In microscopy, image quality is not only limited by optics, but also by how finely the image is sampled.
Even a perfect microscope can produce misleading data if the sampling is inappropriate.
Under-sampling throws away biophysical information that can never be recovered, while over-sampling creates the illusion of higher resolution at the cost of time, light dose, and storage. Understanding image sampling means knowing how much information your experiment actually contains — and how much you really need to record.
Prerequisites
Before starting this lesson, you should be familiar with:
After completing this lesson, learners should be able to:
Explain how spatial and axial sampling discretize a continuous optical image.
Identify under-sampling artifacts such as aliasing and loss of structural information.
Recognize over-sampling and its practical costs (data volume, phototoxicity, acquisition time).
Understand the optimal sampling depends on the biological question.
Concept map
graph TD
A[Continuous Optical Image] -->|Sampling| B[Discrete Pixels & Voxels]
B --> C{Sampling Regime}
C -->|Under-sampling| D[Aliasing & Information Loss]
C -->|Optimal sampling| E[Faithful Representation]
C -->|Over-sampling| F[Redundant Data]
F --> G[Longer Acquisition / Photobleaching]
D --> H[Biased Measurements]
Figure
Fluorescent nuclei, acquired at different spatial sampling; scale bar 5 micrometer. Left: Intranuclear structures can be investigated; dividing cell can be clearly identified. Middle: Intracellular structures are not visible but the number of nuclei could still be measured. Right: Nuclei in the top left start to blur into one object, rendering cell counting challenging. **TODO: Replace this by a proper image**
A good way to explore what one can still see at different spatial samplings is to acquire an image with very fine sampling and then downsample it in a software. This can inform you how you would then acquire more images with optimal sampling of the microscope.
Open the image (TODO: A heavily oversampled image could be good to start, maybe confocal image with 5 nm pixel size)
Perform a sequence of down-sampling operations and observe which biological structures you can still observe at the various sampling levels
Show activity for:
ImageJ Macro
TODO
Assessment
Fill in the blanks
If the pixel size is larger than half the width of the PSF, the image is _____-sampled and fine spatial detail is _____.
Over-sampling does not improve true optical resolution, but it increases _____, _____, and _____.
