CALM logo

Confocal and Advanced Light Microscopy Facility

CMVM, QMRI, UNIVERSITY OF EDINBURGH

UoE logo
subglobal1 link | subglobal1 link | subglobal1 link | subglobal1 link | subglobal1 link | subglobal1 link | subglobal1 link
subglobal2 link | subglobal2 link | subglobal2 link | subglobal2 link | subglobal2 link | subglobal2 link | subglobal2 link
subglobal3 link | subglobal3 link | subglobal3 link | subglobal3 link | subglobal3 link | subglobal3 link | subglobal3 link
subglobal4 link | subglobal4 link | subglobal4 link | subglobal4 link | subglobal4 link | subglobal4 link | subglobal4 link
subglobal5 link | subglobal5 link | subglobal5 link | subglobal5 link | subglobal5 link | subglobal5 link | subglobal5 link
subglobal6 link | subglobal6 link | subglobal6 link | subglobal6 link | subglobal6 link | subglobal6 link | subglobal6 link
subglobal7 link | subglobal7 link | subglobal7 link | subglobal7 link | subglobal7 link | subglobal7 link | subglobal7 link
subglobal8 link | subglobal8 link | subglobal8 link | subglobal8 link | subglobal8 link | subglobal8 link | subglobal8 link

Bioimaging

small logo

The application of fluorescence light microscopy

Light microscopic techniques have significantly developed over the past 20 years and now provide an indispensible tool to study molecular events at subcellular level in order to gain temporal and spatial information at high resolution. To achieve optimal results, it is essential to carefully plan and carry out microscopy-based experiments, which requires the understanding of at least the basics of cell biology, sample preparation and fluorescence light microscopy. The following section aims to provide the information necessary to carry out imaging experiments and gives useful links to imaging-related knowledge bases.

Cell biology

Prerequisite for a successful imaging experiment is to grow the cell specimen under optimal conditions. Besides factors that determine the growth conditions such as nutritional supplements, pH buffering and osmolarity of the medium, the substrate the cells or tissues are growing on should be suitable for both optimal tissue culturing and light microscopy. If glass is used as a substrate, its thickness and refractive index should be taken into consideration. The choice of substrate and cultivation vessel in some cases also dictates the microscope system you need to use. For instance, if tissue slices are growing on tissue culture mesh inserts that cannot be penetrated by light, the use of an upright microscope base with a dipping lens is essential for live cell expeiments. The existing CALM facility does not have an upright microscope, but access to other facilities within the University with the appropriate systems can be arranged (see IMPACT).

Since many cell types do not optimally adhere to glass surfaces, coating of the glass with adhesion-enhancing substrates might be an option. However, potential autofluorescence and diffraction caused by these subtrates should be carefully considered and tested beforehand. Parallel control samples for viability assays are recommended to monitor the condition of the specimen in culture. This is particularly important for live cell experiments, in which it is crucial to measure the impact of phototoxicity and environmental factors on the cell specimen.

Comprehensive information about tissue culture can be found in the Sigma-Aldrich on-line manual under Tissue Culture, Technical information (page 326-377).

Sample preparation

The preparation of samples for microscopy from single cells, tissues or whole organisms is crucial for obtaining optimal imaging results. In the appropriate linked pages the different steps of sample preparation such as fixation, permeabilisation, immuno-fluorescence labelling etc will be discussed. Alternatively, recommendations for sample preparation for live cell experiments are given (see more ).

Choice of fluorophores

For fluorescence microscopy the choice of fluorophores used for labelling specific molecules, structures or organelles of cells is crucial and dependent on the application and the microscope system used. For the simultaneous detection of several, differently labelled components in the same specimen, the following technical specifications determine how many different fluorophores can be spectrally separated: the band or line of excitation light, the optical filter or slit sets and the number and type of detectors available (see more). It is also dependent on the type of experiment, e.g. working with live cells or fixed samples. Both confocal laser scanning system in the CALM facility have three different PMT detectors that can be used to record three independent channels. In addition, the Zeiss LSM 510 META system allows recording of more than three channels simultaneously using the spectral unmixing technique.

In general, for detection of specific, endogenous proteins in fixed samples, indirect immunofluorescence labelling with secondary antibody conjugates is recommended. A large range of synthetic dyes that label specific cell organelles, compartments or molecules are available and most are suitable for live cell imaging. These include fluorescent indicators for measuring intracellular parameters such as ion concentrations, pH and membrane potentials. The expression of exogenous fluorescent fusion proteins is the method of choice for live cell imaging and fluorescence lifetime measurements (FLIM). A potential drawback of this method is the steric effect of the relatively large fluorophore component in the fusion protein and the potentially negative effect of the over-expression of these fusion proteins and both effects should be taken into consideration when planning experiments. A whole range of bioindicators based on fluorescent proteins is available to measure intracellular parameters such as ion concentrations, pH, redox state, protein activities etc.

Alternatively, the use of genetically encoded small tags in connection with small dyes (e.g. expression of the FLASH motif visualised with the application of biarsenic reagents) would be an option, but practical limitations should be taken into account. Most recently, the use of antibody-tagged quantum dots has been introduced. However, technical problems caused by the relatively large size of the coated quantum dots and strong 'blinking' significantly limits the use for potential biological application .

Live cell imaging

To study biological processes in intact cells at a high spatial and temporal resolution, live cell imaging is the method of choice. Most critical for all live cell imaging experiments is the phototoxicity caused by excitation light sources. Thus, the golden rule for these experiments is to keep the irradiation of the sample as short and with the lowest power input as possible. Phototoxicity should be monitored by including parallel viability controls. Two main groups of fluorescent markers are generally used for the labelling of specific molecules, structures or organelles of cells: synthetic fluorophores and genetically encoded fluorescent proteins (see also above). For more details see live cell imaging.

Protocols for techniques such as transfection, viral infection or microinjection to introduce material into cells will be added to this website in due course.

Image acquisition

The parameters for optimal image acquisition are part of the training provided by the CALM facility for the use of the facility systems. If you have questions beyond the training, please contact Rolly Wiegand (calm.head@ed.ac.uk).

Image restoration, analysis and quantitation

To fully exploit scientific images, raw pictures require post-acquisition restoration and quantitation to extract experimental data. The first stage the bioinformatic extension of the CALM facility now provides a high-end workstation for image processing and analysis equipped with the following software packages .

Zeiss Ltd. provide a free software to visualise images from Zeiss microscopes and TIF files as well as opening Zeiss image databases. It can be downloaded from the Zeiss website, following the links LSM > Further information/download > Free Zeiss LSM Image Browser.

For image data quantitation a very versatile, free software ImageJ provided by the NIH is available from the ImageJ website. This website also includes links for downloading a detailed manual and a large range of plugins.

For advanced image analysis and visualisation we provide a copy of the Improvision software package Volocity 4. Please find more information and a copy of the software handbook on the Improvision website.

Last update:29 June 2010

About Us | Site Map | Contact Us | Disclaimer | Web design and contents by Rolly Wiegand © 2006 - 2010 University of Edinburgh

Top of page