Linearity Settings for your Imaging System

We’ve seen it time and time again…. imaging systems are not installed by manufacturers in a linear state, or user settings on the camera software have been modified and, unknowingly, created a non-linear state. Why does linearity matter?

CHROMACAL is not camera software, so CHROMACAL cannot correct the settings within your imaging system. However, after you capture an image of the CHROMACAL slide and open the image in CHROMACAL, the software can diagnose the state of your imaging system. For the quality and consistency of the image data, this is critical for you to know before your specimen images are captured. Ideally, you want the CHROMACAL diagnostic result for linearity to be either “Good” or “Within Tolerance.”

Presented below is General Guidance for setting and using your imaging system. In addition, Specific Guidance is provided on an assortment of common camera software.

General Guidance:

  • White balance once at beginning of imaging session, then do not adjust light intensity on the microscope again during that same session.
  • Use camera exposure or shutter control to adjust image brightness.
  • Use a histogram within the capture software to ensure all color channels are within range (saturation = lost data!).
  • Do not adjust contrast, saturation, hue or gamma unless such guidance is provided below.

Specific Guidance:

Note: CHROMACAL is camera equipment agnostic.  The table below provides guidance on common camera software.

Camera Software and Version Camera
The following camera / software combinations generally deliver linear images, but click “View Info” to confirm settings:
Lumenera Infinity Capture v6.x Lumenera Infinity1, Infinity2, Infinity3
View Info
Lumenera Infinity Analyze v6.x Lumenera Infinity1, Infinity2, Infinity3
Motic Images Plus v2.x Moticam 3
QImaging QCam v2.x QImaging QIClick with QImaging RGB filter
QImaging QCapture Pro v6.x, v7.x QImaging QIClick with QImaging RGB filter
The following camera / software combinations generally require additional acquisition settings adjustments, so View Info for specific details:
Diagnostic Imaging SPOT Basic and Advanced SPOT Idea, SPOT Insight2, SPOT Insight4, SPOT Flex
View Info
Leica LAS v2.x Leica DFC290
View Info
Nikon NIS-Elements v4.x Nikon DS-Fi1 / DS-Fi2 / DS-Ri2
View Info
Nikon NIS-Elements v3.x Nikon DS-Fi1
View Info
Olympus cellSens (Entry, Standard, Dimension) Olympus DP25
View Info
Olympus DP27
View Info
Olympus DP73
View Info
Zeiss AxioVision (release 4.x) Zeiss AxioCam MRc5, AxioCam 503 color, AxioCam 506 color
View Info
Zeiss Zen (v2012, v2), Zen Lite (v2012, vBlack and Blue) Zeiss AxioCam MRc5, AxioCam 503 color, AxioCam 506 color
View Info
CHROMACAL cannot be used with the following cameras (generally, the acquisition settings of the cameras cannot be adjusted to capture linear images):
Lumenera InfinityHD

Contact us at to share your experiences with other imaging systems.

Checking Linearity within the CHROMACAL Image Software:

After opening your CHROMACAL slide image in the CHROMAL Image software, perform the following basic checks:

  • Be sure that brightest white patch in the CHROMACAL color matrix is located in the lower right-hand corner of the CHROMACAL Calibration Profile window. If not, use the Cropping Tools (Flip Tools) to rotate the matrix until properly oriented.
  • The CHROMACAL color matrix should also be aligned horizontally; if adjustment is needed, use the Cropping Tools (Rotate Tools) to correct.
  • Once the color matrix is in proper orientation, check that the grid squares are properly aligned within each color circle in the matrix. If not, use the adjustments available as you move your cursor over each square.

After the above steps, check your linearity diagnostic result. If the linearity result is “Good” or “Within Tolerance”, you can proceed with color calibration.

If the linearity result is “Poor”, the issue is likely caused by the settings in your camera or camera acquisition software, or the capability of your camera. To correct, follow the General Guidance and Specific Guidance provided above.

Why does the linearity of my imaging system matter?

In microscopy, the relative darkness of stained cells and tissue is an indication of the greater (or lesser) effect on the tissue and cells. If an image is linear, the darkness/brightness levels (“grey levels”) have equal increments from complete black to white; therefore, an evaluation of relative intensity is valid. If an image is non-linear, the grey levels are not equal, and therefore the result can be deceiving visual data. Moreover, when these images are measured for darkness levels, the data is inaccurate!

Many journals require that non-linear images be reported as an alteration of gamma. When non-linear images are published without the necessary citation, authors do not comply with publisher’s requirements, and they unintentionally commit “scientific misconduct.”

Without ensuring a linear baseline, the images produced by your digital microscopy systems are based on the capabilities (and limitations) of the underlying equipment, and also subject to color interpretations as independently defined by each hardware/camera manufacturer. Further, there is image variability introduced by the microscope and camera software settings, and these user-controlled parameters can often be modified without awareness by other users of the same equipment.

Without ensuring a linear baseline and establishing a color standard, the consistency and reproducibility of your images is unknown, which means that the integrity of the image color data (which is scientific data) cannot be assumed and it should not be relied upon.

By providing a linear diagnostic tool and a color standard, CHROMACAL delivers high-quality images with the consistency and reproducibility demanded by science.