Imaging thick biological samples

When imaging thick samples, one needs to be aware that intensities are influenced by many factors, making intensity quantification in general very difficult. Measured intensities can be affected so much that even object shape measurements can become difficult. Already, the nature of the PSF makes measurements along the Z-direction different than in the XY plane, biological specimens can be more or less opaque (scattering, refracting index mismatch, absorption) making measurements deeper in the sample challenging. 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 and conclusions.

Prerequisites
Before starting this lesson, you should be familiar with:

Digital image basics

Point spread function

Optical sectioning

Learning Objectives
After completing this lesson, learners should be able to:

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

Concept map

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

Figure

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.

Activities

Confocal signal at membranes and inside a large specimen

Open the zebrafish embryo confocal image
Inspect the image along various axes and appreciate the following:
- Membranes appear with more contrast if they run along the optical axis
- Signal intensities become weaker to in the specimen’s interior, due to scattering and absorption
Appreciate the cell segmentation (and morphometry) in the specimen interior are challenging/impossible
Appreciate that intensity quantifications under such circumstances are highly challenging/impossible

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

Assessment

Fill in the blanks

In fluorescence microscopy the signal quality typically ___ when imaging deep inside a specimen.
In confocal microscopy elongated structures that align with the z-axis typically appear ___ than elongated structures that align with the x or y-axis.

Solution

decreases

brighter

Follow-up material

Recommended follow-up modules:

Object intensity measurements

Spherical aberrations

Learn more: