Microscopy (BOOK)
Chapter 9 pp 529-556
Visualising Cells
Looking at cells in the light microscope
Schleiden and Schwann proposed that all plant and animal tissues were aggregates of individual cells,
this is called the cell doctrine.
Bacteria and mitochondria are about 500 nm and are generally the smallest objects whose shape can
clearly be discerned in the light microscope. Objects smaller than this are obscured by the effects
resulting from the wavelike nature of light as it passes through the lenses of the microscope. Light
beams interfere with one another and cause optical diffraction effects. If two trains of waves
reaching the same point with matching crests and troughs (see image below) they are in phase and
will reinforce each other. When they are out phase they will cancel each other partly or entirely.
The interaction of light with an objects changes this way of phase relationship.
The limiting separation at which two objects appear distinct is called the limit of resolution. The limit
of resolution depends on the wavelength and the numerical aperture (diafragma) of the lens system.
The refractive index is the ratio of the speed of light in a vacuum to the speed of light in a particular
transparant medium.
It is not possible in a conventional light microscope to resolve two objects that are separated by less
than 200 nm, they will appear as a single object.
In the normal bright-field microscope, light passing through a cell in culture forms the image directly.
In dark-field microscopy the fact that light rays can be scattered in all directions by small objects is
exploited. In this form of microscopy, no other light than light from the object can be seen, so it is
possible to see particles which can’t be seen with normal light.
The phase-contrast microscope and the differential-interference-contrast microscope increase the
phase differences (the phase of light waves is changed according to the cells particles refractive
index), thereby creating an image of the cells structure.
Digital techniques
The human eye cannot see well in extremely dim light and it cannot perceive small differences in
light intensity against a bright background. To increase this ability we can attach a sensitive digital
camera to a microscope which detect light by means of charge-coupled devices (CCDs) or high
sensitivity complementary metal-oxide semiconductor (CMOS) sensors.
Intact tissues
Tissues are mostly cut into thin transparent slices called sections before examining. To preserve the
cells within these sections, they must be treated with a fixative which lock and stabilise proteins into
position. A microtome is a machine which makes tissue sections by operating like a meat slicer.
Because most cells have little content (about 70% water by weight) there is still not much to see
under the light microscope. There are three main approaches to working with thin tissue section that
reveal the differences in types of molecules present.
, Firstly, sections can be stained with organic dyes that have some specific affinity for particular sub
cellular components.
Secondly, sectioned tissues can be used to visualise specific patterns of differential gene expression.
This is particularly effective when used in conjunction with fluorescent probes.
Thirdly, generally and ideally applicable for localising proteins of interest also depend on using
fluorescent probes and markers.
Fluorescence microscopy
Fluorescent molecules absorb light at one wavelength, and emit it at another longer wavelength. If
this is viewed through a filter which only allows light of the emitted wavelength to pass, it will glow
against a dark background. The fluorescent dyes used for staining cells are visualised with a
fluorescence microscope in which the illuminating light is passed through two filters; one filers the
light before it reaches the specimen and the other filters the light obtained from the specimen. The
first only allows the absorbed wavelength, and the second only the emitted wavelength.
Fluorescein emits an intense green fluorescence when exited with blue light.
Rhodamine emits deep red fluorescence when exited with green-yellow light.
Use of antibodies
Each unique antibody has a different binding site for a specific target molecule called an antigen.
When labelled with fluorescent dyes, they can be used to locate specific molecules.
Although a marker molecule such as a fluorescent dye can be linked directly to an antibody (the
primary antibody) a stronger signal is achieved by using an unlabelled primary antibody and then
detecting it with group of labelled secondary antibodies that bind to it, this is called indirect
immunocytochemistry.
Optical microscope
Information about the third dimension (3D) is lost through sectioning. Because an optical microscope
is focuses on a particular focal plane, all other parts above and below the plain of focus are ‘’out-of-
focus’’. There are two ways to solve this problem:
Firstly by taking a serie of optical sections (light from the plane, the other light is rejected) at diffent
depths and storing them in a computer, a 3D image can be reconstructed.
Another approach is called image deconvolution. To understand we need to know what the point
spread function is. This is the blurred image of a point source due to the lens system which produces
a small blurred disc as the image of a point light source with increased blurring if the point source lies
above or below the focal plane.
Chapter 9 pp 529-556
Visualising Cells
Looking at cells in the light microscope
Schleiden and Schwann proposed that all plant and animal tissues were aggregates of individual cells,
this is called the cell doctrine.
Bacteria and mitochondria are about 500 nm and are generally the smallest objects whose shape can
clearly be discerned in the light microscope. Objects smaller than this are obscured by the effects
resulting from the wavelike nature of light as it passes through the lenses of the microscope. Light
beams interfere with one another and cause optical diffraction effects. If two trains of waves
reaching the same point with matching crests and troughs (see image below) they are in phase and
will reinforce each other. When they are out phase they will cancel each other partly or entirely.
The interaction of light with an objects changes this way of phase relationship.
The limiting separation at which two objects appear distinct is called the limit of resolution. The limit
of resolution depends on the wavelength and the numerical aperture (diafragma) of the lens system.
The refractive index is the ratio of the speed of light in a vacuum to the speed of light in a particular
transparant medium.
It is not possible in a conventional light microscope to resolve two objects that are separated by less
than 200 nm, they will appear as a single object.
In the normal bright-field microscope, light passing through a cell in culture forms the image directly.
In dark-field microscopy the fact that light rays can be scattered in all directions by small objects is
exploited. In this form of microscopy, no other light than light from the object can be seen, so it is
possible to see particles which can’t be seen with normal light.
The phase-contrast microscope and the differential-interference-contrast microscope increase the
phase differences (the phase of light waves is changed according to the cells particles refractive
index), thereby creating an image of the cells structure.
Digital techniques
The human eye cannot see well in extremely dim light and it cannot perceive small differences in
light intensity against a bright background. To increase this ability we can attach a sensitive digital
camera to a microscope which detect light by means of charge-coupled devices (CCDs) or high
sensitivity complementary metal-oxide semiconductor (CMOS) sensors.
Intact tissues
Tissues are mostly cut into thin transparent slices called sections before examining. To preserve the
cells within these sections, they must be treated with a fixative which lock and stabilise proteins into
position. A microtome is a machine which makes tissue sections by operating like a meat slicer.
Because most cells have little content (about 70% water by weight) there is still not much to see
under the light microscope. There are three main approaches to working with thin tissue section that
reveal the differences in types of molecules present.
, Firstly, sections can be stained with organic dyes that have some specific affinity for particular sub
cellular components.
Secondly, sectioned tissues can be used to visualise specific patterns of differential gene expression.
This is particularly effective when used in conjunction with fluorescent probes.
Thirdly, generally and ideally applicable for localising proteins of interest also depend on using
fluorescent probes and markers.
Fluorescence microscopy
Fluorescent molecules absorb light at one wavelength, and emit it at another longer wavelength. If
this is viewed through a filter which only allows light of the emitted wavelength to pass, it will glow
against a dark background. The fluorescent dyes used for staining cells are visualised with a
fluorescence microscope in which the illuminating light is passed through two filters; one filers the
light before it reaches the specimen and the other filters the light obtained from the specimen. The
first only allows the absorbed wavelength, and the second only the emitted wavelength.
Fluorescein emits an intense green fluorescence when exited with blue light.
Rhodamine emits deep red fluorescence when exited with green-yellow light.
Use of antibodies
Each unique antibody has a different binding site for a specific target molecule called an antigen.
When labelled with fluorescent dyes, they can be used to locate specific molecules.
Although a marker molecule such as a fluorescent dye can be linked directly to an antibody (the
primary antibody) a stronger signal is achieved by using an unlabelled primary antibody and then
detecting it with group of labelled secondary antibodies that bind to it, this is called indirect
immunocytochemistry.
Optical microscope
Information about the third dimension (3D) is lost through sectioning. Because an optical microscope
is focuses on a particular focal plane, all other parts above and below the plain of focus are ‘’out-of-
focus’’. There are two ways to solve this problem:
Firstly by taking a serie of optical sections (light from the plane, the other light is rejected) at diffent
depths and storing them in a computer, a 3D image can be reconstructed.
Another approach is called image deconvolution. To understand we need to know what the point
spread function is. This is the blurred image of a point source due to the lens system which produces
a small blurred disc as the image of a point light source with increased blurring if the point source lies
above or below the focal plane.
