Processing

The Processing Panel provides the opportunity to enhance images during focus and acquisition by incorporating image-processing operations during or immediately after image Capture. The operations may be at frame rate for some Capture Devices, or may be applied as a sequential step for others. Some operations require multiple images to be acquired and combined before the result is displayed.

Image Processing Operations

To select an Image Processing operation, first expand the Process Pane and then select the Operation Type. Rolling Average and Frame Integration are used for noise reduction. Use the image arithmetic functions like Shade Correction, Background Subtraction or Image Subtraction to remove artifacts from the incoming image. Clicking Capture1 will initiate image capture with the selected image processing operations applied.

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Rolling Average

Rolling (or Temporal) Averaging works by first capturing a single live image, and then adding sequential live images to the original image weighted by a fraction of their intensity. The effect of this is to create a rolling average of the image coming from the capture device over time.

Images viewed using Rolling averaging are optimized over a period of time related to the weighting factor. If a large weighting factor (e.g. ¼) is used, each new image has a big impact on the image being accumulated. A small weighting factor (e.g. 1/64) allows images resulting in a very low noise component to be built up over a longer period of time. Typically for low noise images a small weighting factor can be used (e.g. 4), but for extremely noisy images a large weighting factor can be used to good effect (e.g. 30 - 60). If the subject moves during accumulation of the image, it will appear to fade away, and then reappear at its new location. This makes Rolling Averaging unsuitable for moving objects because the display seems sluggish and blurred.

As a guide to the time required to completely optimize a live image using Rolling Average, the weighting factor approximates the number of frames necessary to average, e.g. a weighting factor of 1/64 will require approximately 64 frames to produce a fully averaged image. This may be an under-estimate when moving from a bright object to dark objects, as the image intensity change from weighting a dark object is less than a light object. The best criterion is the visual one, if the noise speckle in the image has been removed sufficiently to make the image look good, then click the Abort button in the Capture Pane. The numerical weighting value can be entered in the adjacent slot in the form.

Frame Integrate

A selected number of images are acquired and the accumulated image intensities are displayed. The number of images used (1 - 50) may be changed in the box on the right labeled Frames.

This has two effects:

The image intensity can be significantly increased, which is useful for very dim images, which cannot be increased by other means.
The image noise can be somewhat smoothed as we are combining a number of images each with random noise, that will cancel itself out and produce more of a typical result than a single image would. The integration process only updates the display every N frames, giving a stop-action feel to the display.

Care should be taken with the number of frames to integrate. Large numbers of frames can take a long time to acquire, especially for cameras that have a slow exposure or data transfer rate. If too many images are integrated, the image itself may saturate at the bright end of the scale, reducing the image data that can be discriminated. In this case, use Auto Integrate first, which will sense the approximate number of frames to use to generate a good image.

Auto Integrate

Auto Integration is similar to Frame Integration in that a number of images are accumulated, but Auto Integrate will automatically stop when a value greater than the bit-depth of the capture device is detected. The number of frames is reported in the numerical value slot to indicate how many frames were used. If Auto Integrate is selected, each new image will be integrated on the basis of its image content. This may affect the accuracy of thresholding as set in the Identify Menu.

Shade Correction

If the illumination source for an image presents a bright non-uniform background, as is typical in bright field microscopy, applying a correction for this shading may be helpful for producing an image better suited for measurements.

The Shade Correction algorithm calculates a correction image in which each pixel's value is a positive integer which, when added to the corresponding pixel in the shaded image, increases intensity value of darker pixels (presumably more shaded) by a greater amount than the intensities of lighter pixels (which are presumed to have been more strongly illuminated).

Note: For Image Correction or Arithmetic, the user must first choose a source or background image. The image may be the current image saved in a buffer or one previously saved to disk. To use the current image, make sure Processing is OFF, select Buffer, click Capture and then select Shade Correction, Background Subtraction or Image Subtraction. Use the same method when using an image from Disk.

Hint: Enable Processing ON for correction image when you would like to capture a correction image using Rolling Average or Frame Integration. When you are ready to capture the correction image, select Rolling Average and enter the number of frames, enable Processing ON for correction image and then click the Capture button to the right of Buffer. The captured averaged image is stored in the buffer and ready to use a correction image.

Background Subtraction

If the illumination source for an image presents a dark non-uniform background, as is typical in fluorescence microscopy, it may be necessary to apply an image background subtraction to produce an image suitable for measurement.

Typically used in fluorescence microscopy, a background subtraction can be used when the image presents a dark non-uniform background. To perform a background subtraction click Live, bring the sample into focus and then move the stage off of the sample so that only the background is visible. Next, follow the steps below, when finished move the stage to bring the sample into view and the background subtraction is applied.

Hint: HCImage remembers the capture settings from the previous session, if background subtraction was left enabled, Process ON will be displayed in the Camera Control panel. The display image may appear distorted or black. Processing dialog

Note: This is different from an Image Subtraction which scales the subtracted intensities between +127 to -127 into the range of 0 to 255, indicating loss or gain of intensity.

Image Subtraction

If it is necessary to measure the difference between two images, from two different time intervals, or an experiment versus a control, we may need to generate an image showing intensity loss or gain. Using Image Subtraction, an image of the first instant is stored in memory, and subsequent live images from the Capture Device are subtracted from it before the result is stored in the display. This technique can be used for full gray scale image alignment between images.

When subtracting two images with gray levels from 0 to 255, we can arrive at gray levels of -255 to +255. In Image Subtraction, these gray levels are scaled into the resulting image by scaling gray level -255 equal to 0 (Black) and gray level +255 equal to +255 (white). This means a gray level 127, is equal to mid gray and reflects no change between the two images.

Note this is different than Background Subtraction which does a simple subtraction of the background intensities from the captured image then truncates negative values to zero.

In order to process the incoming live image with Image Subtraction, it may be necessary to miss some frames, and so the number of frames per second (fps) of the live image may be reduced. Use this option only if live Image Subtraction is necessary. If it is possible to subtract the images later, capture both images and then use Capture1 to capture the images available in the Enhance Menu, where they may be subtracted also.

Note: For Image Arithmetic, the user must first choose a source image. The image can be from Disk or the Current Image in the display. If the Current Image is chosen, it becomes the Buffered Image.

Subtraction/Addition

Split Image Registration is used in conjunction with the Optical Insights Dual View. The processed image in the display is split in two parts A & B. The left side displays (A - B) and the right side displays (A + B). This processing feature is not available in HCImage Live.