Cell Biology Lecture No. 3: Isolation & Analysis Of Organelles & Molecules
Wednesday January 16 , 2013
LECTURE 2 CONT’D
-Confocal fluorescence microscopy illuminates only a focal plane of the specimen and achieves
greater clarity than conventional fluorescence microscopy, which produces a blurry image due
to fluorescence detection above and below the focal plane.
-Deconvolution is a computational restoration method (using the point spread function
algorithm) that provides an alternative means of producing high-resolution images. In
deconvolution microscopy, a series of images of an object are taken at different focal planes
called a Z-stack with a conventional fluorescence microscope or confocal microscope. Unlike
confocal techniques, which physically remove the out-of-focus emission information by means
of a pinhole, deconvolution is a mathematical processing method in which computations for the
acquired stacks reassign the diffracted light to its original location. As the emitted signal is
collected in its entirety by means of a highly sensitive digital camera, post-deconvolution images
may in some cases provide higher resolution than confocal microscopy.
Fluorescence Resonance Energy Transfer (FRET):
-Fluorescence Resonance Energy Transfer (FRET) is used as method to measure protein
interactions in live cells. If no protein interaction occurs then the excitation of cyan fluorescent
protein (CFP) will only result in cyan fluorescence (480 nm). However, if protein interaction
occurs then the excitation of CFP will result in yellow fluorescence (535 nm) of another protein.
The crucial concept to grasp is that a non-optimal excitation wavelength from the interaction is
used to excite the YFP, not the microscope being used. If there is an interaction, the use of two
filter sets will detect the two possible wavelengths of the fluorescent proteins.
From Fluorescence To Electron Microscopy:
-Though fluorescence microscopy has many advantages (uses live cells, sees cellular
structures), it is somewhat limited by its resolution, partly because of the limited properties of
light microscopy (between 300 and 700nm). Electron microscopy offers a major advantage with
high resolution and high magnification by using electron particle wavelengths as small as 1 nm
(great resolution). Some limitations of electron microscopy however are the fact that dead cells
(frozen, then embedded through sample preparation and coated in metal that doesn’t allow
electrons through) are used. Essentials Of Electron Microscopy:
-As light is no longer used in this from of microscopy, heated wire filaments are used as the
source for electrons, which will “fly off” the filament and attract to positively charged anions.
Glass is no longer used for electrons as it was for light, instead magnetic fields or magnetic
condensers are used to focus the electron particles on the specimen. In order to see contrast,
the specimen is stained with electron-dense heavy metals such as lead uranium and OsO 4
(these don’t allow electrons to pass through).
Types Of Electron Microscopes:
-There are two types of electron microscopes: Scanning electron microscopes (SEM) and
transmission electron microscopes (TEM). In transmission electron microscopes, images are
formed from electrons that pass through a specimen (cross-section of a cell). Electrons are
emitted from a filament and accelerated in an electric field. A condenser lens focuses the
electron beam on the sample, the objective and projector lenses focus the electrons that pass
through the specimen and project them onto a viewing screen or other detector. In a scanning
electron microscope, images are formed from electrons that are scattered from a metal-coated
specimen. To coat samples, heavy metals such as platinum and gold are used. SEM produces
images that appear to be three-dimensional. It is important that the entire tube in both TEM and
SEM is maintained under an ultrahigh vacuum because atoms in the air would absorb electrons.
Resolution Of Transmission Electron Microscopes (TEM):
-In electron microscopes, the equation for resolution (D = 0.61 λ / α) does not account for N
(refraction) as light is replaced by electrons in a vacuum. Also sin α is simply α since the
electron scatter is almost zero. Electron microscopy produces a fine resolution (very low D
value) and a 2000 times greater magnification than light microscopy.
Sample Preparation For TEM & Image Formation:
-Cells must be fixed, embedded in plastic, and use a sectioning apparatus to make very thin
sections that are later captured on a copper grid. The sections are then stained with a heavy
metal and placed within the vacuum of the electron microscope where electrons will be
projected through them.
-When electrons hit the specimen and are deflected due to metal deposits, they will appear as
dark spots on the final image. Electrons that are unobstructed are focused by lenses onto a
phosphorescent screen. This screed possesses crystals that are excited by the electrons and
give off energy as visible light. The black and white image is made up of shadows where
electrons failed to penetrate.
-Here, an antibody linked to gold readily binds to catalase (a detoxifying enzyme found in
peroxisomes). Protein A is used simply to help gold link to the antibody. So wherever catalase is inside the cell, the antibody will recognize it, bind and have gold indirectly connected to the
desired enzyme. This targeting is an example of immunoelectron microscopy, which a fo