Question

Answer

When SmartCapture was originally designed, it was assumed that there was always going to be something in the image to capture. To this end, SmartCapture's automatic exposure and automatic normalisation will conspire to reveal something in the image even when nothing is there!

Overriding Auto Exposure

Consider a sample where there is Dapi metaphase and a Fitc labeled probe. With the automatic exposure enabled, SmartCapture will keep increasing the exposure time until the camera has an optimum exposure for each of the Dapi and Fitc channels. Obviously if there are no Fitc probes present, the exposure algorithm will simply keep increasing the exposure time for the Fitc until it finds an optimum exposure for the residual background noise in the Fitc channel.

The obvious answer here, is do disable automatic exposure where you have the possibility of signals being absent in a given channel. That just leaves the question of what manual exposure should you use? Fortunately this is easy to establish. Just take a picture of a sample that does have the signal present using automatic exposure. Once the picture has been taken, the exposure values used for that picture will be recorded in the capture settings palette. All that is necessary now is to switch off the automatic exposure. and then these exposure values can be reused for the subsequent captures. A good tip is to half the recorded exposure time. Doing this will help ensure that subsequent pictures, even if the sample is a little brighter, will result in underexposed pictures rather that overexposed pictures. Due to the capabilities of your camera, underexposure is far better than overexposure.

Overriding Auto Normalisation

There is a second opportunity for SmartCapture to enhance a captured picture that works against you when there is no fluorescence subject matter present. If you are using an integrating camera like the Sensys or Nu200, the camera will be recording the image with up to 4096 levels of brightness between darkest black and brightest white. SmartCapture by default, scales this down so that there are only 256 levels of brightness. For most circumstance this provides more than enough graduation and it results in images that can be stored in half as much disk space. The algorithm it uses to scale the 4094 (exact number depending on camera) levels to 256 levels is to take the minimum value of the captured data and map this to 0, and the maximum value of the captured data and map this 256. All values in between the captured minimum and maximum are mapped proportionately. In this case, even if we have a fixed exposure time, and there is no signal present in the image, this automatic normalisation causes the image to be scaled in such away that the brightest part of the low level background gets to be represented at full brightness!

The answer is to disable the "Save as 8 bit" option in the capture palette

The Recipe

If you want to take pictures of samples in which fluorescence signals may be completely absent follow this recipe:

1. Open the Capture Settings Palette and make sure that the the auto exposure and 'Save as 8 bit' are checked
   
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2. Locate an example that has a presence of signals in each fluorescence plane.
3. Take a picture.
4. In the capture settings palette, switch off auto exposure for all of the planes to be captured, and type in exposure times that are approximately half of that which was recorded for the previous exposure.
5. Switch off 'Save as 8 bit' in the capture settings palette
   
   
   
   
6. Now you can proceed to capture your remaining images with these manual capture settings.

The Final Step

When you come to view the images, take a look at the histograms in the image palette. Locate an image that has signals present in all planes. Adjust the sliders for each of the channels to achieve the desired image balance. Make a note of where the image slider positions are and then use these same positions for all subsequent images.

A Helping Hand with the Final Step

SmartCapture has a script that can copy the settings from the image palette and apply them to all the images in the open filmstrip. The only problem here is that it copies the normalisation levels in a way as to make all of the pictures have a similar appearance. A modified version of this script that uses the absolute numerical value of the normalisation levels is given here. Download this script and install it on script button. See the FAQ on Scriptable Button Palette to learn how to install scripts. Open an image which has signal present on all channels and adjust the sliders as required. Then with this image still open run this script and the normalisation levels should be set correctly for every image in the filmstrip!

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