Transcript
G:BOX Chemi HR16 Gel Documentation and Analysis System
Quick Guide-Image Capture Image capture and enhancement is carried out within
GeneSnap
(if the program does not open automatically simply double-click on the GeneSnap desktop icon). You will now see the GeneSnap User box.
1) Log in
Type into the space your name and click the OK button. This is now your configuration and any changes you make to the way you use or set-up the program will be stored as defaults under your name. Each user can enter a name or title for their own individual user preferences so that whenever you start G:BOX Chemi HR16 and enter your name at the log-in stage the system adapts for your preferred settings. In future you only need to select your name from the drop down list. GeneSnap will now fully open and you will see an image capture window appear. To ensure the best performance of the camera allow 30mins for cooling. If the image capture window is not visible at any time, click on the camera icon at the top of the screen.
2) View "live" image
To obtain a live image, click on the green live button within the image capture window. The image capture window will now show the current camera view. Open the cabinet and place the sample on the UV transilluminator or visible light converter. Upon clicking on the green live button, the capture window updates and will display the virtual camera controls. Using the mouse, click on any of the buttons to control the chosen function (see below). Use the lighting control drop down menu to select which light source comes on when the image is live. Select ‘No Light’ for chemiluminescence (alternatively choose the Chemi configuration). For UV excitable dyes select Transilluminator (alternatively select the Gel configuration). For visible light converter applications select Transilluminator. If fitted, use the filter wheel drop down menu to select the appropriate filter for your application. For chemiluminescent imaging select ‘No filter’ to maximise light entering the camera (No filter will be automatically selected if the Chemi configuration has been chosen in the previous step). If a filter slider has been fitted move the slider to the appropriate position (left blank, middle Etbr/UV filter, right custom filter). For UV excitable dyes choose the appropriate filter (refer to Application note 10 on the Syngene website for choice of optimal filters). For visible light converter applications select the no filter. By simply clicking the Zoom +/- buttons you can decide which part of the image you wish to capture. For maximum resolution, zoom in on the area of interest and only capture the part of the sample showing tracks/bands. For ease of use it is suggested that the well line is at the top of the image when looking at gels. Click the Iris +/- buttons to alter the camera aperture and allow more or less light to reach the camera sensor. Set the iris to a point where you can view all the bands on the gel. (Don't worry if some are still a little faint as this can be rectified later). Clicking the Focus +/- buttons will allow you to make fine focus adjustments. For zoom, iris and focus changes you can use the large or small buttons. By clicking on a large button the setting will be driven continuously, whereas a single click on a small button will result in the smallest setting change. Note the smallest setting change will not necessarily result in a single digit change.
3) Select lighting
4) Select filter (from filter slider or filter wheel)
5) Adjust zoom
6) Adjust iris
7) Adjust focus
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The *focus indicator shows when the focus condition (amount of contrast) in the picture is increasing or decreasing. If the contents of the picture change suddenly, it stops indicating for a period of one second and appears ‘grey’. If the focus condition is improving the bands in the indicator will move upwards. Bands will move downwards if the focus condition is getting worse. If the focus condition is the best that has been measured, the indicator shows a ‘bright’ green colour. This ‘bright’ colour automatically appears at the beginning of focusing as the camera has not seen a better condition so far. If the focus condition is below the best seen the indicator shows a ‘pale’ green colour. When the camera is live start adjusting the focus and the bands will move upwards as the focus is improving. Once the focus has gone through the optimum condition the bands will start to go downwards and appear ‘pale’ green. Now turn the focus back to return to the optimum setting and when it appears ‘bright’ green you have achieved the best focus and the image can be grabbed. To re-set the focus click on the Re-set icon (eye) at the top of the focus indicator. Bright Grey
8) Focus indicator
Pale
Grabbing the image Once you are completely happy with the image in the window, click on the red button to freeze the image and shut down the camera and lighting.
9) Grabbing the image
An additional indication on this control panel is the darkroom "door open" warning. The pictorial of the darkroom will show if the darkroom door is open or closed.
Further controls Exposure time - Above we "grabbed" an image in real time using the standard exposure setting of the camera. However, in some instances you may wish to extend the exposure time to resolve fainter bands or collect chemiluminescent images. In this case, click the arrows on the ‘Exposure time’ bar to increase (or decrease) the exposure time. The window shows how much exposure time you have set. The arrows on the right allow you to change the units of the exposure time.
Neutral Fielding (NF) This allows the capture of images corrected for uneven illumination. See Neutral Fielding Quick Guide for more details.
Binning and Increased Effective Pixels The G:BOX Chemi HR16 has a resolution of 1.4 million pixels. Binning or merging pixels will increase the sensitivity of the camera to low light levels. GeneSnap offers multiple levels of binning - no binning, 2x2 and 4x4 binning. Each level of binning will result in a corresponding decrease in resolution, and this must be considered when selecting which level to use. *In addition,selecting effective pixels up to 5.5 million pixels is available. Each level of effective pixels will result in an increase in image size and this must be considered when selecting which level to use.
10) Set exposure time
11) NF
12) Binning and Increased Effective Pixels
Please note that 1x1 binning is equivalent to no binning
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Saturation - Clicking the Show Saturation icon when you have a live image will show if the camera is over exposed.
13) Saturation detection
Red coloured shading indicates overexposure of white. Blue coloured shading indicates underexposure. The optimum exposure time is one showing no red shading. The G:BOX Chemi HR16 has a very wide dynamic range which means that higher levels of light can be detected by the camera before it reaches saturation point. To check for saturation on a grabbed image, click on the Histogram icon.
A graph will be displayed indicating which grey shades have been detected by the camera, from 0 4096 (or 0 - 65535 if EDR has been used). Providing that all areas of the image are within this range, it is within the linear range of the camera, and quantification may be performed with confidence. Please note that for dyes that have a lower dynamic range compared to the camera e.g. silver or coomassie then it will not be possible to utilise the full dynamic range of the camera. Auto exposure - G:BOX Chemi HR16 has a unique auto exposure system leaving the software to decide the optimum camera settings. The image will have no saturated areas and is fully quantifiable. This setting should only be used when capturing images of fluorescent samples. It is especially useful when the gel is dark or if the bands are very faint. To activate this function simply click on the AUTO EXPOSE button.
14) Auto exposure
Depending upon the gel this can take several seconds until the camera has found the optimum settings (a progress indicator shows the automatic exposure control is operating). Once it has completed the process, the "grabbed" image is displayed in the image window and the lighting is automatically switched off. Chemiluminescence imaging tips Place the sample on the centre of the UV transilluminator or visible light converter. It is recommended that you place the membrane between two sheets of acetate film.
15) Chemiluminescence imaging tips
Ensure that ‘No light’ and ‘No filter’ are selected, and that the iris setting is open to the maximum (alternatively choose the Chemi configuration). Set the zoom so that the sample fills the image area. You may wish to select one of the binning modes - see above. Now select the ‘Series Capture’ button from the image capture window.
Select the required number of frames and whether or not these should be added (adding is recommended). Exposure time may be entered individually for each frame or the same time used throughout. A single long exposure may be set if required. GeneSnap will display your series of images - simply select that which is most appropriate, which should be the one with the largest dynamic range but containing no saturation.
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Printing - When an image is "grabbed”, the PRINT icon is available. Click this to send a copy of the image to the thermal printer for a hard copy output.
16) Printing
Saving the image - You have the choice of either selecting FILE/SAVE AS or FILE/EXPORT. In the first case, the image can be saved in the Syngene secure SGD format for true GLP. With the second option, the image can be saved in a range of formats including TIF, BMP, GIF, PCX, TGA, WPG, and JPEG. Images can then be exported to other software packages.
17) Saving
UV Safety override -There are occasions when you may want the UV transilluminator to remain on when the door is open, in order to cut bands out of the gel. Therefore, the safety interlock will need to be overridden. This is achieved by pushing in the black safety interlock switch for 2-3 secs (a click will be heard), which is located approximately 2cms below the green LED.
18) UV safety override
Note - Please take adequate precautions to ensure that the UV light cannot enter your eyes or anyone else’s by wearing the appropriate protective eye or face shield. Note - The interlock will be re-set when the door is closed. * If both the focus indicator and increased effective pixel features are not present then install GeneTools software as these features are licenced with the analysis software.
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