Often underestimated, the sample preparation and the quality of the imaging specimen is the most crucial part of an imaging experiment. It seems obvious that it is impossible to acquire a good image from a poor sample. The following will try to highlight a few guidelines and pitfalls of sample prearation and is in no way comprehensive. You can find more details in the appropriate literature.
Biological specimens and experimental and physiological conditions underlie great variations and the experimental design of an imaging experiment needs to address this adequately. Therefore, adapted protocols are only guidelines and individual protocols for experiments should be optimised.
As for all scientific experiments, controls are absolutely essential for bioimaging to determine intrinsic and experimental background, the quality of the sample labelling etc.
The most common application for light microscopy is the image acquisition from fixed samples. For this, cells, tissue sections or whole small specimens are preserved by fixation, treated, permeabilised, fluorescence-labelled and finally mounted. For more information about each indiviual step, please use the links on the left of this page. A typical protocol for indirect immunofluorescence labelling can be found here. For more detailed protocols please refer to the literature or the IHC World website.
Primary and secondary antibodies or conjugates should be used as recommended by the suppliers or the literature. However, many primary antibodies have been raised against peptides or fragments of larger proteins, which resemble more a denatured epitope(s) of the original protein. If this is the case, an epitope retrieval techniques, which include an additional denaturation step, might improve the antibody binding. The IHC World website offers various protocols.
To study dynamic processes in intact cells, live cell imaging has become another essential field pf applications of light microscopy. Exogenous expression of fluorescent proteins or the use of live cell dyes allow the visualisation of cells, subcellular structures of even single molecules in live cells or tissues. The control of the physiological conditions of the specimen on the microscope are essential for a successful live cell experiment, and therefore it is crucial to control the viability and environmental parameters (temperature, gas supply, humidity, light exposure, pH and osmolarity of the medium, etc). Particular focus should lie on the photo-toxic effect of the excitation light, and the doses used should be kept at a minimum. Depending on the experiment, the use of water-corrected objectives are highly recommended. The CALM facility is equipped for live cell imaging and for detailed information, please contact Rolly Wiegand (calm.head@ed.ac.uk).