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
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
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.
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.
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
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.
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
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 can cause problems in fluorescence microscopy. In most
fluorescence microscopy, fluorescent stains are applied to the samples to stain
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:
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.