Seminar highlights: Novel developments in confocal microscopy

Published on
November 30, 2017

About 60 participants showed interest in the latest developments in Fluorescence Microscopy during the seminar on 20 November. The working and applications of the Spinning disk microscope, Confocal microscope and the Single particle tracking were the topics that were presented. One of the areas highlighted was in improving resolution of scans and images, e.g. by using mathematical restoration techniques and easier use through computer optimising settings and conditions.

In the introduction Daniëlla Stijnen outlined the activities and aims of WUR’s Shared Research Facilities. activities, such as stimulating facility sharing, supporting accessibility of research equipment within WUR and investing in advanced research equipment.

Spinning Disk Microscope

Following this introduction, Marcel Janson showed in his presentation how the Spinning Disk Microscope (SDM) is used to visualise motion in live cells or soft matter, e.g. colloidial systems. Advantages of SDM are the faster image acquisition speed (up to about 100 images/second if needed) and in particular a decreased rate of photo bleaching.

SDM can be combined with FRAP, Photo activation, and sub-cellular photo ablation .but is less well suited for spectral-analysis or fluorescence life time measurements. Spinning Disk microscopy is optimised to perform live cell imaging but the scanning confocal layout (CLSM) offers more flexibility to increase spatial resolution.

Confocal Microscopy

Jan Willem Borst’s presentation put the Leica SP 8 Multimode Confocal Microscope (MMCM) central. The MMCM uses different lasers, has a sensitive detection and an improved resolution through pinhole technology. Multiple fluorophores (fluorescent chemical compounds) are available for qualitative or quantitative analysis. The output scans can be combined to form a 3D image of the studied structure.

Developments in these techniques tackle the resolution ‘problem’, as the lower limit of the microscopy is ~200 nm. Through mathematical restoration the resolution can be improved and by making use of optimal computer settings the visibility of expression levels of proteins can be increased.

Super-resolution Microscopy

Johannes Hohlbein also focused on image resolution during his presentation. Super-resolution Microscopy allows images to be taken with a higher resolution, thereby making it possible to localise a single molecule at ~10-20 nm. Different techniques (e.g., PALM and STORM) can be used to make several close-by fluorophores to emit light several times giving the opportunity to track the mobility of a single molecule.

At the end of the seminar the audiences’ questions were related to opportunities fluorescence microscopy has for different research fields. To find out more about the different techniques and microscopes please take a look at the presentations. Or take a look at the Shared Research Facilities website for more facilities in the fields of Imaging, NGS and Mass spectroscopy.