LE 1 Introduction to microscopy
Resolution is the closest distance at which two dots can be distinguished
from each other or resolved. The resolving power of the microscope depends
on the width of the cone of illumination and therefore on both the condenser
and the objective lens. The condenser lens focuses a cone of light rays onto
each point of the specimen, the objective lens collects a cone of ray lights
to create an image. Resolution is not the same as magnification.
Magnification is the ability to make small objects seem larger. (magnification
≠ resolution)
1 LIGHT MICROSCOPY
Light microscopy uses visible light and a system of lenses to generate magnified images
of small objects. The magnification power of optical microscopes is typically limited to
around 1000x times because of the limited resolving power of visible light. When using light
microscopy often different specific techniques are used to localize specific structures.
Examples of such techniques are:
- Enzyme histochemistry, localization of activity
- Autoradiography, localization of specific events
- In situ hybridization, localization of DNA/RNA.
uses labeled complementary DNA, RNA or probes to localize a specific sequence
in a proportion or section of tissue (in situ). Hybridization consists of four steps;
pretreatment, hybridization, detection and visualization.
- Immunohistochemistry, localization of specific antigens
can either be a one-step or two-step process. In the one-step process the binding
antibody is fluorescently labeled (direct). In the two-step process the binding
antibody is not fluorescently labeled, but a second antibody is used which is
labeled (indirect).
1.1 PHASE-CONTRAST MICROSCOPY
Phase-contrast microscopy is a type of light microscopy that converts phase shifts in light
passing through a transparent specimen to change brightness within the image. Phase shifts
themselves are invisible, but become visible when shown as brightness variations. Phase-
contrast microscopy is particularly important in biology. It reveals many cellular structures
that are not visible with a simpler bright-field microscope. It is one of the few methods
available to quantify cellular structure and components that does not use fluorescence.
2 ELECTRON MICROSCOPY
Electron microscopy uses a beam of
accelerated electrons as source of illumination.
The contrast of these microscopes is produced
by the scattering of electrons from the beam
by atoms in the sample (electron scattering).
The bigger the atom, the more electrons it
has, and the more likely it is to repel and
deflect electrons from the beam. The image is
,created by the electrons that are not deflected. Electron microscopes have a higher
resolving power than light microscopes and can reveal the structure of smaller objects.
Also with electron microscopy different techniques can be used for specific localization:
- Enzyme cytochemistry, localization of activity
- EM-autoradiography, localization of specific events
- In situ hybridization, localization of DNA/RNA
- Immunocytochemistry, localization of specific antigens
uses e.g. fluorescent antibodies or immunogold labeling, either in a direct or
indirect fashion.
There are pros and cons when using ultrastructural localization at the EM-level. Pros are high
structural detail and high resolution. Cons are time consuming, skills and in principal no live
cell imaging. An ‘in between’ method would be high resolution fluorescence. This technique
is much faster, however it also has a lower resolution.
2.1 CRYOSECTIONING
The frozen section procedure, or cryosection, is a pathological laboratory procedure to
perform rapid microscopic analysis of a specimen. The histology slice is cut very thin and
frozen rapidly. The technique can for example be used e.g. for the examination of tissue
while surgery of a patient is taking place.
3 FLUORESCENCE MICROSCOPY
Fluorescence microscopy uses fluorescence to generate an image. The specimen is
illuminated with light of a specific wavelength (or wavelengths) which is absorbed by the
fluorophores, causing them to emit light of longer wavelengths. In principle fluorescence
microscopy needs three things: probes (in vivo, e.g. GFP), techniques (FRET, FRAP, FLIM,
etc.) and equipment.
Absorption of light rises a fluorochrome molecule to an
excited state of higher energy content, the molecule
remains in this excited state for only a very short period
of time (nsec range). The way back to the basic energy
level is accompanied by the emission of light, called
fluorescence. Due to internal energy dissipation, the
emitted light always has a longer wavelength (= lower
energy) than the exciting light, this difference is called
the Stokes shift. The quantity of emitted light is very
small compared to the quantity of excitation light.
3.1 EQUIPMENT
Optical filters can be used to
selectively transmit light of
different wavelengths. An example
of such a filter is a short pass
filter (SP) which attenuates longer
wavelengths and passes shorter
wavelengths (UV). On the contrary
there is the long pass filter (LP)
, which attenuates shorter wavelengths and passes longer wavelengths (visible/IR). Other
types of filters are band pass filters which only transmit a certain wavelength band and
block others. The width of such a filter is expressed in the wavelength range it lets through.
Such a filter can for example be made by combining the SP and LP filter.
3.1.1 Multi-color detection
Using wide fluorescence microscopy also two colors can be detected at the same time. This
can either be done using conventional detection where two excitation wavelengths are
used simultaneously. This however leads to limited emission signal detection and cross talk
due to overlap of emission signals. Another way to detect more than one color is
multitracking. This technique uses two excitation wavelengths at different times. This
causes excitation dependent emission and efficient emission signal detection. Since the
wavelengths are not used simultaneously, cross talk is prevented. The signal can be
improved by using long pass instead of band-detection.
3.1.2 Objectives and cameras
Numerical aperture defines the resolving power and the luminosity of an objective:
numerical aperature ( n . A . )=n∗sin α , where n equals the refractive index of the optical
medium between front lens and coverslip and α is equal to one-half of the objective angular
aperture. The n.A. can thus be increased by increasing the angular aperture. This can be
done by moving the specimen closer to the lens. The n.A. can also be increased by
increasing the refractive index (n).
Single lenses have different focal distances for different wavelengths. This color error can be
corrected by a combination of the type of glass and coatings, in order of color error
elimination: achromats, fluorite objectives, apochromats (fully color corrected for up to 7
wavelengths).
The point spread function describes the response of an imaging system to a point source
or point object.
Next to objectives also cameras are used in microscopy. An example of such a camera is a
microscope charge-coupled device (CCD) which is a camera that attaches to a
microscope and takes still pictures or movies of the specimen being observed. Wide-field
CCD based microscopy can be used as setup for fast, high-resolution live imaging.
1.22 λ
The lateral resolution can be calculated by r lateral = .
2∗NA
The integrity of images uses bits. One bit can store 2 grey values, resulting in a binary
image. An n number of bits results in 2n grey values.
Flat field correction is a technique used to improve quality in digital imaging. It cancels
the effects of imaging artifacts caused by variations in the pixel-to-pixel sensitivity of the
detector and by distortions in the optical path. It is often a standard calibration procedure.
3.2 TECHNIQUES
3.2.1 Confocal laser scanning microscopy (CLSM)
CLSM is an optical imaging technique used for increasing optical resolution and contrast of a
micrograph by means of using a spatial pinhole to block out-of-focus light in image
