iXon EMCCD Camera Settings
As described in the Image Mode and Feature Location section, device settings can be moved between Channel, Global, Image Mode and Advanced. Therefore, the settings described here may be found in a different location depending on the customisation / user of your Fusion installation.
Channel Settings
Exposure Time - this is the time that light will be collected on the sensor. Lower values will provide higher speed, but lower signal:noise / image quality / brightness / less total laser exposure to the sample. A starting value for this might be 100 ms.
Global Settings
EM Gain - Higher values will generally provide better signal:noise / brightness. A suggested starting value is 100. This is because the approximate read noise of an EMCCD is 100 electrons. The purpose of EM Gain is to multiply the signal before readout, therefore effectively eliminating read noise. The maximum is 300, but can be extended to 1000 using the EM Advanced option in the Advanced features (see Advanced Settings section below). The specification sheet supplied with your Andor camera will provide exact values for the pixel noise (read out noise).
Overlap - (Default OFF) When enabled, the exposure time and readout will happen in parallel, increasing the speed of acquisition. Note that this feature is only used in a Finite Burst protocol. In all other cases, it will not be used. When enabled and a Finite Burst protocol is used, the exposure time will be limited by the Readout Time (see Advanced Settings below).
Frame Averaging - (Default 1) When set to values higher than 1, this will produce an image of the average of a number of frames. Its main purpose is to reduce spurious noise in the image. However, it would be better to increase the Exposure Time (unless this would lead to saturation of the image). Both options will decrease the speed, but increase the signal:noise / image quality / more total laser exposure to the sample.
Pre-defined AOI - (Default - full chip size) Allows the selection of a smaller AOI (Area of Interest) for imaging. This will reduce the size of the image (both on-screen and in total data size); allow lower exposure times to be used. It will not affect the area of the sample being illuminated - only the area being imaged.
Symmetrical Binning - (Default: ON) When on, the X and Y values for binning will the same.
Binning - (Default 1x1) Binning allows the image to be reduced in XY resolution by combining (binning) pixels on the camera chip. Increasing values will lead to lower resolution images, smaller data sizes and shorter exposure times to be used. Binning will also increase the signal:noise of the image (for example, using 2x2 binning will increase the signal:noise by a factor of four. Note that this same improvement does not apply for sCMOS cameras - see Zyla sCMOS settings for more information). Fusion will automatically adjust the exposure time to maintain the image intensity. See Scale Exposure with Binning option in the Advanced Settings section (below).
Advanced Settings
Sync Exposure Time - (Default ON) When used with a confocal system, such as the Andor Dragonfly, this option will set the exposure time to match the image scanning speed (based on the rotation of the spinning disk). Assuming a default rotation speed of 6000 rpm on Dragonfly, this will cause the exposure time to be rounded to the nearest 2.5 ms. Switching this setting off may lead to image artefacts and/or scan lines.
Readout Time (ms) - This is a read-only value and shown for information only. It is most useful when using Overlap in a Finite Burst Protocol (see Global Settings section above). Because Overlap will limit the exposure time to be no lower than the Readout Time, it can be useful to see this value. If Finite Burst is frequently used, this setting could be moved to the Global Settings section for easily visibility. See the Image Mode and Feature Location section for more information.
EM Advanced - (Default OFF) As described in the Global Settings section, this will allow EM Gain values greater than 300 to be used.
Pre Amp Gain - (Default highest value available) The Pre Amp Gain is most easily thought of as a conversion factor. Higher values will result in less photons being needed to produce a count, or intensity value, on the screen. For fluorescence microscopy, the light levels are typically quite low, therefore a high Pre Amp Gain is advised. The specification sheet supplied with your Andor camera will provide figures for how many photons are used per count for each Pre Amp Gain.
Is Full Sensor - (Default ON) This is linked / related to the Pre-defined AOI setting (see Global Settings section above) and the AOI settings (see below). When ON, it will reset the camera back to full chip mode.
AOI Width / Height / Left / Bottom - (Default is full chip resolution) These settings allow the AOI to be adjusted by single pixels. See also the Pre-defined AOI (Global Settings, above)
Scale Exposure with Binning - (Default ON) As described in the Global Settings section (see above), this will automatically adjust the exposure time when the binning is changed.
Fan Mode - (Default ON (Full) This allows the cooling fan in the camera to be slowed / stopped. It is strongly advised not to adjust this setting unless water-cooling is fitted to the camera. Changing this setting when using only air cooling may result in the camera overheating. It is unlikely to damage the camera, but will result in an overheating alarm to be triggered, requiring the software to be restarted and leaving the camera to cool before restarting.
Temperature Set Point - (Default -50 C) This allows the camera's target cooling temperature to be changed. Similar to Fan Mode, it is not recommended to adjust this as lower values may result in the camera overheating. However, for advanced users, lower temperatures will result in lower dark current noise, which can be useful at long exposure times (e.g. 1 second or more).
Output Amplifier - (Default Electron Multiplying) - This feature will depend on the model of camera being used. Some cameras provide additional readout modes, allowing the EM Gain circuitry to be bypassed. This can be useful if EM Gain is not needed. Similar to the Temperature Set Point, this feature will generally only be used by advanced users who require very long exposure times.
Horizontal Shift Speed - (Default - Highest available). In general, the highest value available should be used. Advanced users who require longer exposure times may find that decreasing the shift speed will reduce the read out noise. Lowering the Shift Speed will dramatically increase the Readout Time.
Vertical Shift Speed - (Default - Second largest value available). Numbers in square brackets indicate that this speed is outside of the operating specification of the sensor. However, due to the advanced electronics used in Andor cameras, most cameras will operate correctly using the largest number in square brackets. This is usually the second largest (slowest) value. Lower values will increase the vertical shift speed, which can dramatically reduce the Readout Time, particularly when using Binning or reduced AOIs. However, increasing the vertical shift speed (using lower values) may result in image artefacts such as "tearing" or "blurring", particularly towards the centre of the image. These effects may be resolved by adjusting the Vertical Clock Voltage (see next).
Vertical Clock Voltage - (Default NORMAL). When increasing the Vertical Shift Speed to achieve lower Readout Times (especially with Binning and smaller AOIs) and therefore allow shorter exposure times and higher acquisition speeds, it may be necessary to increase the Vertical Clock Voltage to resolve the image artefacts described in the Vertical Shift Speed section above. However, increasing the Vertical Clock Voltage will increase the likelihood of spurious noise (generally single pixel bright spots) appearing in the image.
Trigger Mode - (Default NORMAL). This is an advanced feature that allows the camera's Ext Trigger input pin / cable to either Start an acquisition, or take a single Exposure.
External Trigger Timeout - (Default 10 seconds). Related to the Trigger Mode, this is the length of time the software will allow before producing an error message if it does not receive a trigger on the camera's Ext Trigger pin / cable.
Mapping Minimum Value - (Default - depends on the camera). Related to the Mapping of the image. This sets a minimum value that is allowed when using the mapping tools to control the on-screen brightness. It has no effect on the raw data / intensity of the image.
Mapping Minimum Range- (Default - depends on the camera). Related to the Mapping of the image. This sets a minimum range that is allowed when using the mapping tools to control the on-screen brightness. It has no effect on the raw data / intensity of the image.
Shutter Open - (Default ON). Allows the camera's shutter (if available) to be manually opened and closed. This is potentially useful for capturing an image where no light is falling on the sensor.