CTAC / ICBR SOP : Controlling the Light Captured During a Fluorescent Scan Using the IVIS Software Control Panel

 

Title: Controlling the Light Captured During a Fluorescent Scan using the IVIS Software (Living Image™) Control Panel

Materials Required:

  • Computer
  • Living Image™ software

Purpose:

To detail the exposure, F stop and lamp intensity controls and their effects on light detection during a scan for fluorescence.

Software component identification:

Light Controls

The elements that control the amount of light that can be detected are indicated in yellow.  These are, left to right:

Exposure Time:

This is the length of time set for the scan by the user.  Using the UP and DOWN arrows, the value may be changed by increments of 1.00.  More discrete values can be entered manually, by highlighting the numerals intended for change and overwriting them.

The unit of measure may also be changed by toggling the drop down control to toggle between seconds and minutes, dependent on the expected signal strength.

F Stop:

This value is a standard photography term to refer to the size of the aperture for the camera, which is like the iris in the human eye.  Its function is to admit or reduce the amount of light entering the camera.

A low F Stop value is a wider, more open aperture, which will capture more of the available light.  This is useful when the signal strength is too low.

A high F Stop value is a narrower, more restrictive aperture which will capture less of the available light.  This is useful when the signal strength is too high.

F stop

As can be seen, the capture area is different for each setting.  The most open setting of the aperture is F1.  All other aperture settings are relative to this standard.  Therefore, each is a Factor (F) of division relative to F1.  F2 is 50% of the capture area of F1.  F4 is 25% of the capture area of F1.  F8 is 12.5% of the capture area of F1.

Lamp Level

The lamp providing the light spectrum can be set to 4 positions, of which 3 are user selectable.

Off – ensures lamp is off

High – ensures lamp setting is on high intensity

Low – ensures lamp setting is on low intensity

Inspect – for instrument service – CTAC staff only.

 

Relationships

In order to optimize scanning, the inter-relationship between exposure, F stop and light intensity should be understood.

Exposure – determines the length of time photons are able to be detected.

F stop – determines the amount of photons that can be captured over a given unit of time.

Lamp level – determines the rate at which photons are made available for detection.

The best analogy for remembering this interdependency is to consider a plumbing system.

Light Control example

The PUMP is analogous to the Lamp Level – this determines the RATE at which photons are transmitted into the detection chamber from the instrument source.  It has controls to have it OFF, or at one of two different SPEEDS.

The pump’s output can be directed into one of four different widths of pipe.  This is analogous to the control of the F stop, where each F stop value is a different pipe width.  The bigger the diameter of the pipe (F stop), the more photons can flow through for a given length of time.

The VALVE in the system is analogous to the EXPOSURE control, and represents the amount of time that the photons are allowed to flow.

All of these elements are INTERDEPENDENT, and can have their values changed in different ways to arrive at the same level of energy capture.  Imagine if a scan is completed with a given set of parameters. If the scan is repeated but light is halved in intensity, and the exposure is doubled, a similar number of photons will be detected at the endpoint.  Understanding the interdependence allows the operator more flexibility in controlling the available light available for the camera to capture.

Transillumination:

While this is also a light control, it is the toggle for transillumination, which is where the excitation light source is located below the stage.  This is not a control for light level or intensity, only for light direction.