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EasyRatioPro
Fluorescence Imaging

 
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Fluorescence Imaging
 
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Fluorescence Imaging
 

Software

EasyRatioPro software represents the ultimate workhorse platform for professional live cell imaging, setting the standard for reliability, efficiency, and integration with a multi-wavelength illuminator.

Software Features

Software Plug-Ins

About the Software

From fixed cell preparations to dynamic events, EasyRatioPro software represents the ultimate, expandable platform for professional live cell imaging applications. There is no other system that delivers the superb quality, flexibility, and breadth of creative options of EasyRatioPro software.

EasyRatioPro Fluorescence Imaging Software

Key Features

Peace of Mind

Day to Day Routines

Non-Destructive

Modular Design

EasyRatioPro

Session Files
Session file format keeps track of all image data, parameters for the entire system.

Acquire Template
Common experiments can be saved as a template and run with one button operation.

Data Integrity
Non-destructive real time plug-ins can be added. Your data is always safe on hard disk.

Plug-Ins
real time OR post acquisition plug-ins can be added at any time.

Key Feature: PTI's PAE (PTI Acquisition Engine) streams imaging data to hard disk for acquisition and playback. This gives the user the benefit of running experiments without worrying about the internal RAM limitation of the computer. You can high-speed stream up to terabytes of hard disk storage. Competitive systems can only offer RAM based high speed streaming, limited to 2 GB of RAM.

Key Feature: PTI knows how valuable your data is, which is why we use non-destructive image processing. Processing Plug-ins can be applied during record or playback mode. The raw data integrity is kept, allowing the researcher to experiment with threshold masking to enhance the image data with peace of mind that their data remains in its original form on the hard drive.

Key Feature: EasyRatioPro software also allows for the maximum flexibility for your future needs with our modular software architecture. Plug-ins can be added to the system to increase functionality. This feature allows researchers to get started inexpensively and then add functionality later as their research grows.

Peace of Mind
An experiment session file keeps track of all imaging data on the hard disk RAID or server. Allowing you to never worry about where you saved your image data. The session keeps track of all image data, region of interest data, wavelength, channels, exposure times, and all vital controls.

Day to day routines
Session templates for common experiments are provided and you can create your own templates. Ready to run common experiments “out of the box” with minimal input from the user. Just push record and go!

Other Features of the software:

  • Square, ellipse, freehand, and linear profile drawn area of interest photometry in real-time or in post acquisition.
  • Trace math analysis functions such as anti-log, average, combine, XY combine, differentiate, integrate, and linear fit, peak finder.
  • Real-time generation of user defined event markers and event journaling.
  • Real-time non-destructive plug-ins for ratio-metric calculation, thresholding, and background subtraction.
  • Export of image data to popular formats such as avi, bmp, jpg, tif, png.
  • Control of up to 16 excitation, emission, or derived channels.
  • “WARP DRIVE” human user interface control for dedicated control over exposure, gain, electron multiplier, mute, solo, select, record, window functions, jog/shuttle, play, rewind, fast forward, stop, time code, looping and much more.

You need an EasyRatioPro if:
Collection and analysis of ratio-metric imaging data for Calcium, pH and intracellular ion imaging are important to you. PTI has poured all of our 20 years of ratio-metric imaging expertise into this new package. The package is modular and expandable offering plug-ins for different applications. It allows researchers get “control back” into their research by reducing the learning curve associated with complex general purpose imaging platforms. This allows researchers to focus on their research application specifically, rather than a complex system with difficult learning curve.

EasyRatioPro Plug-Ins
Plug-Ins are special purpose software components that provide additional image processing functionality to EasyRatioPro. The plug-ins included with EasyRatioPro provide a comprehensive set of digital image processing functions that include Background Subtract, Fast Image Math, Inverse, Math Operations, ROI Calculation and Threshold. The number and variety of plug-ins that you can use simultaneously in a session are limited by the amount of processing power available on the CPU. The more powerful your computer, the greater the number and variety of plug-ins that you can use simultaneously. Because of this dependence on the CPU or host processing, the more plug-ins you use concurrently in a session, the greater the impact it will have on other aspects of your system’s performance, such as the acquisition speed, playback speed, number of channels and processing latency.

 

Background Subtract

The Background Subtract plug-in enables the user to make corrections to acquired images due to uneven illumination or optical imperfections. Background subtraction should only be applied on fluorescence images, not on transmitted light images. Use background subtraction whenever there is debris in the field of view, uneven illumination or high background autofluorescence.

Applying the BGSubtract Plug-in
To use the BGSubtract plug-in, apply it to an insert on the desired channel, record enable and solo the channel, then enable the Master Record button on the transport. This will place the system into "Live Mode". Place your cells on the stage and adjust the camera exposure and gain until you have the desired image. Then remove the cells or move the slide to an area where there are no cells and click on the "Snap Background" button in the plug-in window. This will store the image in the background subtract buffer. Each time the snap background button is clicked, another image is averaged with the previous image in the background subtract buffer. Place the cells back into the field of view. The plug-in will begin to subtract the background once the play button is enabled and the experiment begins. There are two subtraction formulas available. Use "Background Subtract" for fluorescence images and use "Divide flat field" for bright-field images. Background Images can be saved and loaded to the hard disk for future use via the "Save BG Image" and the "Load BG Image" buttons. The Offset parameter allows a positive or negative offset to be applied to the image. This adds or subtracts the offset value for each pixel in the image.

EasyRatioPro Fluorescence Imaging Software

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Calibration Plug-In

Fluorescence Imaging Software

The calibration is a plug-in that can be applied to a channel insert before an ROI calculation plug-in. It will take the intensity or Ratio values of an ROI and then reference them to the selected calibration. The output will then be scaled or "calibrated" against this reference file, and plotted into the Graph window. The calibration file must be created prior to using the calibration plug-in by using the calibration editor. Any number of calibration files can be created by the calibration editor, but only one calibration file can be selected per calibration plug-in. Also, only one calibration plug-in can be used per channel.

Applying the calibration plug-in

Simply apply this plug-in to an insert on a selected channel. Click on "Load New Data" and navigate to your calibration file you saved in the calibration editor. The calibration data status will show loaded if the file loads correctly and the calibration file type will show either "Intensity" or "Ratio" depending on the original calibration selected in the calibration editor. The calibration plug-in can be moved up or down in the insert of the channel. To ensure the system calibrates the ROI data, be sure the calibration plug-in is BEFORE the ROI calculation Plug-in. This is very important as the ROI averages are derived from the calibration function to calibrate the ROI trace.

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Composite Plug-In

The composite plug in enables the user to create an RGB color composite image or sequence from up to three different excitation channels in a session. Any channel can be selected for Red, Green and blue respectively. The image pixels can be adjusted in the X, Y directions to correct for any image offset due to microscope optics. The Gamma, Brightness and Contrast can be adjusted for each channel and the composite channel to enhance the image. It can be used during record or playback. The composite plug-in is processor intensive, therefore it is recommended to be used during playback only if a fast camera acquisition rate is required. Since the composite plug-in generates a new image, it is recommended to use this plug-in on a derived channel only. The actual data is generated internally by the CPU as an Alpha blended channel with three layers at the original camera bit depth. The Image window shows an RGB24 Image for display purposes. If the channel is record enabled, it is the Alpha blended channel that is written to disk and saved with the session. If the users wishes to export the Color composite RGB24 image to tiff, the Authoring Tools can be used to export any portion of the sequence to individual tiff files or a multi-paged tiff file.

Applying the Composite Plug-In

To use the composite plug-in , you should have more that one excitation channel. For this example we will have three excitation channels, DAPI, FITC and CY5. The microscope will have a triple pass cube to allow the excitation monochromator to alternate the wavelengths for DAPI, FITC, CY5. Next, create a derived channel and apply the composite plug-in to this channel. Click on the setting button of the composite plugin and the composite controls can be viewed.

Fluorescence Imaging Software

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Fast Image Math

EasyRatioPro Fluorescence Imaging Software

The Fast Image Math Plug-in is optimized to be used during real-time acquisition. Its algorithms are designed to take advantage of modern CPU and the plug-in offers the following options for processing:

  • Ratio Type FF – Float ratio using float range check
  • Ratio Type DD – Double ratio using double range check
  • Ratio Type FI – Float ratio using integer range check
  • Ratio Type DI – Double ratio using integer range check
  • Addition
  • Subtraction

Depending on the CPU type (Intel P4 or AMD) a user can choose from the different ratio algorithms to determine which method should be employed during their experiment. If the enable status check box is activated, the statistics will reveal the processing time required in mS for this plug-in algorithm. Simply choose the fasted one for your system.

The Fast Image Math plug-in requires a channel source 1 and source 2 to provide input to the processing function. The Channel Source 1 and Source 2 drop down menus will list all the channels in the current session. The Parameters field shows Rmin and Rmax. Adjusting these parameters will scale the 3 pixel range to these upper and lower values. This is useful to help visualize the ratio image.

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Inverse

The Inverse plug-in inverts the image such that white pixels become black and black pixels become white. The formula for this plug-in is for every pixel:

new pixel value = max. pixel value - current pixel value

There are no user adjustable parameters for the Inverse plug-in

EasyRatioPro Fluorescence Imaging Software

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Math Operations

EasyRatioPro Fluorescence Imaging Software

The Math Operations plug-in is a general purpose plug-in that allows the following operations:

  • Add
  • Excitation Ratio
  • Subtract

In order to operate this plug-in, a source 1 channel is required. The source 2 can be either a channel or a constant. The parameters and status field are similar in operation to the Fast Ratio Math plug-in parameters. This plug-in uses more processing time that the Fast Ratio Math plug-in, therefore it is not recommended for use during acquisition. It is better to be used during playback.

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Palette

The palette plug-in allows an individual channel to be Pseudo colored with a user selected palette. This plug-in flags the global palette in the image window to change to the palette reflected in the channel plugin. A common use would be to set channel one palette to green for a FITC dye and channel two to blue for a DAPI dye. If the global palette is set to another palette, once a channel is soloed, the channel palette will override the global palette. The start and end range adjustments can be made to adjust the palette rage over the dynamic range of the image. This will allow the enhancement of the image by pseudo coloring only the used dynamic range of the camera.

Fluorescence Imaging Software

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ROI Calculation

EasyRatioPro Fluorescence Imaging Software

The ROI Calculation plug-in is a plug-in that is used to activate the ability for a channel to read the pixel values of images in acquisition or playback. The ROI Calculation plug-in must be applied to any channels for which you wish to report the pixel values into the Graph or ROI List windows. You must put a Fast Math plug-in with a Ratio operation before the ROI plug-in in a Ratio channel.

In order for ROI Calculation plotting to occur, the user must select the channel and then draw one of the ROI shapes onto the image in the Image window. If you select more than one channel, then the same ROI shapes will be applied to each of these channels. The graph window will then show a trace in the graph legend, ready to plot data when the experiment begins. ROI Calculation plug-ins can be added at any time during recording or playback. Only one ROI calculation plug-in is allowed per channel.

ROI Mode

Average: – This mode will average the pixel intensity values in each ROI for the selected channels.

Median: – Not implemented at this time.

Area: – This mode will give the number of pixels in each ROI for the selected channels.

ROI Options

Use all Pixels except in Area Mode: In Area Mode do not use Zero-Pixels – You can suppress background pixels by applying a threshold plug-in before the ROI plug-in. Then these zero-pixels will not be counted in the area value. This is useful to display changes in the size of active areas. The average will be the average of all pixels in the ROI.

Use All Pixels in any Mode: – The area of an ROI will remain the same. The average will be the average of all pixels in the ROI.

Do not use Zero-Pixels in any mode: – Zero-Pixels will not be used in the calculation of the average or area values.

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Threshold

The Threshold plug-in can be applied to any channel or derived channel. It is commonly used to suppress the background by mapping any pixels below the user defined threshold value to zero. It has two controls

Lower Threshold –- This maps all pixels below this value to zero. It is commonly used to suppress the background.

Upper Threshold – This maps all pixels above this value to zero. It is commonly used to suppress a "hot spot" or high intensity location to zero.

The Threshold plug-in can be applied at any time during recording or playback. Keep in mind this plug-in will take processing time, so it is not recommended for a high speed acquisition, as inserting this plug-in will slow down the rate of acquisition.

EasyRatioPro Fluorescence Imaging Software

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Software Features

Quick Operation

Quick Operation EasyRatioPro has a hot palette with ten buttons that are mapped to common application templates. These templates have been factory defined as:

  1. FITC – single excitation channel at 488 nm.
  2. DAPI – single excitation channel at 365 nm.
  3. CY3 – single excitation channel at 550 nm.
  4. Fura2 – dual excitation channels at 340 and 380 nm, plus one derived ratio channel to monitor the Ca++ ratio. ROI Pug-Ins on all channels.
  5. BCECF – dual excitation channels at 440 and 485 nm, plus one derived ratio channel to monitor the the pH ratio. ROI Pug-Ins on all channels.
  6. Fura2/BCECF – quad excitation channels at 340, 380, 440 and 485 nm, plus two derived ratio channels to monitor the Ca++ ratio and the pH ratio. ROI Pug-Ins on all channels.
  7. GFP – single excitation channel at 470 nm to monitor changes in GFP fluorescence.
  8. SBFI – dual excitation channels at 340 and 380 nm, plus one derived ratio channel to monitor the sodium ratio. ROI Pug-Ins on all channels.

These templates reside in the C:\Program Files\PTI\EZRatioPro\Templates folder. The templates can be user modified (e.g., window positions and parameters) by saving any changes to a template.

To use the templates, simply click on a template button. You will be asked to define a directory in which to store the sequences. You will then be prompted to rename the session file. Simply choose a name and location for your session. You may now record enable the channels, start your experiment, and create ROIs. The experiment will continue to record until the Stop button is pressed.

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Channel Window

After the hardware is defined, select File \ New Session from the main menu and enter the name of the session. Then select File \ New Channel. This opens a dialog to define the type of channel (excitation or derived) and number of channels. The dialog looks like the following:

EasyRatioPro Fluorescence Imaging Software

EasyRatioPro Fluorescence Imaging Software

Once the "Create" button is pushed, the Channels window creates a "Channel Object" like the example below.

The left hand side of the above object is created with the first channel and gives a summary of all channels.

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Bank Selection

To reduce overcrowding on the monitor, a maximum of four channels is shown at at any time. If you have more than four channels, then channels 5–8 will be shown by clicking on Bank 2, Bank 3 shows channels 9–12, and Bank 4 shows channels 13–16.

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Channels

This field shows the names and types (excitation or derived) of all channels in the current session.

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Groups

This field shows "-- All --" and any named groups of channels. Clicking on "All" selects all channels in all banks; clicking on a group name selects only those channels in the group.

The Channels window is a dynamic controller array of channel strips similar to an audio mixer. It dynamically reflects the channels defined by the user, showing only what the user has defined as excitation and derived channels.

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Moving from the bottom of the channel to the top, the objects and functions are defined as follows:

ERP Fluorescence Imaging SoftwareChannel Select button

Single-clicking this button toggles the selection of this channel for renaming, ROI creation or grouping. Multiple channels can be selected. Selected channels are shown by darker colored buttons. This selection also works in conjunction with the creation of an ROI. If an ROI is created, the ROI will be generated for each selected channel that has an ROI plug-in. Then the Graph Window shows a trace for each selected channel. Double-clicking this button allows the user to rename the selected channel. This is the same as selecting "Rename Selected Channels" from the File menu.

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ERP Fluorescence Imaging SoftwareCamera Exposure Time

This field shows the camera exposure time in units of 10 µs and allows editing of the value manually with a keyboard entry. The exposure time range is 0.0 to 0.65535 s.

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ERP Fluorescence Imaging Software

Channel Fader Control

The camera exposure time can also be controlled by moving the channel fader. The fader increases the exposure time linearly as the fader is pushed upwards. The vertical bar to the right is not implemented at this time.

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ERP Fluorescence Imaging SoftwareAutoexposure

Above the exposure time field, is the auto exposure time button. Autoexposure is not implemented at this time. The camera excitation will move to the wavelength defined by the channel and will auto expose and enter the recommended exposure time in the exposure time field. This is done by acquiring a camera image, calculating the average value of the image intensity, and repeating until the average image intensity reaches 50% of the camera's dynamic range. The exposure fader of Warp Drive (explained later) will be updated to reflect this exposure time.

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ERP Fluorescence Imaging SoftwareGain

Below the auto expose button, is the camera gain fader. Moving this fader from left to right increases the gain of the CCD camera or the EMCCD camera electron multiplier. The range is whatever gain the camera can supply scaled to 0 to 100%.

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ERP Fluorescence Imaging SoftwareCamera Binning

The next controller encountered is the camera binning control. This shows the elections available from the camera, querying the camera to see what binning the camera supports, and then showing the values available for the user to select which will populate this. X and Y values are linked to maintain the image aspect ratio.

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ERP Fluorescence Imaging SoftwareSolo

The Solo button works in conjunction with the image display. If the Solo button is pressed, the button is highlighted (yellow), and the Image window will only show the soloed channel. All other channels will be muted. This allows the user to view any channel during the experiment or during playback. During the experiment (Record mode), the illuminator switches between wavelengths, but the Image window shows only the soloed channel. During Play mode, the Solo condition will show only the soloed channel select reading from hard disk. If the master record button on the Transport window is enabled, pushing the Solo button sends the illuminator to the solo wavelength and the Image window shows the live camera image. This allows the soloed channel to be used to focus the image.

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ERP Fluorescence Imaging SoftwareMute

The mute button works in conjunction with the Image window. If a channel is muted, the channel becomes grayed out and it will NOT be displayed in the Image window. Mute will become more important when the Image window can show more than one image. To unmute a channel, click the mute button again. Mute does not override Solo. Mute works in both Record and Playback.

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ERP Fluorescence Imaging SoftwareRecord Enable

This activates recording for the selected channel. When a channel is placed in record mode, the record button turns red and the channel will be prepared to write images into it's sequence file on disk. Any channels with record mode activated are written to disk once the master record and play button are depressed. This is similar to a VCR tape recorder.

For excitation channels, Record saves only the raw image data before plug-in processing, even though the Image Window shows the result of processing by the plug-ins. For derived channels, Record saves image data created as a result of plug-in processing. Thus, during an experiment, if you enable record only on a derived channel you will lose the original raw data.

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ERP Fluorescence Imaging SoftwareChannel Setup

A channel MUST be set up before the acquisition can occur. Clicking the Setup button opens the setup dialog.

EasyRatioPro

The user enters the wavelength by typing into the text field. Step size and End wavelength are grayed out if excitation scan is NOT selected. The bandwidth will be for motorized slits. Each Channel strip can have a separate bandwidth if desired. If Stream scan is selected, the excitation scan will stream into memory to allow high speed scanning. In this manner, the experiment acquires a series of excitation scans, vs. time points. The excitation monochromator will scan the desired range at each time point. If 3D is enabled, the piezo driver controls the z-focus and collects the user defined z planes.

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Derived Channels

ERP Fluorescence Imaging Software

You must have at least one excitation channel before a derived channel can be created.

Derived channels look similar to excitation or emission channels except they do not have controls for exposure time or gain, since they do not control the camera. Record, Solo and Mute functions work similar to Excitation channels.

The derived channel Setup button is used to select an excitation channel's timecode source.

ERP Fluorescence Imaging Software

The derived channel by default uses the time source of the channel left of the derived channel. This will show the derived channel's data was calculated when the frame of the channel to the left was collected. The excitation monochromator will always collect channels from the left channel to the right channel in the channel window.

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Channel Plug Ins

Channel Inserts

EasyRatioPro Fluorescence Imaging SoftwareThe channel inserts section of the Channel object allows up to five Image processing plug-ins to be inserted into the image data stream. Plug-ins route the signal from the channel, through the plug-in process of your choice, then automatically return it to the same channel. Plug-ins can be used on both excitation channels and derived channels, can be made active or inactive (bypassed), and will operate in both record and playback mode.

Plug-Ins can be assigned to any excitation or derived channel and can be added or removed in real-time. The Plug-in Window can be opened by clicking on the channel window insert selector, , by selecting Display, Show Plug-In Window on the menu, or by selecting the PLUGIN button on the PTI Warp Drive. This will show a Plug-in Window similar to the following:

Fluorescence Imaging Software

ERP

The left-hand section is common to all plug-ins.

ERP Fluorescence Imaging Software This field indicates the channel the plug-in is applied to. A drop down menu lists all the channels defined by the user. If another channel is selected the plug-in window will change to reflect that selected channel.

ERP Fluorescence Imaging SoftwareThis field is the name of the plug-in applied to this insert. If this button is clicked and held, a drop down menu will show a list of all the installed plug-ins. If the user selects a plug-in from the list and clicks the ADD button, the plug-in will be added and activated on the selected channel and the plug-in interface controls will change.

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ERP Fluorescence Imaging SoftwareThis field lists the plug-ins that have been inserted, up to five per channel. If another insert is selected, the Plug-in Window will change to reflect the plug-in for the selected insert.

ERP Fluorescence Imaging SoftwareUnchecking the enable box will bypass the processing of the plug-in on the selected channel.

Fluorescence Imaging SoftwareThe Up and Down buttons allow the plug-in processing order to be changed. Select a plug-in and then click the Up or Down button to raise or lower the order of the plug-in process.

ERP Fluorescence Imaging SoftwareClicking this button enables all plugins in the insert list.

ERP Fluorescence Imaging SoftwareClicking this button bypasses the processing of all plug-ins on the insert list.

ERP Fluorescence Imaging SoftwareClicking on this button removes the selected plug-in from the insert list.

ERP Fluorescence Imaging SoftwareClicking on this button removes all plug-ins from the insert list.

ERP Fluorescence Imaging SoftwareCloses the Plug-In Window.

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Making Plug-Ins Inactive

You can make plug-ins inactive in order to free up resources for other plug-ins and processing. When a plug-in is inactive, it retains it's assignment, position, and settings. However, it will not pass video into it and does not consume processing resources.

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To toggle a plug-in active or inactive

Click on the check box next to the plug-in name in the plug-in list field. A check mark indicates the plug-in is active.

- or -

Click on the plug-in name in the channel insert of the channels window. A [M] will show in front of the plug-in text to denote that the plug-in is bypassed.

EasyRatioPro Fluorescence Imaging Software

The right-hand section of the Plug-In Window is different for each plug-in depending on the parameters that need to be set for each type of process. For example, the right side of the Threshold Plug-In is below.

EasyRatioPro Fluorescence Imaging Software

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Chaining Plug-Ins

Since each channel has five inserts, it is possible to add five plug-ins. The output of the plug-in on insert A will be processed first, the output of insert A is then fed to insert B and so on. An example would be to have an excitation ratio created in Derived 1 channel using the Excitation Ratio Plug-in on insert A, then insert B would have the ROI Calculation Plug-In to create a trace in the Graph Window of the average intensity of an ROI.

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Transport Operations

ERP Fluorescence Imaging Software

The transport control defaults to open when a new session is started. The transport can be hidden from the Windows menu by selecting "Hide Transport" (Ctrl+Alt+3); this menu entry then changes to "Show Transport" if the transport is hidden. The transport functions are as follows:

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EasyRatioPro Fluorescence Imaging SoftwareReturn to zero

This button returns the transport to the beginning of experiment or time zero.

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ERP Fluorescence Imaging SoftwareRewind

Clicking and holding this button rewinds the transport. Releasing the button stops rewinding.

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ERP Fluorescence Imaging SoftwareStop

Clicking on this button stops the transport, in play or record mode.

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ERP Fluorescence Imaging SoftwarePlay

Clicking on this button begins playing the recorded images from disk, or if the transport is record enabled, begins recording images to hard disk.

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ERP Fluorescence Imaging SoftwareFast Forward

Clicking and holding this button fast forwards the transport. Releasing the button stops fast forward.

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ERP Fluorescence Imaging SoftwareGo to END

Clicking on the End button takes the transport to the end of the experiment.

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ERP Fluorescence Imaging SoftwareRecord

Clicking on this button activates the master record. The button will turn red. Channels’ individual record mode buttons must be activated to determine which channels will be recorded. Play can then be activated and recording will commence to hard disk.

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Region Selections

When the user defines a region in the data trace window, these co-ordinates are filled. These regions determine the range where the playback can be looped and also the region of the experiment that can be "Bounced to Movie". I.e., if the user wishes to create a short movie of a section of the experiment, the defined area is highlighted in the trace window, and the File command "Bounce to Movie" creates a move of this selected region. The movie will be an AVI or QuickTime movie.

EasyRatioPro Fluorescence Imaging Software

The elapsed time counter displays the elapsed time since the experiment began. The upper counter displays time in hours: min: sec, the lower counter in frames.

ERP Fluorescence Imaging Software

Playback speed sets the playback speed that the sequencer plays back. It can be increased or decreased. It may have to be decreased when using a slower PC. This playback speed can also be adjusted using the Warp Drive remote control shuttle ring (see the Warp Drive section).

ERP Fluorescence Imaging Software

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Image Window

EasyRatioPro Fluorescence Imaging Software

The Image window is a single window that displays the camera image. The image displayed depends on the Solo and Mute buttons status on the channel strips.

For example if the 340 nm channel is soloed, the Image window displays the 340 nm image. If the ratio is soloed, the Image window displays the derived 1 ratio image.

This keeps the interface uncluttered and the application easy to use. The Image window intelligently zooms or shrinks the image to fit into the full size of the workspace provided. Small binned images are auto zoomed to the workspace and large 1 x 1 binned images are shrunk to fit into this workspace.

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Timebased Options

Fluorescence Imaging Software

The Timebase Options allows the user to dynamically change the acquisition speed during the experiment. A user should be able to start acquiring at 1 image per second then change it to some other value and the system speed will change to accommodate to user's request, if possible. This dynamic nature is very important. Data points generated from this acquisition will be stamped to reflect the users selection. If the user selects one image per second each data point will be stamped with one-second intervals.

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Authoring Tools

The Authoring Tools tab allows the export of the image window. It outputs a what you see is what you get (WYSIWYG) RGB image to TIFF format. It will export frames as long as the play button is activated once the exported has been initialized.

Fluorescence Imaging Software

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Bin./Area

Fluorescence Imaging Software

This tab allows for setting the camera binning and offset parameters.

Binning reduces the image size by grouping equal numbers of adjacent pixels in the X and Y directions. Increasing binning increases camera sensitivity at the expense of camera spatial resolution. X and Y binning are kept equal to maintain the aspect ratio.

Offset X and Y define in pixels the top left corner of the active region.

Width and Height define the size in pixels of the active region of the image.

Full Frame defaults the width and height to the full image frame defined by the binning parameter. Offset X and Y are also defaulted to 0.

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Calibration Editor Setup

Fluorescence Imaging Software

The Calibration Editor can be accessed by going to the Display Menu and selecting Calibration Setup. The editor window shows the calibrated values on the Y-axis vs. the Raw intensity on the X-axis if Intensity is chosen, or Ratio on the X-axis if Ratio is chosen.

In order to use the calibration editor, a series of data points must be entered manually, captured from a region of interest or grabbed from the data cursor (DC) of the graph window.

The following example will describe the procedure to enter values manually.

  1. After performing a titration with a single wavelength dye, you should have a list of values on the intensity and the concentration of each sample. Click on the New Point button. This will open a dialog like this:Fluorescence Imaging Software
    Simply enter the measured value in the Measured field, and the calibrated value in the Calibrated field. You may also enter a comment in the Comment field. Once "OK" is pushed, the x, y co-ordinates will show up in the calibration equation graph field. Continue by adding more points. The fitting algorithms require a minimum of six points to be entered for the 3rd, fourth and fifth order polynomial functions.
  2. After the points have been entered, select a fitting method such as 3rd order polynomial and click on the FIT button. A fitted curve function with statistics will be generated from the entered data. Continue selecting different methods like 3rd, 4th or 5th order until the curve function fits the data the best and the MSE value reports the lowest value possible.
  3. Save the calibration to a file by selecting the Save button and give the file a distinct name, for example "Fluo4 intensity calibration". This calibration can then be loaded into a channels "calibration" plug-in. See the Plugin guide for operation of the calibration plug-in.

Variations of the above method
The calibration editor allows the measured values to be captured or grabbed from the data cursor of the graph window. In order to accomplish this, simply select a trace in the trace graph window, select the data cursor tool and move the data cursor to the desired point using the left or right arrows on the keyboard. Once the point is selected, click on the Capture DC button to capture the data cursor values into the equation editor measured field.

You may also extract values from an Active ROI that has been drawn onto the Image window. To do this draw an ROI on the area of interest of the image. Then select the ROI from the drop down list, choose options Min, Max or Average. Then click on the Capture ROI button to capture the value into the measured field.

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Grynkiewicz & Grynkiewicz pH Equation parameters

If the Grynkiewicz or Grynkiewicz pH curve fitting methods are selected, an addition parameter field will become available for parameters of this equation.

Fluorescence Imaging Software

Grynkiewicz equation Clicking on the Params button will open a dialog in which to enter all the above parameters.

Fluorescence Imaging Software

The parameters are defined as follows:
Kd – dye dissociation constant.
Rmin – Ratio minimum (usually acquired by adding EGTA to Fura2 loaded cells)
Rmax – Ratio Maximum (usually acquired by adding Ionomycin to Fura2 loaded cells)
Beta – The fluorescence intensity of Sf2/Sb2 where Sf2 = the selected region corresponding to the ionfree form of the dye in the denominator curve (contemporal with Rmin) and Sb2 = selected region corresponding to the ion-bound form of the dye in the denominator curve (contemporal with Rmax).
V – intracellular viscosity for water is close to 1.0.
These text boxes are editable.

Select a data point in an ROI trace, and click on Capture DC. This will capture that value and place it in the Acquired value text box (editable). Click on the Set acquired value button beside a paramater to enter the acquired value into that text box.

After entering the Grynkiewicz fit parameters, clicking on the Fit button will fit the Rmin and Rmax values, and a function will be created to define the equation.

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Grynkiewicz pH equation

Clicking on the Params button will open a dialog in which to enter all the above parameters.

Fluorescence Imaging Software

Fluorescence Imaging Software

The parameters are defined as follows:
pKa – dye dissociation constant
Rmin – Ratio minimum
Rmax – Ratio Maximum
Beta – The fluorescence intensity of Sf2/Sb2 where Sf2 = the selected region corresponding to the ionfree form of the dye in the denominator curve (contemporal with Rmin) and Sb2 = selected region corresponding to the ion-bound form of the dye in the denominator curve (contemporal with Rmax). These text boxes are editable.

Select a data point in an ROI trace, and click on Capture DC. This will capture that value and place it in the Acquired value text box (editable). Click on the Set acquired value button beside a paramater to enter the acquired value into that text box.

After entering the Grynkiewicz pH fit parameters, clicking on the Fit button will fit the Rmin and Rmax values, and a function will be created to define the equation..

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ROI Tools

Easy Ratio Pro Fluorescence Imaging Software

The ROI Tools section allows the user to click on the tools to define and draw ROIs onto the image. The ROIs defined create one ROI per selected channel. For example, if channels 1, 2 and Der1 are selected by clicking on a channel's Select button, when clicking on an ROI tool and drawing an ROI, three ROIs are created, one for each channel. These ROIs will show up as trace data in the graph/sequencer window. Available ROI shapes are rectangle, ellipse, and free hand. In order for an ROI to be activated, an ROI calculation Plug-in MUST be applied to the channels of interest. ROI Calculation plug-ins may be added or removed during record or playback buy using the Plug-In window's Add or Remove buttons.

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Color Bar

EasyRatioPro Fluorescence Imaging SoftwareThe color bar area of the display shows the grayscale values, or pseudo color values that have been mapped onto the image.

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Image Enhancement

ERP Fluorescence Imaging Software

These controls allow for setting of the brightness, contrast and gamma settings.

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Sequence Graph Window

The sequence graph window plays an important part in the EasyRatioPro interface. Not only does it show ROI data traces, but it also shows the locate/event markers as well. It also allows the user to select defined regions for looping the sequencer and selecting regions to bounce to movie.

ERP Fluorescence Imaging Software

A sequencer vertical bar shows the location of the sequencer time during record and playback mode. Once user defined ROI's have been selected, pushing play will show data traces for the selected ROI's in the graph window. The user can zoom the graph window in or out during record or playback mode. The data window will follow the menu scroll selections. This window also permits mathematical analysis of the ROI data traces.

A full description of the commands available in the Graph Window is given in the Graph Window manual.

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Event/ Journal

ERP Fluorescence Imaging Software From the Events Journal menu a user is able to open, create, delete, search, output and transfer events. Events can be created on the fly during an experiment in both record and playback mode. Search Events and Transfer Events to Excel are not implemented; for Output Events to Log File – use Graph Window, right-click on a session, Export Acquisition (should be Export Session).

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Create Events

EasyRatioPro Fluorescence Imaging Software

When an event is created, either by selecting "Create Event" in the Events/Journal menu, hitting Enter on the keyboard, "J" on the transport, or by the remote control interface, this dialog box opens to allow the user to enter information about the event. Events can be a marker or a selection; an event has a name and allows comments.

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Events Window

ERP Fluorescence Imaging Software

The Events Window is above. It has a list of events that are numbered, the user defined name of the event, time the event occurs, and comments carried over from the create events dialog. The event names and times can be edited within this dialog by double clicking or deleted by selecting the events and choosing "Delete Event" from the Event/Journal menu.

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Program Menus

File Menu

EasyRatioPro Fluorescence Imaging Software

New Session - This allows the user to create a new session. A session is an experiment.

Import Sequence into Session - This allows the user to import a sequence from another experiment into the current session. This is useful to compare results from day to day.

Open Session/Template – Opens a previously saved session or template.

Close Session – Closes the existing session.

Save Session - Saves the current session.

Save Session As – Allows the user to save the session under a new name. Also allows the user to save a session template.

Bounce to Disk – Looks at the current user selection range and soloed channels. Based on this information a movie can be created using the windows AVI or possibly QuickTime movie formats.

New Channel – This opens the new channel dialog to allow user to add channels to the channel window.

Rename Selected Channels – Opens the Rename Selected Channels dialog to allow the user to rename the channels.

Group Selected Channels – Groups the selected channels. If fader channel 1 is moved, fader channel 2 will follow its movements. Exposure time and Gain should be able to be grouped.

Delete Selected Channels – Deletes the selected channels in the channels window. For example a user could delete the derived 1 channel if that no longer wish to create a derived channel.

Information – Allows the user to enter information about their experiment into the session.

Page Setup – (Not Implemented).

Print – (Not implemented).

Quit – Quits EasyRatioPro.

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View Menu

ERP Fluorescence Imaging SoftwareToolbar – Toggles ON/OFF the Toolbar with predefined sessions.

Status Bar – Toggles ON/OFF the Status Bar.

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Operations Menu

ERP Fluorescence Imaging SoftwareLoop playback – (Not implemented) Activates the loop playback mode where the sequence will loop the selected region. The region can be user selected.

Scroll options – (Not implemented)

No auto-scrolling – Will not update the sequence graph window during the experiment

Scroll after playback – Will update the sequence graph when playback has stopped

Page Scroll during playback – Will scroll the sequence graph window during playback.

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Setups Menu

Hardware Setup – Opens the hardware setup window to define interface and excitation device and camera/grabber settings.

Camera/Grabber Options – Opens the Camera/Grabber Options dialog to set digital camera parameters.

Disk Allocation – Defines the hard disk used for the excitation channels record to disk.

Peripherals –- Sets the location to define the operations of the Warp Drive remote control interface. (Not implemented here - use Hardware Setup)

Preferences – Opens the preferences dialog that will be a tabbed dialog for the user to store program preferences. (Not implemented)

ERP Fluorescence Imaging Software

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Events Journal

ERP Fluorescence Imaging Software

Open Event Window –- Opens the event window.

Create Event –- Opens the create event window to allow the user to create an event.

Delete Events – Deletes the selected event(s) in the event window.

Search Events –- Not Implemented

Output Event to Log File –- Not Implemented. However, saving a session includes saving the events; in the Graph Window, Exporting an acquisition saves the ROI trace data and the events.

Transfer Events to Excel Not Implemented

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R.O.I. Menu

Load – Opens a load dialog box and allow the user to load a previously saved ROI file.

Save –- Opens a Save As dialog box to save the ROI as a file.

Add > Rectangle – Allows the user to draw a rectangle onto the image and create ROIs based on the channels currently selected.

Add > Ellipse – same as Add > Rectangle except created ellipse ROIs.

Add > Freehand –- Same as Add > Rectangle except creates freehand ROIs

Edit – Allows editing of the selected ROI.

Clear – Clears all selected ROI's from the Image window.

ERP Fluorescence Imaging Software

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Display Menu

ERP Fluorescence Imaging Software

These commands open and bring the respective window to the forefront (on top of other windows if there is overlap). The number keys are those above the QWERTY keyboard; the number pad will not work here.

Show Channels Window – Show the channels window.

Show Graph Window – Show the Graph window.

Show Transport Window – Show the transport window.

Show Image Window – Show the Image window.

Show Plug-in Window –- Show the plug- in control window. This allows the user to control Plug-In functions.

Annotation – Show the annotation window.

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Windows Menu

These commands toggle between hiding and showing the respective window (on top of other windows if there is overlap). The number keys are those above the QWERTY keyboard; the number pad will not work here.

Hide Channels –- Toggles between Hide and Show channels window.

Hide Graph – Toggles between Hide and Show Graph window.

Hide Transport – Toggles between Hide and Show Transport window

Hide Image – Toggles between Hide and Show Image window

Hide Plug-ins –- Toggles between Hide and Show Plug-ins window

Show Session Setup –- Opens the Show Session Setup Window.

Hide Clocks – Toggles between Hide and Show the Counter Window.

Show Marker locations – Opens the marker location window.

Show Disk Space - Opens the Disk Space status window.

ERP Fluorescence Imaging Software

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