Adjusting Fluorescent Images

Processing a fluorescent image mainly consists of adjusting the background and the brightness to improve the quality of the image. Cytosketch provides tools that make adjusting fluorescent images substantially faster, easier and more consistent.

The adjustments to the image are non-destructive, this means that the adjustments do not degrade the quality of the image and it means that you can always reset the adjustments to the original values.

This section describes the various ways to adjust fluorescent images in Cytosketch.

Table of Content

Fluorescent Image Controls Overview

This section provides a quick overview of the controls that are available in the Adjust Image box when a fluorescent image is selected. Move the mouse over the screen shot below to see a short description of the individual controls or over the description to see the corresponding controls on the screen shot.

Fluorescent Tool merge heatmap Color smooth background brightness channel reset auto apply
  1. Indicates the selected image as being a merged image.

  2. Determines if the selected images are displayed as a heat map. The heat map is useful for better visualizing co-expression in three and four color images, because each color in the heat map represents a different combination of co-expression.

    See the images to the right for an example of the difference between displaying the image as a heat map and as a fluorescent image. Heat Map Off Heat Map On
    Fluorescent Image Heatmap Image
  3. Button to change the color of the channel.

  4. Button to toggle smooth brightness. By default smooth brightness is on so that images adjusted in Cytosketch look the same as in Photoshop.

    You should turn the smooth brightness off, if you want to quantify the fluorescent. Have a look at the two images to the right for the difference between smooth brightness turned on and off.

    		Smooth On Icon Smooth Off Icon
    Smooth On Icon Smooth On Icon
  5. Controls to adjust the background of the selected images.
  6. Controls to adjust the brightness of the selected images
  7. Button to toggle the visibility of the channel.
  8. Resets the adjustment values back to the original values.
  9. Automatically adjusts the background and the brightness of the selected images.
  10. Applies the current adjustment values to the rest of the selected images.

Automatically Adjusting Images

It's easy - select the images to adjust and click the Auto button Auto in the Adjust Image box or press Ctrl + G simultaneously.

Automatically Adjusting Brightness

The brightness of a fluorescent image is generally increased to make weak fluorescence signal visible. This section describes how to adjust the brightness using the automatic brightness function.

  1. Select the images to adjust, go to Image and then select Auto Brightness or press Ctrl + B simultaneously. The brightness of all visible channels is automatically increased.
  2. To further increase the brightness use the Area Brightness Tool Brightness Tool or fine tune each channel individually using the controllers in the control box.
  3. Brightness Controls

Adjusting Brightness using Area Brightness Tool

If the automatic brightness function did not increase the signal well enough, follow the steps below to further increase the brightness using the Brightness Tool Brightness Tool.

  1. Activate the Brightness Tool by clicking the Brightness Tool button Brightness Tool or pressing the B key.
  2. Move the cursor to the area that needs to be brighter.
  3. Click on this area and the brightness of all visible channels is increased based on the brightest pixel in the selected area.
  4. To fine tune each channel individually use the controllers in the control box.
  5. Brightness Controls

Tip:

To easily adjust the brightness with the area brightness tool, only work with one channel at a time. Click the eye icons Eye in the Adjust Image box to turn channels on and off.

Automatically Adjusting Background

This section describes how to remove background fluorescent using the automatic background function.

  1. Select the images to adjust, go to Image and then select Auto Background or press Ctrl + T simultaneously. The background of all visible channels is automatically adjusted.
  2. To further remove background use the Area Background Tool Background Tool or fine tune each channel individually using the controllers in the control box.
  3. Background Controls

Adjusting Background using Area Background Tool

If the automatic background function did not remove the background well enough, follow the steps below to further remove background using the Background Tool Background Tool.

  1. Activate the Background Tool by clicking the Background Tool button Background Tool or pressing the T key.
  2. Move the cursor to the area with background fluorescence.
  3. Click on this area and the background of all visible channels is removed.
  4. To fine tune each channel individually use the controllers in the control box.
  5. Background Controls

Tip:

To easily adjust the background with the area background tool, only work with one channel at a time. Click the eye icons Eye in the Adjust Image box to turn channels on and off.

How to Get Back to the Original Image

  1. Select the image to reset.
  2. Click Reset in the Adjust Image box and the adjustments values are reset to the original values.

Changing the Color of a Channel

  1. Select the image by using the selection tool Selection Tool.
  2. Click the Color button Changing Color in the Adjust Image box and select a new color from the list.
  3. Color

Turning Individual Channels On and Off

  1. Select the image by using the selection tool Selection Tool.
  2. Click the Eye icon Eye in the Adjust Image box to turn the channels on and off.
  3. Channels

Tip:

To quickly turn channels on and off use the keyboard shortcuts. You can find a list of all available shortcuts on the Reference Sheet page.