Sample preparation artefacts

When preparing samples for imaging, a series of steps to preserve and label the structure of interest are applied. These protocols are rarely perfect and depending on the type of fixation and labelling one obtain variable results. For example, certain structure may change their appearance, the biological sample may shrink, or some of the labels may not penetrate the sample. It is important to be aware how the sample preparation can affect the final imaging result.

Prerequisites

Before starting this lesson, you should be familiar with:

• Digital image basics

Learning Objectives

After completing this lesson, learners should be able to:

• Understand that sample preparation will affect the final imaging result

• Understand which type of artefacts can come from sample preparation

Concept map

Figure

Activities

Asses sample preparation

Compare different fixation protocols with respect to integrity of the structure and dimensions of the structure. Use orthogonal views if necessary. All samples have been stained for DNA (Hoechst, 405 excitation), Microtubules (primary and secondary antibody, 488nm excitation), Actin (Phalloidin, 561 nm excitation).

1. Methanol fixation xyzc_16bit__DNA_MT_actin_methanol.tif
2. Formaldehyde fixation xyzc_16bit__DNA_MT_actin_FA.tif

3. Formaldehyde and Glutaraldehyde fixation xyzc_16bit__DNA_MT_actin_FA_GA.tif

   What you expect to see

   1. Methanol fixation
      • Observation: Good staining of channel 1 (DNA), good staining in channel 2 (MTs) with nearly no background, no clear staining in channel 3 (actin). Along Z one can observe that the cell is flattened compared to the other protocols
      • Explanation: Alcohols (like ethanol and methanol) are coagulant fixatives that preserve cells by dehydrating them and precipitating proteins. This causes a collapse of the cell that is visible by comparing the nuclei along Z for the different protocols. Phalloidin only binds to the native structure of actin filaments and alcohols dissolve F-actin, therefore no actin labelling is visible
   2. Formaldehye fixation
      • Observation: Good staining of channel 1 (DNA), MTs appear disturbed and broken, good staining of actin
      • Explanation: In this sample the fixation via formaldehyde may have been too slow causing a deoplymerization of MTs. This image shows an extreme example, typically the disruption of MTs is much more subtle.
   3. Formaldehye and Glutaraldehyde fixation
      • Observation: All 3 structures look as expected. There seems to be more cytoplasmic background in the MTs channel than for the other fixation protocols.
      • Explanation: This protocol uses a small amount of Glutaraldehyde, a fixative also used in electron microscopy. GA crosslinks proteins faster (2-aldehydes groups) and so better fixes structures before they disassemble. It can create more autofluorescence with insufficient quenching. This may explain the observed background in the MTs channel.

ImageJ Macro

run("Close All")
open('https://github.com/NEUBIAS/training-resources/raw/master/image_data/sample_preparation/xyzc_16bit__DNA_MT_actin_methanol.tif')
open('https://github.com/NEUBIAS/training-resources/raw/master/image_data/sample_preparation/xyzc_16bit__DNA_MT_actin_FA.tif')
open('https://github.com/NEUBIAS/training-resources/raw/master/image_data/sample_preparation/xyzc_16bit__DNA_MT_actin_FA_GA.tif')

Assessment

True or False

1. Sample preparation has no effect on the imaging result.
2. The optical resolution depends on the sample preparation.
3. The signal to background depends on the sample preparation.

Solution

1. False
2. False
3. True

Follow-up material

Recommended follow-up modules:

Learn more: