and STK file formats Introduction gt Supported Hardware Cameras Andor Ixon and Luca and others supported by Andor SDK library Princeton I Pentamax Photometrics Coolsnap and Cascade and others supported by PVCAM library Q Imaging Rolera XR Retiga and other supported by QImaging library PCO Pixelfly Hamamatsu Orca ER Orca 285 C4880 81 Image EM and others supported by Hamamatsu DCAM library Analog video cameras using Data Translation or National Instruments frame grabbers Cameras supported by National Instruments IMAQakx library Thorlabs DCx Serie
40. citation Wavelength SequenCesS eceecsseseescsseseteeseescseeseesceeesecseeaesecaesecaeeaeeecseeaeeeeeeeseeaeaeeneaeees 38 MY AS ANO dd Ob 41 Z AXis POSO a A A AA AS 42 Recording Image Sia deso 43 Image A cs As tase sa dlen es es a E ne aad Savas Wy a acd E RRAN 45 Image Cap AAA A 46 Display Contrast Mage 47 Aoi a f 91 ate COMEN o a e 48 XY Slag SCO FO ass seer cca ton tea soonest ce acta toca e uted taten tte ges a E E 41 ESE FOCUS CONE Oly ernes sivecsaeionctactocusehtal aden e oca 50 Signals Monitor Seal Test Signals Monitor Seal Test cornada 51 Current amp Voltage Re E o eine datas Celia hatte 53 Holding Voltage and Test PulsesSia ixbci See cone eee A Uae AA 54 Patch Clamp Amplifier Settings siine sees ene da 55 Stimulus Protocols Creating Stimulus POLO CONS iss nd cdas 56 Voltage Stimulus Stimulus Waveforms cc eceecsseseescseesceseseesescesecaeeaesecaeescseeseesceeesecaesaeaecaeeaeereaeeeees 59 Digital Stim lus Waveform S nit ted piece Dieses daeteiehinds essed poe Gere ented eae 62 Viewing Measuring Recorded Images PRECHIONIS A A Mace fee ae GREG IEA ASE LN SN ll ACI SMES aoe 63 VIEWING Image RECON nuria AA AS AAA 65 TIMES VV INOW scien SA A ANS 67 Viewing Line Scan Record Senses An 69 TON BINGING EQUAatiONS SENS EI AA AAA Ee AGE 71 Analysis Time Course Analysis Time COURSE ANOVA acinar A a Galo Rhee 72 Ploting Time Co rSeS nnonser ee ease E A viet EEE nee ease 73 Changing RIDE Axes QO ADEN Sit
41. cked the contrast can be adjusted for individual panels by clicking on a panel to select it then clicking the Best or Full Range button Display contrast can also be adjusted manually by selected the Sliders tab and adjusting the Brightness and Contrast slider bars to achieve the desired image contrast Display Contrast Sliders Contrast 4 Jo Brightness Dn m Recording gt Images Signals gt Shading Correction The shading correction function can be used to cancel out uneven illumination intensity across the image by subtracting a smoothed background image a Shading Correction Correction Enabled Acquire Backg Image Image block size 10 Images averaged Hormalise to Mean Exrndbrdiarm inl To apply shading correction 1 Set the of number pixels n x n in a smoothing block in the Image block size box default 10 Set the the number of images to be averaged in the Images Averaged box default 1 Select the grey level to which the corrected image is normalised from in the Normalise to list Select Mean Min or Max to normalise to the mean minimum or maximum grey level within the uncorrected image or Zero to normalise about zero default Mean Click the Acquire Backg Image button to acquire a background image Select the Correction Enabled option to apply the shading correction to live images Shading correction using the background image
42. creen m Contrast 340 10 _ ES Green scale y Display Contrast sliders Full Range Best 149 5484 Auto adjust Jv All image panels M 6xs d only The Grey Scale option maps image intensity as shades of grey Green Blue and Red Scale maps intensities into shades of these colours False Colour maps intensities in a rainbow of colours ranging from minimum to maximum in the sequence black blue green yellow red white The range of pixel intensities mapped into the display colour range is displayed in the Display Contrast tab To achieve optimal image contrast click the Best button to adjust the intensity range to the range of pixel intensities within the image Click the Full Range button to set the display to the full range of intensities supported by the camera The display can be set to a specific intensity range by entering the required range into the intensity range display box and pressing the lt Enter gt key When the Auto adjust option is ticked the display contrast is automatically adjusted to the optimal setting as frames as acquired The 6 x s d only option is ticked the optimal contrast range is set to 3 standard deviations on either side of the mean image intensity and to the minimum and maximum intensities within the image when unticked When the All image panels option is ticked the contrast is set for all image panels of multi wavelength files simultaneously Unti
43. d the new time course graph to an existing Y axis by selecting its Axis No from the list and clicking the Add To Plot button instead The displayed plot can be printed File Print Graph or copied to the Windows clipboard as data Edit Copy Graph Data or as an image Edit Copy Graph as Image A plot is selected for printing copying by clicking on it Selected plot is outlined in red Analysis gt Time Course Analysis gt Changing Plot Axes amp Labels The X and Y axis range and labels of the graphs can be modified by clicking the Set Axes button to open the Customise Graph dialog box x Plot Plot 4 EX3 10 gt X Axis Y Axis C Automatic Automatic Manual Manual Min jo Max 296 4 Tick 59 28 Scale Linear y Min fo Max 200 Tick so Scale Linear y Labels XAxis lis Y Axis EX3 10 PE Update _ Update All V Lines Markers V Show Line Labels Select the graph when more than one exists in the plot to be modified from the Plot list Axis limits and tick spacing are initially set to default values based upon the range of the data The axis limits can be changed by entering new values for into Min Max and Tick spacing boxes for the X and Y axes An axis can be made Linear or Logarithmic by selecting the option from its Scale list The X and Y axis labels can be changed by editing the entries in the X Axis and Y Axis labels box
44. do a ie 75 Event Detection amp Analysis Plotting Waveform Measurement o 76 Averaging Events seenen O a tudes neta A 79 Exporting Detected EV ntS usas rats airada 80 Fluorescence DISPARA AAA EI 81 View ing Detected Events ii A A die 83 Detecting Events curia 85 Event Detection amp AnalySi Si A eid ae eA aden 87 Spectral Analysis Plotting Fluorescence Excitation Spectra cccccccesesssssssssesesesseseeescseseeseseseeseseeecseeeseeesecsenenseseeeeaes 88 Plotting Fluorescence Time Course at Selected Wavelengths oooocccnnnnnncnnnnonnccnnconnccncnnnncnos 89 Pixel Intensity Histogram Pixel Intensity Historia 90 Line Profile Displaying a Line Profil s ass A A ae E 92 ra ls AAA a eae aE A C EI S Rauseneterniaae cere ta aa 93 File Import Export Creating E 95 Exporting IMSS dels 97 Exporting Analogue Sal Sissi 98 Exporting ROP Time COURSES nad 99 Importing IMSS AS AAA 100 Printing amp Copying Graphs Printing Gee NS as ots cSt ds tl oie 101 Copying a Graph as Data to the Clip ios 102 Copy a Graph as an Image to the Clipboard oocooccicnccicnociccicconnaciccnncanononononononn cn noro nooo cono nn co noncnnnos 103 Strathclyde Electrophysiology Software WinFluor V3 7 6 Fluorescence Image amp Electrophysiology Acquisition Program c John Dempster University of Strathclyde 2002 2015 Introduction gt Main Features WinFluor is a combined image and analogue signal acquisition and analysis program whi
45. dth 20 ms OIP Chan vout 0 120 8068 Amplifier 0 5 10 15 20 25 ms Amp Gain Ch 0 A ia JV Auto scale Voltage gt y Current Holding Holding Pipette Cen Divide Factors 99 7 my 121 9 pA Vout 0 10 Pulse Pulse Resistance Vout 1 10 X 99 mv 212pA 469 0 MOhm oct Ga estimate from _Reset 00 09 E Peak Exp Amp Current and voltage channels The Channels group shows the analogue input channels containing the patch clamp current and voltage signals Channels Current eho Im y Voltage cn 1 Ym y Amplifier 1 2 C When two patch clamp amplifiers are configured the seal test pulse and cell resistance calculation can be switched between amplifiers by selecting Amplifier 1 or Amplifier 2 Display scaling The vertical display magnification is automatically adjusted to maintain a visible image of the test pulse within the display area Automatic scaling can be disabled by un checking the Auto scale check box allowing the vertical magnification for each channel to be expanded to a selected region by moving the mouse to the upper limit of the region pressing the left mouse button drawing a rectangle to indicate the region and releasing the mouse button The vertical magnification can also be adjusted using the buttons at the right edge of each plot Recording gt Signals Monitor Seal Test gt Current amp Voltage Readout A readout
46. e Export Image Series to open the window Export Images File to be Exported E Data 150409_003 IDR Frames Image Wavelengths Format All Frames Whole Image All wavelengths e TIFF C Range All ROIs MetaMorph STK 1 990 C ROI f Intoricavod BioRad PIC Ron y 4 G Separate files Skip 0 Matching Frames frame count ox Cancel C Single wavelength Exs00 10 To export a series of images 1 2 Format Select the format of the export file BioRad PIC MetaMorph STK or TIFF format Frames Select the All Frames option to export all images in the data file or Range and enter a selected the range of frames Enter the number of frames to be skipped for each frame exported in the Skip frames box optional Image Select the Whole Image option to export the whole image or All ROIs to export a series of rectangular sub images defined by the currently defined regions of interest or ROI to export the single ROI image selected from the ROI list Wavelengths For multi wavelength data files select All wavelengths to export all wavelength image types selecting Interleaved to store them in a single file or Separate files to store each wavelength types in a separate file when exporting to separate files select the Match frame count option to create files with the same number of frames matched to the nearest frame in each file To export one waveleng
47. e lens focus can be adjusted from the Image and Image amp Signals recording windows and Z stacks containing sections at a series of Z positions can be recorded Support for an voltage controlled stage or piezo lens focus controllers can be configured using the Z Stage settings in the XY Z Stage page l0lxi Camera Light Source Stimulus Outputs Analog Inputs amp Timing XY Z Stage Z Stage Enabled Z Stage Control Voltage Output None y m Calibration Voltage Range Limits Position yam Cc Minimum 4oy Position 2 10um nov Maximum 4ov Min Step Size 0 05 um Max Step Time 0 ms Excitation off during step End exposure at step m XY Stage i XY Stage Type Thoriabs MLS203 1 0 mm Motors X max 410 mm X Motor Serial Number 94828021 Y min 0 mm Y Motor Serial Number 34325022 Y max 75 mm Enable Tick the Enabled option to enable Z position control in WinFluor s Image and Image amp Signals recording windows Z Stage Control Voltage Output The Z Stage Control Voltage Output selects the analog output channel to be used to control the lens stage positioner This analog output is connected to the voltage control input of the positioner Voltage Range Limits The Minimum and Maximum fields set the lower and upper limits of the lens positioner analog input voltage working range Calibration The Position 1 and Position 2 pair of P
48. e cycle of the wavelengths within the sequence is produced When a divide factor of N N gt 1 is applied to one or more of the wavelengths these wavelengths are applied at 7 N the rate of the others For instance a sequence of 3 wavelengths 0 1 2 where O has a divide factor of 5 and the rest 1 0 480 10 nm 5 1 340 10 nm 1 2 380010 nm 1 produces the sequence 0121212121201212121212 Note Only two divide factor values are allowed with a sequence one of which must be 1 the other less than 100 Those wavelengths with a divide factor greater than one are always placed at the beginning of the sequence Spectrum The spectrum options define the spectral sequence applied when the Spectrum excitation option is selected in the Live Images window m Spectrum Range 400 00 500 00 nm Step Size 10 nm Bandwidth 10 nm Em Fiter o To define a spectral sequence enter the beginning and end in nm of the range of wavelengths to be applied into the Range box Enter the amount in nm that the wavelength is to be shifted after each frame is acquired in the Step Size box Enter the width in nm of the passband to be used in the Bandwidth box Select the emission filter to be used in the Em Filter box ignored if no emission filter is available Recording gt Images Signals gt XY Stage Control When a motorised XY stage is available select Setup gt XY Stage Control to open the XY Stage control panel
49. e displayed within the image panel Images Zoom 50 y 40 Zoom factors of 25 800 are currently supported The magnified region of the image displayed within the image panel can be panned horizontally and vertically within the image Adding markers Marker text can be added to each image by entering text into the marker text box and clicking the Add Marker button The marker text appears at the bottom right of the image and also on the time course plot Up to 20 marker text labels can be added to each file Marker text can also be edited by opening the data file Properties box File Properties and editing the text in the Markers table Markers Add Marker marker text here Viewing Measuring Recorded Images gt Time Course Window The Time Course Plots window displays the time course of the fluorescence intensity at a selected region of interest in the image and or analogue signals 10 x Display Y Fluorescence ROI RoI y T Ratio 488 25 y 550 30 v 060 00658 0005 Excl 1 Threshold Y AD Channels 80000 a Vim B D m B E005 E 88 501 mv 200 a Im 20 D pm a 300 a 15259 p 8000 a Ch 2 B DO my a 10000 o O mv 1 2 3 4 5 Bs El 3 gt FixZeroLeve Secs Mins 4 6235s p Fluorescence Select the Fluorescence option to display the time course of the fluorescence intensity of a selected the region of interes
50. e plotted in the histogram from the Source list which contains a list of currently active image display windows Images Window selects images stored on the currently open data file Record Window selects the live image If the image source is a multi wavelength sequence select the wavelength frame to be displayed from the Frame Type list Select the Whole Image option to display the intensity histogram for all pixels within the the image To restrict the histogram to a region of interest within the image select the ROI option and choose an ROI number from the list Note The ROI option is not available when the image source is live images Click the Full Range button to set the range of intensities plotted in the histogram to full intensity range of the camera Click the Auto Scale button to restrict the intensity range to the min max range of values actually within the image The histogram can also be set to a user defined range by entering values into the Low and High histogram range boxes then pressing the lt Enter gt key Customising graph axes and labels The X and Y axis range and labels of the histogram graph can be customised by clicking the Set Axes button to open the Customise Histogram dialog box Set Axes Range Labels x X Axis Y Axis Automatic Automatic Manual Manual Min 339 Min fo Max 2613 Max 200 Tick 4549 Tick so Scale Linear Scale Linear gt Labels X Axis Pixel Inte
51. e x Frames ROI Wavelength m Format All Frames C All e 480 nm y Axon Range ROI C Text Ratio C EDR 1 1008 R011 y 480 nm y C Igor Wave Subtract ROI L IAA C CFS OK Cancel H 480 nm y C A Excl Threshold To export the fluorescence time courses 1 Export Format Select the format of the export file Axon for the Axon ABF file format readable by PCLAMP EDR for the Strathclyde WinEDR file format or Text to export the data as tables of tab delimited ASCII text Igor Wave to export the data to an Igor binary wave format file or CED CES format 2 Frames Select the All Frames option to export all frames or Range and enter a selected frame range to be exported 3 ROI Select the ROI s to be exported Select All to export all ROI time courses or ROI to export the ROI selected in the adjacent list 4 Wavelength For multi wavelength file select the specific wavelength to be exported or Ratio to export the ratio of two wavelengths Enter the lowest acceptable intensity level for the ratio denominator wavelength in the Excl Threshold box Ratios with denominators below this level are set to zero 5 Click the OK button to export the ROI time courses File Import Export gt Importing Images To import a series of images into a WinFluor data file select File Import Images To open to the dialog box Import File 21x Look in E image
52. egin Amplitude the amplitude of each pulse in the series Duration the duration of each pulse Pulse interval within train determines the time interval between pulses Number of pulses defines the number of pulses in the train This element can be used to produce a series of stimuli to observe the effect of repeated application of a stimulus at a high rate It can also be used to produce a train of pre conditioning stimuli for a subsequent test waveform Voltage ramp This element produces a linear voltage ramp between two voltage levels It is defined by 4 parameters Initial delay the delay period before the series of pulses begin Start at amplitude the voltage level at the start of the ramp End at amplitude the voltage level at the end of the ramp Ramp duration the time taken for the voltage to slew between the start and end amplitudes Voltage ramps provide a means of rapidly generating the steady state current voltage relationship for an ionic conductance Note that the ramp generated by the computer is not truly linear but consists of a staircase of fine steps These steps can be smoothed out by low pass filtering the voltage stimulus signal before it is fed into the patch clamp Digitised analogue waveform Digitised analogue waveforms which have been previously acquired by WinFluor or synthesised by another program can be used as a waveform element To insert a digitised waveform into the protocol
53. el from the Vout 0 list or select None to disable it Note When a patch clamp is in use Vout 0 is typically used to provide the command voltage signal To select an analogue output channel for Voltage stimulus waveforms 1 or 2 select a DAC channel from the Vout 1 or Vout 2 list or select None to disable it Digital Outputs To select a range of digital pulse stimulus channels select the first digital output line from the Digital Outputs Range Start list and the last digital output from the End list or select None to disable digital outputs Photo Stimulus The Photo Stimulus settings configure WinFluor s laser stimulated point photolysis feature currently only available when used with Prairie Technology Ultima microscope Galvos Select the analog outputs used to control laser position from the X and Y Galvo Control list boxes Attenuators Select the analog outputs used to control laser attenuators Pockel Cells from the Attenuator Control Channels 1 3 list boxes Shutter Select the analog outputs used to control laser safety shutter from the Shutter Control Channel 1 Enter the shutter opening time into the Latency field and select whether the shutter is opened by an 5V signal level Active High or OV Active Low Meter Select the signal level meter from Meter Input list box Getting Started gt XY Z Stages gt Z Stage Controller Setup When a voltage controlled piezo stage or lens focus controller is availabl
54. electing the required equation from the Equation list and if necessary modifying any of the parameters A selected equation can be deleted by clicking the Delete Equation button Analysis gt Time Course Analysis gt Time Course Analysis The time course analysis window plots graphs of the time course of the mean image intensity within the regions of interest defined in the Images window Relative changes in intensity F FO ratios can be computed and also intensity ratios and ionic concentration plots for multi wavelength recordings Select Analysis Time Course Analysis to open the time course analysis window Frame No Pasar 11008 oS CED PEER Time 12058 Display 480 nm Zoom 100 Display Contrast stiders Full Range Best 114 870 0 0600 Auto adjust JV All image panels TF 6xs d only Shading Correction I Correction Enabled Regions of Interest _Delete_ airois y Save ROIs to File Load ROIs from File _ Edit ROI Table Add to New Plot i 4 Add to Plot fi y 3 0103 1 4641 Add all ROIs 2 5103 g 2 0x103 6 o 1 5x103 2 103 cos 500 All Points 560 t 28 789 E i 101 201 Time Units s Secs Mins o Q9 O ho Le 0 Analysis gt Time Course Analysis gt Plotting Time Courses To plot the time course of changes in image intensity 1 Define one or more regions of interest within the image displayed in the Images window 2 Select a re
55. es X Y graphs can be plotted as a line unconnected markers or both by ticking the Lines and or Markers tick boxes Ticking the Show Line Labels check box displays a label at the beginning of each line displaying the ROI or channel number associated with the line Click the Update button to update the graph after changes have been made or Update All to update all graphs when more than one exists within the plot Analysis gt Event Detection amp Analysis gt Plotting Waveform Measurements A series of waveform measurements can be computed from the detect event records and plotted on the Plot Graphs page 1 Before plotting a graph switch to the View Events page and use the waveform measurement cursors to define the region of each event record to be analysed Event Detection amp Analysis JE Detect events View Events average Events Plot Graphs Event No as MB Rejected 7 r Region of Interest ROI fRo1 1 gt AO 1397 _ 3s 5 iuil E E SS 05 1 Fluorescence Display Fluorescence v Excit ch 480 nm 39 776 mv 1 Superimpose Events Enable 7 al 258 Dl 2 If you want to measure a specific point on each event waveform Display Cursor measurement drag the green measurement cursor to the point to be measured 3 The pair of analysis region cursors grey a cursors linked by a horizontal bar define the region o
56. f the displayed event record to be measured Points outside this region are ignored for peak and time course measurements They are initially set to include the whole record Move these cursors if you want to limit analysis to a specific region of the record 4 If you want to exclude an event from a measurement plot tick the event s Rejected box 5 Change to the Plot Graphs page lx Detect events View Events Average Events Plot Graphs 3 0x108 2 5103 2 0103 X Axis Variable E E Event No y o F 15x103 From 480 nm y ye D o 103 Y Axis Variable 500 Peak y From 480 nm y Polarity 03 Positive 0 2 4 6 8 C Negative Absolute Event No Select the range of events to be plotted either by selecting All Events or by selecting Range and entering the Select the type of measurement see Table 1 below and the signal to be measured and plotted on the X axis of 1 below and the signal to be measured and plotted on the Y axis of 6 range of events Note Events marked as Rejected are not included in the plot 7 the graph from the X Axis variable lists 8 Select the type of measurement see Table the graph from the Y Axis variable lists 9 Click the Plot Graph button to plot the selected graph 10 To modify the graph axes and labels click the Set Axes button The displayed graph can be printed File Print Graph or copied to the Windows clipboard as data Edit Copy
57. factors for the current and voltage analogue channels must be entered manually To do this select Manual Gain Entry in the Amplifier list and enter the appropriate calibration information into the channel calibration table The Channel calibration table contains the name units and calibration factor for each analogue input channel used by WinFluor There are 3 entries in the table for each analogue channel Channel calibration table Ch Name YViUnits Units o Mi o 0001 pa 1 Ym 0 01 my Names contains a 1 4 letter name used to identify the source of the channel e g Vm Im Units defines the measurement units of the signal e g mV pA etc V Units defines the scaling factors relating the voltage level at the inputs of the A D converter in V to the actual signal levels in each channel in the channel units For instance if the membrane voltage output of your patch clamp supplies a signal which is 10X the measured membrane potential of the cell and the units have been defined as mV then the appropriate V Units setting is 0 01 since the patch clamp voltage output is 0 01 Volts per mV In the case of patch clamp current channels the V Units value is determined by the current gain setting which is usually a switchable value e g if the current output was set at 0 5 mV pA and the channel units were pA the V Units settings would be 0 0005 The patch clamp command voltage divide factor the scaling factor between the applied c
58. ge Capture Exposure 100 ms o Min 33 ms CCD Area 512 x 512 Full CCD area Pixel Use rectangle Binning Set CCD area 1 Gain fi w Contrast Grey scale Display Contrast Sliders Full Range 1128 5340 7 Auto adjust M 6xs d only Shading Correction Correction Enabled Acquire Backg Image r Excitation Light Con amp off 340 10 I Wavelength JJ fo 340 10 X 3312 um 207 Y 3120 um 195 1 2213 Set Laser Intensity Display calibration bar Y Z Axis Position 48 5 um Ez a Br Recording gt Image gt Image Capture The Image Capture group of settings define the size and location of the image to be captured and the time interval between frames M Image Capture Exposure 40 ms kieren Min 33 ms CCD Area 512 x 512 Full CCD area piye Use rectangle Binning Set CCD area 1 Gain E Exposure Interval Enter the required time interval in ms between successive image frames into the Exposure Interval box The shortest valid inter frame interval is indicated below the entry box This will vary depending upon capture region size and CCD binning factor CCD Area If the camera supports CCD sub region readout the CCD area controls allow you to acquire the image from a defined sub region within the CCD To use the full imagi
59. gion of interest or analogue signal to be plotted from the Source ROI Channel list Optional To subtract the intensity of a background region of interest select an ROI from the Background ROI list otherwise leave it blank ROI Channel Source fon y Background y Fluorescence Display Fluorescence v Excit ch 480 nm y 3 Select the range of frames to be plotted either by selecting All Frames or selecting Range and specifying a range of frames Plot Range All Points Range 71008 4 Select the time units for the plot Secs or Mins Time Units Secs Mins 5 The Fluorescence Display option determines how the fluorescence signal from the ROI selected in Source is displayed in terms of either the raw fluorescence intensity in pixel grey scale units the fractional change relative to a standard level the ratio at two different wavelengths or the computed ion concentration Select the Fluorescence option to display the raw fluorescence signal If the file contains multi wavelength images select the required wavelength from the list Fluorescence Display Fluorescence i Excit ch 480 nm y or Select the Fluorescence dE F0 option to display the fluorescence signal as a fraction of a predefined baseline fluorescence level FO Fluorescence Display Fluorescence dF FO Excit ch 480 nm y FO frame 4 1 C FO 1 0 F FO FO C FFO
60. he A D converter s input voltage range By changing the voltage range you can adapt the sensitivity of the A D converter to best match the amplitude of the signals from your experiment Amplifier WinFluor supports up to two patch clamp amplifiers Select the type of the first attached patch clamp amplifier from the Amplifier 1 list If the amplifier provides gain and or mode telegraph signals check that the gain and mode telegraph outputs are connected to the analogue input channels specified in the Gain Tel and Mode Tel boxes Typically the gain telegraph output is connected to analogue input 7 and the mode telegraph to input 6 Note Not all amplifiers require telegraph channels either because they do not support telegraphs or transfer the telegraph data via USB or serial connections The command voltage signals for Amplifiers 1 is provided by analogue stimulus channel Vout 0 Check that the analogue output channel associated with Vout 0 is connected to the command voltage input of the patch clamp The Vout 0 Divide Factor the scaling factor between the applied command voltage and resulting cell membrane potential set by the patch clamp is set automatically when the amplifier type is selected If a second patch clamp is available enter its settings in the Amplifier 2 group Manual patch clamp settings If the patch clamp amplifier is not one supported by WinFluor or if it does not have a gain telegraph facility then the calibration
61. he Light Source Setup dialog box Select Set Laser Intensity to set the intensity of a laser source light source this option currently only applies to the Optoscan Lasers TIRF light source option Recording gt Images Signals gt Excitation Wavelength Sequences The excitation emission light editor allows up to 20 fluorescence excitation emission wavelength combinations to be defined for selection by the Excitation Light control in the Record Image and Record Images amp Signals windows Excitation emission wavelength sequences of up to 9 of these wavelengths can be defined and applied sequentially A multi wavelength fluorescent light source is required monochromator LED filter wheel for excitation wavelength control and and filter wheel in the emission light path for emission light bandwidth control To configure the light wavelengths and sequences select Setup Excitation Light Wavelengths to display the Excitation Light Setup dialog box Excitation Light Setup loj x m Multiwavelength sequences Excitation emission wavelength table Sequence 1 i R Sequence 0 480 nm 10 nm 50 0 500 520 1 550 nm 5 nm 100 1 600 650 0 480 10 nm 1 2 Onm 0 nm 100 0 1 550 5 nm 1 3 Onm 100 0 4 Onm Onm 100 0 5 0nm 0 nm 100 0 6 Onm 0 nm 100 0 7 Onm 0 nm 100 0 8 0 nm 0 nm 100 0 9 Onm 0 nm 100 0 se a fi p0 00m O nm 100 0 Divide Mo 11 Onm 0 nm 100 0 12 Onm 0 nm 100 0 Clear
62. he Time Lapse option to record images intermittently at intervals defined by the Time Lapse Interval box Recording Record Stop Mode Recording Period 603 Time lapse interval 30s Select the T Lapse Burst option to both record images intermittently at intervals defined by the Time Lapse Interval box and to acquire high speed bursts of images at the rate defined by the camera Exposure Interval Enter the duration of the high speed burst into the Burst Duration box and the interval between bursts into Burst Interval Recording Record Stop Mode T Lapse Burst y Recording Period 5508s Time lapse interval 204s Burst Duration hooo Burst Interval ro 6 Enter any experiment identification information into the Expt box 7 Click the Record button to begin recording During recording the number of frames acquired is indicated in the status line at the bottom of the program window Recording is terminated when the required number of frames have been acquired or when the Stop button is clicked Markers To add marker information to the file during the recording enter the text into the Markers text box and click the Add Marker button HS Recording gt Image gt Record Image To open a window displaying live camera images Select Record Image to display the window Record Image Images Zoom Save Image xe Ima
63. hor Except where otherwise specified no warranty is implied by either the author or the University of Strathclyde concerning the fitness of the software for any purpose The software is supplied as found and the user is advised to verify that the software functions appropriately for the purposes that they choose to use it An acknowledgement of the use of the software in publications to which it has contributed would be gratefully appreciated by the author John Dempster Strathclyde Institute for Pharmacy amp Biomedical Sciences University of Strathclyde 161 Cathedral St Glasgow G4 ONR Scotland Tel 0 141 548 2320 Fax 0 141 552 2562 E mail j dempster strath ac uk Getting Started gt Installing WinFluor To install WinFluor 1 2 Install the National Instruments NI DAQ software supplied with your NI interface card s Install PCI or attach USB the NI interface card s to your computer If you have more than one PCI card installed connect the cards together using an RTSI bus cable Check that the card s are detected when the computer is re started Install the driver software and support library supplied with your camera Note For Andor cameras the Andor Software Development Kit library must be installed If the camera uses a specialised interface board install it in the computer Check that the camera is configured and functioning correctly using the test software supplied with the camera Install
64. ile Images F My Data File_D18 I1DR Frame No 1 198 KE gt Time 30s Display 340 10 Green scale ba Display Contrast stiders Full Range _ Best 122 4522 M Auto adjust fv Allimage panels TF 6xs d only Shading Correction I Correction Enabled Regions of Interest _Delete_ fairois gt Save ROIs to File Load ROIs from File _ Edit ROI Table Markers Add Marker ROIs N 0 ee E oragontoimase FM Display calibration bar A graph of the time course of pixel intensity from a selected region of interest ROI within the displayed image one for each image pane in multiwavelength sequence is displayed in the time course window Analogue signals if they have been acquired are also displayed in this area Display Calibration Bar Checking this option displays a calibration bar at the bottom left of each display pane The size of the calibration bar and lens magnification from which it is derived is defined in the Camera Settings dialog box Selecting dis playing frames Individual image frames can be selected for display using the frame selection slider bar or by directly entering the required frame number into the Frame No display box Frames can be played forwards and backward in sequence using the playback control buttons Frame No i8763 m ml gt m ajm gt Time 2s The Zoom option can be used to magnify or reduce the region of the imag
65. ion maps image intensity as shades of grey Green Blue and Red Scale maps intensities into shades of these colours False Colour maps intensities in a rainbow of colours ranging from minimum to maximum in the sequence black blue green yellow red white The Zoom option can be used to magnify or reduce the region of the image displayed within the image panel Zoom factors of 25 800 are currently supported The magnified region of the image displayed within the image panel can be panned horizontally and vertically within the image The range of pixel intensities mapped into the display colour range is displayed in the Display Contrast tab To achieve optimal image contrast click the Best button to adjust the intensity range to the range of pixel intensities within the image Click the Full Range button to set the display to the full range of intensities supported by the camera The display can be set to a specific intensity range by entering the required range into the intensity range display box and pressing the lt Enter gt key When the Auto adjust option is ticked the display contrast is automatically adjusted to the optimal setting as frames as acquired The 6 x s d only option is ticked the optimal contrast range is set to 3 standard deviations on either side of the mean image intensity and to the minimum and maximum intensities within the image when unticked When the All image panels option is ticked the contrast is set for all im
66. ition and boundaries Existing ROIs can be moved resized by clicking on them Removing Regions of Interest To remove an ROI from the image select the ROI number from the deletion list and click the Delete button To remove all ROIs click Delete All Regions of Interest Delete 201 Cam Dido ta Cila Saving Loading Regions of Interest Save ROIs to File _ Load ROIs from File ROls from File ___EditROITable ROI Table The current set of ROIs can be saved to a ROI settings file by clicking the Save ROIs to File button and entering a file name Settings can be reloaded from an ROI settings file by clicking the Load ROIs from File button Click Edit ROI Table to display the Edit Regions of Interest table where centre width and height of the ROI can be edited Note ROI settings are stored in tab text format files with ROI extensions One ROI is stored per line with the format Shape lt tab gt Centre X position lt tab gt Centre Y position lt tab gt Width lt tab gt Height lt tab gt Polyline XO lt tab gt Polyline YO lt tab gt Polyline Xn lt tab gt Polyline Yn where Shape indicates the type of ROI 0 Point 1 Rectangle 2 Line 3 Polyline 4 Polygon Viewing Measuring Recorded Images gt Viewing Image Recordings To open and view a WinFluor data file containing a series of stored images amp signals select File Open Data File to open the Open Data File dialog box then select the required IDR f
67. meters correspond with the known values of the model Recording gt Signals Monitor Seal Test gt Holding Voltage and Test Pulses You can control the holding voltage applied to the cell and the amplitude and duration of a test voltage pulse by selecting one of three available test pulses Pulse 1 2 3 m Test Pulse Pulse 1 F3 l Y Hold 100 mY Amplitude 10 mw C Pulse 2 F4 Hold 0 my Amplitude 10 mw Pulse 3 F5 Y Hold 0 my Amplitude DO mY Pulse width 20 ms O P Chan vout 0 y The size of each pulse is set by entering an appropriate value for holding voltage and pulse amplitude into the V Hold or Amplitude box for each pulse The width of all pulses is defined by the pulse width box Y ou can switch between pulses by pressing the function key associated with each pulse Pulse 1 F3 Pulse 1 F4 Pulse 1 F5 The voltage stimulus output to which the pulse is is indicated in the O P Chan list This is normally set automatically to output channel connected to the patch clamp command voltage input Recording gt Signals Monitor Seal Test gt Patch Clamp Amplifier Settings The patch clamp current gain in voltage clamp mode and voltage gain current clamp mode is indicated in the Amplifier group box Amplifier _ Amp 1 Gain Ch D 0 0005 VIpA Divide Factors Vout0 10X Vout 1 10X When using a patch clamp with gain telegraph s
68. ng area of the CCD click the Full CCD Area button To acquire the image from a CCD sub area defined on screen use the mouse to drag in the edges of the capture rectangle superimposed on the live displayed image until it encompasses the region to be acquired then click the Use rectangle button To define the CCD sub area in terms of its X and Y pixel coordinates within the CCD click the Set CCD Area button and enter the required X and Y pixel range into the Set CCD Area dialog box which pops up loxi CCD readout area x pixel range Y pixel range 0 519 OK Cancel Binning If the camera supports CCD pixel binning to bin together blocks of pixels to increase image intensity and or allow faster frame capture rates enter a value greater than one into the Pixel Binning box then press the lt Enter gt key A value of 2 bins together 2x2 blocks of pixels increasing image intensity by 4 Gain Set the camera CCD readout amplifier gain by selecting a gain factor from the Gain list With EMCCD cameras the gain is expressed as a percentage of the maximum electron multiplying gain Recording gt Image gt Display Contrast Image The display colour mapping options determines how pixel intensities within images are displayed on screen Contrast Green scale Display Contrast Sliders Full Range Best 145 5930 J Auto adjust 6x s d only The Grey Scale opt
69. nsity Y Axis No Pixels OOO Trace OK Jv Lines Cancel V Markers Axis limits and tick spacing are initially set to default values based upon the range of the data The axis limits can be changed by entering new values for into Min Max and Tick spacing boxes for the X and Y axes An axis can be made Linear or Logarithmic by selecting the option from its Scale list The X and Y axis labels can be changed by editing the entries in the X Axis and Y Axis labels boxes The histogram Bin Style can be set to empty solid or hatched boxes in a variety of colours When Full Borders option is ticked a solid rectangular border is drawn round each bin Click the Update button to update the graph after changes have been made or Update All to update all graphs when more than one exists within the plot Analysis gt Line Profile gt Displaying a Line Profile The line profile module plots image intensity along the length of a linear ROI defined within an image stored on file It can oper Select Analysis Line Profile to open the Line Profile display window Frame No 871451 i 2 es QDD E Line Profile Edge Track Time 87s pr Frame Type 200 Display Fr Fr O y Grey scale ROI ROM Zoom TRIS Axes Range 150 Display Contrast Sliders Auto Scale C Manual R thresh Full Range Best Set Axes E 100 22 255 Y Auto adjust Line width TE JV All image panels E pixels TF
70. of the cell membrane holding current and voltage and test pulse amplitude appears at the bottom of the monitor window Voltage Current Holding Holding Pipette Cen 99 7 mi 1219 pA Pulse Pulse Resistance 9 9 mv 21 2 pA 469 0 MOhm Ga estimate frorm C Peak Exp Amp During initial formation of a giga seal the Pipette option displays pipette resistance computed from V _ pulse pipette pulse where Vpuise and Ipuise are the steady state voltage and current pulse amplitudes The Cell option displays the cell membrane conductance Gm capacity Cm and access conductance Ga Pipette Cell Ga 6 723 nS Gm 3 31 nS Cm 15 28 pF Ga estimate frorm C Peak Exp Amp computed from eat pulse G pulse K I ulse Yue G Cn t G G where lo is the initial current at the peak of the capacity transient and is the exponential time constant of decay of the capacitance current See Gillis 1995 for details Note If Ga Gn and Cm are to be estimated correctly the patch clamp s pipette series resistance compensation and capacity current cancellation features must be turned off A good test to check if WinFluor is set up with the correct input output connections and channel scaling factors is to attach the model cell supplied with most voltage patch clamps and observe the holding potential and current test pulse amplitude and cell para
71. ommand voltage and resulting cell membrane potential Enter the division factor in the Vout 0 Divide Factor box Typical divide factors are 50 Axon Instruments amplifiers or 10 Heka EPC 7 8 Master Clock Timing To set the timing resolution of the master clock used for cameras light source and analogue input and output synchronisation enter a time interval into the Timing resolution box The default value is 0 1 ms If two or more interface cards are installed in the system and connected together internally using National Instruments RTSI bus cables select RSTI 0 as the Clock synch link option If cards are linked externally using the PFIS line select the PFI 5 option Note The PFI 5 option currently only works NIDAQ Traditional library option Synchronisation using the RTSI bus is to be preferred Getting Started gt Patch Clamp Signal Connections To configure WinFluor to acquire cell current and voltage signals from a patch clamp signal connections must be made between the patch clamp amplifier outputs current voltage gain telegraph and mode telegraph and inputs command voltage and appropriate analogue inputs and outputs on the PCI 6229 analogue interface board The following signals connections are required i Patch Clamp Amplifier h Analogue Interface Inputs Outputs ee Te Current and voltage outputs 1 2 must be connected to interface inputs AIO and All respectively Telegraph connections 3 4 are not be requi
72. on which creates a new ROI at the current ROI 1 position All ROIs except ROI 1 can be deleted by clicking the Delete ROT button Once defined the fluorescence intensity within the ROI can be selected for display in the time course plot by selecting it in Fluorescence ROI list in the time course window The difference between 2 ROIs can be plotted by selecting an ROI in the ROT list Ratio Select the Ratio option to display the time course of the ratio of intensities from a selected pair of wavelength image at the selected ROIs The Display Max box sets the upper limit of the ratio display range The Excl Threshold box defines the minimum fluorescence signal level required for computation of a ratio If either of the two fluorescence signals fall below this value the ratio is set to 0 A D Channels Select the A D Channel option to display analogue signal channels if they are being acquired Dis play The duration of the time course plot can be set by entering a value in seconds into the duration box at the bottom right of the graph Clicking the left and right arrow buttons halves and doubles the display duration The vertical magnification of each time course plot can be expanded to a selected region by moving the mouse to the upper limit of the region pressing the left mouse button drawing a rectangle to indicate the region and releasing the mouse button The vertical magnification can also be adjusted using the buttons at
73. ontroller Setup When a motorised XY stage is available the stage position can be controlled from the XY Stage control panel Setup gt XY Stage and a list of stage positions stepped through when recording in time lapse mode in the Image and Image amp Signals recording window Support for an XY stage can be configured using the XY Stage settings in the XY Z Stage page lx Camera Light Source Stimulus Outputs Analog Inputs amp Timing XY Z Stage Z Stage Enabled Z Stage Control Voltage Output None Position Voltage Position 10 um fov Minimum 40 y Position 2 40 um 10 v Maximum 40 y Calibration Voltage Range Limits Excitation off during step End exposure at step Min Step Size 0 05 um Max Step Time 0 ms m XY Stage lose XY Stage Type Thoriabs MLS203 y x min 0 mm Motors X max 440 mm X Motor Serial Number 94828024 Y m n 0 mm Y Motor Serial Number 94825022 Y max 75 mm XY Stage Type Selects the type motorised stage in use currently only the Thorlabs MLS203 stage is supported c Feb 2015 X Motor Serial Number The serial ID number of the motor controlling the stage X position Y Motor Serial Number The serial ID number of the motor controlling the stage Y position Limits X min Stage X position lower limit mm X max Stage X position upper limit mm Y min Stage Y position lower limit mm Y max Stage
74. or Wave OK Cancel C CFS Select the range of events to be exported either by select All Events or by selecting Range and entering the range of events Events marked as Rejected are not exported Optional Click the Change Output File button to change the name or destination folder of the export file Optional If more one signal channel is available select the channels to be exported Click the OK button to export Analysis gt Event Detection amp Analysis gt Fluorescence Display The Fluorescence Display options determines how the fluorescence signal is displayed in terms of either the raw fluorescence intensity in pixel grey scale units the fractional change relative to a standard level the ratio at two different wavelengths or the computed ion concentration Region of Interest The region of interest within the image from which the fluorescence signal is derived can be selected from the ROI list A background fluorescence signal can be subtracted from the displayed signal by selected a background ROI from the list not applicable to line scan files Region of Interest ROI Roa y Jro1 y 1 Fluorescence Select the Fluorescence option to display the average fluorescence intensity signal computed over the selected ROI If the file contains multiple excitation wavelength channels select the required wavelength from Excit Ch list m Fluorescence Display Fluorescence Y Excit ch
75. osition Voltage fields define the relationship between lens positioner control voltage V and position microns The lens positioner can be calibrated by entering the control voltage and position for a pair of positions near the upper and lower limits of the positioner range Min Step Size The Min Step Size field defines the smallest change in microns in Z position supported by the lens controller Max Step Time the Max Step Time field defines the maximum time taken to move the Z stage between positions Note In time lapse recording mode changes in Z stage position are initiated after the end of each exposure or sequence of exposures when multi wavelength imaging In continuous recording mode the change in Z stage position is initiated Max Step time ms before the end of each exposure or sequence of exposures Excitation off during step When this option is ticked the excitation ligbt source is turned off when the command voltage to the Z positioner is changed when recording a Z stack In continuous recording mode this occurs Max Step Time ms before the end of the camera frame It has no effect in time lapse mode End exposure at step When this option is ticked Z stack recording is enabled and continuous mode recording is selected the camera exposure is shortened by Max Step Time ms so that it terminates before the Z position changes It has no effect in time lapse mode Getting Started gt XY Z Stages gt XY Stage C
76. pboard as data Edit Copy Graph Data or as an image Edit Copy Graph as Image A plot is selected for printing copying by clicking on it Selected plot is outlined in red Viewing Measuring Recorded Images gt Viewing Line Scan Recordings To open and view a WinFluor data file containing a series of stored line scan images amp signals select File Open Data File to open the Open Data File dialog box then select the required IDR file The line scans time series is displayed in the top panel of the View Line Scans windows with the line oriented vertically and time horizontally For multi wavelength files containing more than one line scan image acquired at different emission wavelengths the displayed line scan image can be selected from the Line Scan list Line Scan Frame 0 y A graph of the time course of pixel intensity from a selected point TC on the line is displayed in a time course plot below the image Analogue signals if they are being acquired are also displayed in the analogue signal plot Line Scan Frame 0 Start Line 3573 Line Scan Interval 5 44 ms Initial delay O ms L Display False colour Zoom 50 y Intensity Range Full Range Best Contrast 0 2994 Ua Time Course Pixel gt Pixel 117 No Avgd 5 IV Subtr Backg Background 49 m Plot IV Fluorescence M AD Channels I Ratio J Y Display calibr
77. posure is shortened by lt change time gt to avoid smearing of image Getting Started gt Light Sources gt Cairn Optoscan Monochromator The connections and settings for Cairn Optoscan monochromator attached to a PCI 6229 interface board Note Cairn Reseach can supply a custom input output panel configured to connect the Optoscan to a National Instruments PCI 6229 card Signal connections PCI 6229 A PCI 6229 B PCI 6229 Name 68p 68pin connector connector AOO Camera Ext Trigger sig Camera Ext Trigger gnd Device 1 DIGO Device 1 DAC1 Device 1 DAC3 Device 1 DACO Device 1 DIGI Device 1 DIG7 Device 1 ADCO 7 Getting Started gt Light Sources gt Sutter DG 4 Filter Changer The connections and settings for Sutter DG 4 filter changer attached to a PCI 6229 interface board Camera System Setup Camera exposure trigger EA Device 1 DIGO Wavelength Control Device 1 DIGI CPC peoa CCT Dee Hach e peer Signal Connections PCI 6229 68 way connector Camera Ext Trigger a pe CONAN oma 5 com CONAN ECETIA pp a EEC p PES EA CCE paw fe COM CTC paw ja Co CECI paw e p e sem paw e Cc Getting Started gt Light Sources gt Till or PTI Monochromator Connections and settings for WinFluor system with a PCI 6229 interface board camera and Till monochromator Signal connections PCI 6229 A PCI 6229 68p B PCI 6229 Name connector left 68pin connector right Camera System Setup
78. r copied to the Windows clipboard as data Edit Copy Graph Data or as an image Edit Copy Graph as Image A plot is selected for printing copying by clicking on it Selected plot is outlined in red Analysis gt Event Detection amp Analysis gt Detecting Events To detect events 1 2 Select the Detect Events page in the Event Detection amp Analysis window Select the signal channel to be scanned for events from the Detection Channel list which displays the available analogue and fluorescence channels Analogue channels Select the channel to be scanned from the list Detection Channel i vm v Fluorescence channels Select the region of interest to be used from the ROI list and optionally a background subtraction ROI from the list If more than one excitation wavelength is available select the excitation wavelength channel from the Excit Ch list Detection Channel Fluorescence Region of Interest ROI Ron y Excit Ch 480 nm Fluorescence ratios Select the region of interest to be used from the ROI list and optionally a background subtraction ROI from the list Select the pair of excitation wavelength channels to be ratioed from the Exc Ch Ratio lists Detection Channel Fluorescence ratio Region of Interest ROI Ror Exc 480mm Sh Ratio 480 nm v AN Min Signal 30 Level Display Max 10 1
79. red with patch clamps which do not provide gain and or mode telegraphs or communicate gain information via USB or other communications lines The command voltage input is connected to voltage stimulus channel Vout O which is usually configured as interface digitallanalogue output AOO Table 1 lists the required connections and signal output names for the patch and voltage clamps currently supported by WinFluor Table 1 Patch clamp signal connections Signals Telegraphs Command Voltage O P Axopatch Scaled Output 10 Vm Gain Ext 1D Telegraph Command Axopatch Scaled Output 10 Vm VC mode Mode Gain Ext 200 Im CC mode Telegraph Telegraph Command See Note 1 Multiclamp Scaled Raw See Note 2 Ext 700A Output Output Command Multiclamp Primary Secondary See Note 2 Fxt 700B Output Output Command RK400 Iout Voltage Command Cairn Gain Out Command Pin 11 37 way Gain Command 10 Optopatch X10 Out D socket to Telegraph Out In Dig In 0 Im Vmx10 Gain Telegraph Warner Im Vm x10 Gain PC505B Telegraph Warner I Monitor Vmx10 Gain OC725 Telegraph Curr Warner PCSO1A NPI SECOSLX Current Potential VC Command Output Output Sensitivity Input 10 Monitor AM Systems Output X10 Vm Gain 2400 Telegraph F Note 1 When the Axopatch 200 is switched from voltage to current clamp mode the Scaled Output signal to ADC1 is changed automatically from membrane current to voltage To retain a current signal ADC1 must be
80. s Shading correction using the background image can be toggled on and off as required Recording gt Images Signals gt XY Stage Control When a motorised XY stage is available select Setup gt XY Stage Control to open the XY Stage control panel Stage conte 101 x Stage Position List Stage Position 1 X mm 1 10 000 20 000 2 90 000 60 000 Y mm X E al a E Delete Position Al y Move To h y Coarse Fine Time Lapse Action Ir Incr Stage Position Stage movement The XY stage position can be moved by using the left right and up down arrows or centred by clicking the H button The Coarse and Fine options select the size of the movement step Stage Position List The current XY stage positions can be recorded in the stage position list by clicking the Add Position button Clicking the Move To button moves the stage to the selected position in the list The complete list or individual positions selected in the drop down menu can be deleted from the list by clicking the Delete Position button Time Lapse Action The Incr Stage Position option allows the XY stage position to be controlled during during a time lapse recording sequence in the Record Images amp Signals window When the Incr Stage Position option is selected the XY stage position is incremented to the next position defined in the stage position list between time lapse exposure
81. s cycling through the list of positions for as long as recording continues The image data at each stage position is extracted and stored in a series of Winfluor IDR data files one for each position denoted by the suffixes XY 1 idr X Y 2 idr etc Recording gt Image gt Z Stage Focus Control When a lens stage positioner is available the Z Axis Position group of settings defines the lens focal plane Z position 48 5 um sn I Axis Position The plane of focus can be shifted up our down using the Z Axis Position slider or moved to a specific position by entering the position in microns in the Z Axis Position box and pressing the return key Recording gt Signals Monitor Seal Test gt Signals Monitor Seal Test The Signals Monitor Seal Test window provides a monitor of analogue signals allows the application of seal test pulses to assist the formation of a giga seal and computes pipette resistance cell conductance and capacity Select Record Signals Monitor Seal Test to open the Signals Monitor Seal Test window An oscilloscope monitor showing the current signal on each input channel is displayed Signals Monitor Seal Test a I oj x Channels Voltage cna vm Amplifier 1 2 C Test Pulse gt O Pulse 1 F3 Y Hold 100 mv Amplitude 10 mw Pulse 2 F4 Y Hold 0 my Amplitude 10 mv 70 C Pulse 3 F5 Y Hold omy Vi Amplitude O mv P Me Pulse wi
82. s cameras Light Sources e Cairn Optoscan monochromator e Till amp PTI monochromators e Sutter LS 10 filter wheel e Sutter DG 4 filter changer e Cairn OptoLED and other LED light sources Analog Digital Interface Cards e National Instruments E or M Series interface card can be used for analogue input output and timing The PCI 6229 card or USB 6229 BNC 32 analog inputs and 4 analog outputs is recommended Patch Clamps e Axon Axopatch 1D and 200 and Multiclamp 700A and 700B e Biologic VP500 and RK400 e Cairn Optopatch e WPC 100 e Heka EPC 8 e NPI Turbo Tec 03 Turbo Tec10 SECOSLX e Dagan PCOne 3900A e Warner PC501A B OC725C Introduction gt License Conditions The Strathclyde Electrophysiology Software package is a suite of programs for the acquisition and analysis of electrophysiological signals developed by the author at the Strathclyde Institute for Pharmacy amp Biomedical Sciences University of Strathclyde At the discretion of the author the software is supplied free of charge to academic users and others working for non commercial non profit making organisations Commercial organisations may purchase a license to use the software from the University of Strathclyde contact the author for details The author retains copyright and all rights are reserved The user may use the software freely for their own research but should not sell or pass the software on to others without the permission of the aut
83. s for john dempster E EJ My Recent Documents E Desktop My Documents e SE My Computer mm File name naive x 40 section 1tif tif Open e My Network Files of type TIFF Files TIF y Cancel Places J Open as read only Tnaive x 40 section 1tif tiF i naive x 40 section 2 btif tif le naive x 40 section 4 tif i naive x 40 section 12 tif To import an image series 1 Select the type of file to be imported from Files of Type list 2 Find and select the file to be imported 3 Click the Open button to import the file 4 Data file format which can currently be imported are PIC files as produced by BioRad confocal and 2P microscopes STK MetaMorph STK format files Image Cytology Standard format including the version produced by the Nikon C2 confocal microscope TIF Multi page Tagged Image File Format TIFF files Printing 8 Copying Graphs gt Printing Graphs Hard copies of graphs displayed in a plotting window can be produced by clicking on the graph plot to ensure that it the active window and selecting File Print To open the dialog box Print eee Output to shame Printer Setup Calibration Bars r Typeface Time Times New Roman 340 nm 105 7 Size 10 pts 380 nm 712 3 Line Width 2 pts Show zero levels Jv Use colour Iv Show labels Iv Page Margins Set to 10 range Left 5 0 cem OK Cancel
84. single ended mode the signals are derived from AIO AI7 alone measured relative the AISENSE input The A D Input mode setting must match the type of National Instruments input output box used to connect signals to the interface board The default setting is Differential and this is the only setting that can be used with the BNC 2110 input output panel or USB 6229 BNC device Note If a BNC 2090 19 rack mountable I O panel is in use and Differential mode is selected ensure that all SE DI switches are set to DI If Single Ended mode is selected with a BNC 2090 panel ensure that the SE DI switches are set to DI and the NRSE RSE switch to RSE No channels Set the number of analogue input channels to be acquired in the No channels In Use box typically 2 channels cell membrane current and voltage The number of channels can be increased max 8 channels if additional analogue signals are connected Sampling Interval Set the analogue sampling interval in the Sampling interval box default 0 1 ms Note that setting the sampling interval here changes the master clock timing resolution which defines the precision of image capture timing and waveform generation Voltage Range Select the input voltage range of the analogue digital converter from the Voltage Range list default 10V In order to get an accurate measure of the amplitude of an analogue signal it is important to ensure that it spans a significant proportion 30 50 of t
85. sult Appendix A of your camera s user manual to determine which type of trigger input your camera uses Cameras with optocoupled trigger inputs will have a connection 5VDC power for optocoupler specified for pin 1 in Table 2 Cameras with non optocoupled triggers do not make use of pin 1 and a connection will not be listed in Table 2 A suitable plug Singatron Enterprises part no 62000 EP for connecting to the camera trigger input socket can be obtained from Digikey www digikey com The pin out of the 6 pin mini DIN plug is shown below viewed from rear of plug Getting Started gt Analogue Digital Interface Unit A National Instruments E or M Series multifunction interface unit acts as master clock providing timing and control pulses to coordinate image capture light source wavelength changes analogue signal capture and waveform generation Multiple interface boards synchronised using the National Instruments RTSI bus can be used to increase the number of timing and stimulus waveform generation channels Select the National Instruments software library to be used to communicate with the interface board If an M or X Series interface card is being used e g PCI 6221 PCI 6229 USB 6229 BNC or a combination of M and E Series boards select the NIDAQ MX option A list of installed interface cards available to WinFluor with number of available analogue inputs and outputs channels is displayed Select the NIDAQ Traditional option
86. switched manually from the 10 Vm output of the Axopatch 200 to the Im Output Note 2 WinFluor obtains channel gain information from the Axon Multiclamp commander software Multiclamp Commander must be started up and running before WinFluor is started Getting Started gt Light Sources gt Light Sources The type of light source in use and its settings are configured on the Light Source page ON Camera System Setup 10 x Camera Light Source Stimulus Outputs Analog Inputs amp Timing Z Stage Light Source Shutter Control Shutter closed 340 nm Cairn Optoscan 1200 w wavelength Wavelength Control Outputs Range Opening time 0 ms Start End Device 4 DACO y Device 4 DAC1 y 0 ms period Shutter Control Output None gt Active High Active Low m Emission Filter Filter Control Outputs Start None Y End None v Change Time 100ms Light Source Light Source Specifies the type of computer controllable light source attached to the system Wavelength Control Outputs Range Selects the group of analog or digital output lines used to control light source wavelength Select the first output line in the group from the Start list and the last output line from the End list Shutter Control Shutter closed wavelength Sets the monochromator wavelength used when the excitation Off setting is selected in the
87. t stigers Full Range Best 36 8600 I Auto adjust IV All image panels T 6x s d only Shading Correction I Correction Enabled Regions of Interest _Delete_ au ROls Save ROIs to File Load ROIs from File Edit ROI Table Detection Criteria Postive going events Toe 5 9785 my ev Exceed for 0 01 s Fixed Baseline at 39 531 mv Rolling Average y z ST Period f4 s Mi aox Dead Time fhs Analysis gt Spectral Analysis gt Plotting Fluorescence Excitation Spectra To analyse spectral series data files recorded using the Spectrum excitation light option select Analysis Spectral Analysis to open the spectral window Each file is divided into a series of spectral blocks each block containing a series of images with the excitation light wavelength incremented from the lower to upper limit of the spectrum The spectral analysis modules calculates and displays the intensity from defined regions of interest within the image vs excitation light wavelength throughout the time series _ Spectral Analysis 0 x Spectrum Time Course Spectrum No 11204 Frame 1 0 00 s ROI Rol y y 1 2103 Wavelength Range 340 380 nm 1 1103 Step Size Bandwidth 10 nm 10 nm 1 2103 ROI 1 1 1103 3 10 360 1165 340 348 356 364 372 380 Wavelength nm To display the excitation spectrum for a selected region of interest 1
88. t ROI 1 2 etc Once defined the fluorescence intensity within the ROI can be selected for display in the time course plot by selecting it in Fluorescence ROI list in the time course window The difference between 2 ROIs can be plotted by selecting an ROI in the ROT list Ratio Select the Ratio option to display the time course of the ratio of intensities from a selected pair of wavelength image at the selected ROIs The Display Max box sets the upper limit of the ratio display range The Excl Threshold box defines the minimum fluorescence signal level required for computation of a ratio If either of the two fluorescence signals fall below this value the ratio is set to 0 A D Channels Select the A D Channel option to display analogue signal channels if they are being acquired Dis play The duration of the time course plot can be set by entering a value in seconds into the duration box at the bottom right of the graph Clicking the left and right arrow buttons halves and doubles the display duration The vertical magnification of each time course plot can be expanded to a selected region by moving the mouse to the upper limit of the region pressing the left mouse button drawing a rectangle to indicate the region and releasing the mouse button The vertical magnification can also be adjusted using the buttons at the right edge of each plot Displayed traces can be printed File Print Graph or copied to the Windows cli
89. th type only select Single wavelength and select the wavelength from the list Export File Name Optional Select Change Name to change the name of the export file Click the OK button to export the images File Import Export gt Exporting Analogue Signals To export analogue signals select File Export Analogue Signals to open the window Export Analogue Signals x Output file F My DataWWinFluor Atrial myocyte D PauiCell3_003 Im m abf Change Destination Range Channels Format Whole file V Im Axon Range Jv vm C Text Jo s0s C EDR C Igor Wave C MAT OK Cancel To export the data from one or more analogue signal channel 1 Format Select the format of the export file Axon for the Axon ABF file format readable by PCLAMP EDR for the Strathclyde WinEDR file format or Text to export the data as tables of tab delimited ASCH text Igor Wave to export the data to an Igor binary wave format file 2 Range Select the Whole file option to export all digitised signal data the file OR Range and enter a selected time period to be exported 3 Channels Select the analogue signal channels to be exported 4 Click the OK button to export the signals File Import Export gt Exporting ROI Time Courses To export the fluorescence time course from selected ROI within an image series select File Export ROI Time Course to open the window Export ROI Time Cours
90. the WinFluor program using the setup program downloaded from http spider science strath ac uk sipbs page php show software_imaging Getting Started gt Hardware Configuration To configure WinFluor to work with your system s camera light source and timing hardware open the Camera System Setup dialog box by selecting Setup Camera System Setup MM Camera System Setup i 0 x Camera Light Source Stimulus Outputs Analog Inputs amp Timing Z Stage m Camera Exposure Trigger Output Andor iXon Luca gt Device 1 DIGO Mode C Active High Active Low Electron Multiplying Trigger Delay 1 ms Readout Speed ftomnz y Clear CCD before exposure 7 Post exposure CCD Readout 7 Extra Readout Time 0 ms Calibration Lens magnification 1X Pixel size ieum Calibration bar size 10 um Calibration bar width 4 pixels Tempr Set Point 50 DEGC Interface Cards NI DAQ MX Device 1 USB 6341 8 ADC channels 2 DAC channels C NI DAQ Traditional Reset Devices Auto reset devices Cancel Getting Started gt Cameras gt Cameras The type of camera in use and camera settings are configured on the Camera page lofx Camera Light Source Stimulus Outputs Analog inputs amp Timing XY Z Stage m Camera Exposure Trigger Output _ national Instruments IMAQ v Device 1 PO O Readout Speed ja Active High Active
91. tion of the digital pulse Invert Signal defines whether the digital pulse is an OFF ON or an ON OFF pulse If set to No the digital line is initially OFF OV and switches to ON 5V during the pulse If set to Yes the digital line is initially ON 5V and switches to OFF OV during the pulse The digital pulse element can be used to switch open or close valves controlling the flow of solutions over a cell Multiple digital outputs can be used to simultaneously open one valve while another is closed Family of digital pulse varying in duration This produced a digital pulse on the selected output line with a duration which is incrementable between records It is defined by 5 parameters Initial delay defines the delay before the start of the pulse Starting duration defines the duration of the first pulse in the protocol Increment by defines the amount that the duration is incremented between records Number of increments defines the number of increments in the protocol Note that if there are any voltage waveform elements in use within the protocol the number of increments defined here must be the same Invert Signal defines whether the digital pulse is an OFF ON or an ON OFF pulse If set to No the digital line is initially OFF OV and switches to ON 5V during the pulse If set to Yes the digital line is initially ON 5V and switches to OFF OV during the pulse Train of digital pulses This produces a series of digit
92. upport the Gain value is automatically updated when the gain setting is changed on the patch clamp front panel The voltage output channel Vout 0 or Vout 1 connected to the patch clamp command voltage input is displayed in the V Command O P to list The patch clamp command voltage division factor used to scale the stimulus voltage output to obtain the correct voltage at the cell is indicated in the Divide Factor box The Gain O P to and Divide Factor settings are set automatically when a specific amplifier supported by WinFluor e g Amplifier Axopatch 1D Axopatch 200 etc has been selected as the Amplifier in in Setup Patch Clamp Analogue Inputs dialog box When an unsupported amplifier is in used Amplifier None or Manual Gain Entry appropriate settings must be entered by user Stimulus Protocols gt Creating Stimulus Protocols Stimulus protocols can consist of voltage waveforms on up to 3 voltage output channels Vout 0 2 and 5V TTL digital waveforms on up to 8 TTL digital output channels Note Number of available DAC and digital channels depends on laboratory interface type and available output lines To create a new or edit an existing stimulus protocol file select Setup Stimulus Protocol Editor to open the stimulus protocol editor A diagram of the output waveforms appears in the Waveform display box Stimulus Protocol 1sec stim 10 x tilps Folder IF i Proj New File F Delphi Projects WinFluorwproti open
93. when using E series boards when the older traditional NIDAQ software library is installed instead of the NIDAQ mx library m Interface Cards HI DAQ MX Device 1 USB 6341 8 ADC channels 2 DAC channels NI DAQ Traditional Reset Devices Auto reset devices Getting Started gt Analog Inputs amp Amplifier Settings To set up WinFluor to use an attached patch clamp or other analogue inputs select Setup gt Camera System Setup to display the Camera System Setup dialog box then choose the Analog Inputs amp Amplifiers page EN Camera System Setup l0 x Camera Light Source Stimulus Outputs Analog Inputs amp Amplifiers xy Z Stage Cel Capacity Analog Inputs Input channels Device 1 Al0 3 Input mode Differential Voltage Range 10 v y No Channels In Use 2 Timin g Sampling interval 0 2 ms Board link ers 0 Amplifier 1 Manual gain entry y Input Channels Select the range of analogue input channels to be used for analogue input from the Input Channels list Analog Input mode The A D input mode for the interface device is selected from the Input mode list Analogue input channels can be configured to operate in either differential or single ended input mode In differential mode the input signals are derived from the differences between pairs of inputs AIO AI8 AII AI9 etc In
94. xceed For Enter the period of time that the signal has to remain above the threshold before detection takes place into the Exceed For box Baseline Select the Fixed Baseline option to maintain the baseline and thresholds at a fixed level throughout the event detection scan Select the Rolling Average option to make the baseline follow changes in the signal baseline level averaged over the period of time set by the Period box Dead Time Enter the period of time after detection of an event before another event can be detected into the Dead Time box To avoid detecting an event more than once the dead time value should be longer than the duration of the event being detected but shorter than the time interval between events 5 Click the Detect Events button to detect events within the data file using these criteria Analysis gt Event Detection amp Analysis gt Event Detection amp Analysis The event detection and analysis window allows discrete events voltage steps current pulses fluorescence waves to be located within a continuous recording Detected event waveforms can be superimposed averaged and a range of waveform measurements computed and plotted Select Analysis Event Detection amp Analysis to open the event detection amp analysis window Frame Na 289 1008 4 ajm gt mi Time 14455 Display 480 nm ET 3 EA Zoom 100 y Detect events view Events Average Events Plot Graphs Display Contras

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