Technical Tips:

Microscope Objectives.

Microscope manufacturers now offer a wide range of objectives to meet the needs of a number of different specialized microscopy imaging techniques. The objectives are corrected to compensate for different thickness of the cover glass you are using . They can be designed  to increase the effective working distance of the objective where this is required. They can also be corrected to a varying degree for spherical and chromatic aberrations.
Inscribed on the barrel of each objective lens is some very important information. This information will give you an indication of what the objective has been designed to be used  for and also the quality and resolving capability of that objective lens.

Magnification/ Numerical Aperture

All objective lenses will have this information displayed on the barrel on the lens. The N.A. number is a measure of the light-gathering capacity of the objective lens. The higher the numerical aperture the better that objective is at resolve the fine details of the specimen in the image. The image is usually also brighter with an objective that has a higher N.A.
The higher N.A. objectives are more expensive and are usually corrected for more spherical and chromatic aberrations. The higher the numerical aperture of the objective usually the shorter the effective working distance of the lens.

It is the Numerical Aperature determines the resolution not the magnification. Below are two images  of 0.175um beads. The images were taken with two different x60 objectives one with a 1.4N.A and the other with  a 0.7N.A objective lens.

Numerical Aperture and Resolution above image courtesy of K.Anderson

Fixatives

It is very important that you select the correct method of fixation for the intended technique. The aim of any fixation process is to preserve cell structure. Different fixatives achieve this in different ways.
Antibodies, cellular structures, and samples respond very differently to different fixation methods. You may have to optimize and try out several different fixation processes to fine one that will suit your experiment.

Make sure you are using the proper fixative and blocking agents at the correct temperature.
Use the optimal antibody concentration (you will always need to use a higher concentration of antibody for immunofluorescence than you use for western blots).

Try fixatives such as formaldehyde, paraformaldehyde, ice-cold methanol, ice-cold acetone etc. Also be aware that certain structures, such as the microtubule network, are sensitive to temperature change.


Some microscopy groups have reported that GFP fluorescence is quenched by dehydrating or fixation with ethanol or acetone. This could be resulting from denaturing of the GFP molecule.
Some groups have reported that GFP signal is also greatly reduced by formaldehyde fixation and that long term storage of samples in aldehydes will eventually wipe out the GFP signal and will increase autofluorescence.
Others groups have successfully fixed cells in aldehydes without loss of signal. Please be very careful not to assume that the GFP signal is lost by any of the above fixation methods.

Paraformaldehyde: Aldehydes fix tissue by introducing cross links between different tissue components.

Methanol: This dehydrates, coagulates and precipitates cellular protiens, neucleic acids and carbohydrates. It can be used combined with acetone.

Mounting Media

Liz Black has tested some of the mountants we stock in the Beatson. The hardset mountants seem to cause shrinkage for the cell lines that she is using. Please see attached powerpoint presentations.

Powerpoint 1

Powerpoint 2

It is very important to choose the correct mounting medium for your experiment. One of the major causes of image degradation in microscopy is improper matching of the refractive index of the immersion medium and the mountant.
This miss-matching can results in spherical aberrations and signal loss.
Oil objectives are designed so that the refractive indices of the immersion oil and embedding media are equal (n=1.52).
For water objectives, this index is assumed to be n=1.33.The BAIR facility uses mainly Zeiss 518F oil with a RF value of 1.518.Other oils such as cargil oil is the immersion oil of choice for TIRF and FLIM applications.
 

Flurochromes


Fluorophores are molecules that when absorbing the energy of electromagnetic radiation will jump to a higher energy level (excited state). When some of these molecules return to the ground state they emit radiation. This is known as fluorescence. Fluorophores have special molecular structures and a characteristic excitation and emission spectra. Individual fluorophores are exited within a given wavelength range and will a emit light within a given wavelength range. The emission wavelength will always be longer than the excitation wavelength. Fluorophores are catalogued according to their absorption and fluorescence properties, including the spectral profiles, wavelengths of maximum absorbance and emission, and the fluorescence intensity of the emitted light.

   

 Excitation and emission spectra of EGFP and Cy5                                                                      Single photon excitation and emission

Above image taken from Thermo Fisher Spectral viewer

 

Autofluorescence

Autofluorescence can cause problems in fluorescence microscopy. In most fluorescence microscopy, fluorescent stains are applied to the samples to stain specific structures.
Autofluorescence interferes with detection of the resulting specific fluorescent signals, especially when the signals of interest are very dim. It is not always obvious that autofluorescence has occurred.
Users performing immuno-staining must be aware that autofluorescence is a very common problem in all tissue samples. RBCs and blood vessles tend to show autofluorescence. In some microscopes (confocal microscopes),
it is possible to make use of different spectral emission of the fluorescent markers and the endogenous molecules to exclude most of the autofluorescence.
To preform this properly you will need to have a few controls.

1-An unstained tissue sample with no fluorescent probe.

2-A positive control of the fluorescent dye.
 

Autofluorescence Causes and Cures: http://www.ihcworld.com/autofluorescence.htm


 

Coverslips

Most microscope lenses have a designated 0.17 printed somewhere on the lens. This number indicates the expected glass coverslip thickness that was used to calculate the optical corrections in the lens. Using coverslips of the wrong thickness can add optical aberrations to your image.

 

 

 

 


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Copyright 2009 Beatson Advanced Imaging Reaource
Last modified: 02/25/16