Fluorescence microscopy image formation


Before starting this lesson, you should be familiar with:

Learning Objectives

After completing this lesson, learners should be able to:
  • Understand how the intensities in a digital image that was acquired with a fluorescence microscope are formed

  • Understand how this image formation process has critical influence on the interpretation of intensity measurements

  • Understand that the geometry of your biological specimen can have a large influence on the measured intensities


In bioimage analysis one often wants to quantify the intensities in an image. To do this properly one needs to be aware that these intensities are influenced by many factors, making intensity quantification in general very difficult. Sometimes the measured intensities can be affected so much that even object shape measurements can become difficult. For all those reasons it is very important to understand the reasons for signal distortion! Not knowing those effects can easily lead to wrong measurements.

Concept map

graph TD Light -->|interacs with| Sample -->|forms an| Image


Membranes in a zebrafish embryo, imaged with a confocal microscope. The optical axis runs from bottom to top. One sees that membranes appear more contrasty if they run along the optical axis. One also sees that the signal gets dimmer further inside the specimen. In fact, here only the outer-most cell layer is clearly visible.


Confocal signal at membranes and inside a large specimen

Show activity for:  

ImageJ GUI

  • Open the mentioned image(s)
  • Use Plugins › BigDataViewer › Open Current Image to inspect the image from different angles
    • Use Shift + Y to look along the y-axis
    • Use the mouse wheel to move along the y-axis
    • Appreciate the mentioned effects on the membrane intensities

No optical sectioning in widefield microscopy

Show activity for:  

ImageJ GUI

  • Open the example image
  • Place a ROI around the object in the image
  • Use Image › Stacks › Plot Z-axis Profile to measure the mean intensity at each z-position
  • Compute the change in intensity in percent from the brightest to the dimmest plane: %change = 100% * (max - min) / (max - bg)
    • Do this for both the confocal and the wide-field channel and compare the results
  • Repeat, now using a much larger ROI such that all the blurred wide-field signal is always included in all z-planes


Fill in the blanks

  1. In fluorescence microscopy the signal quality typically ___ when imaging deep inside a specimen.
  2. In confocal microscopy elongated structures that align with the z-axis typically appear ___ than elongated structures that align with the x or y-axis.
  3. In wide-field fluorescence microscopy there is no ___ and thus signal intensity quantifications for one specific z-position are typically not possible.


  1. decreases
  2. brighter
  3. optical sectioning


Follow-up material

Recommended follow-up modules:

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