Biology Reference
In-Depth Information
The term chromosome (colored body) that we still use
today reveals that cellular structures were first discovered
and named according to their appearance (phenotype), and
only later were their functions studied and understood,
mostly by other means. Such a merely descriptive usage of
microscopy evolved to be the quantitative tool that it is
today, suitable to understand the operation of spatially
organized intracellular communication [10] .
Fluorescence microscopy provides an unsurpassed way
to observe the dynamics of molecules inside living systems
and has therefore become a cornerstone of cell biology. In
this technique molecules are specifically labeled with
agents that absorb energy as photons of a given wavelength
and re-emit it as photons of a different, usually red-shifted
wavelength. In immunofluorescence and related methods,
labeling is accomplished by using specific biomolecules
such as antibodies to target fluorescent dyes to a protein of
interest. As an excess of labeled material needs to be
applied to the sample to ensure saturation of binding sites,
washing of unbound material is essential to obtain contrast.
While recent developments in chemical genomics have
provided ways of accomplishing such labeling in living
cells, most labeling methods are not suitable for live cells.
Discovery and tooling of the green fluorescent protein (FP)
was therefore a breakthrough in fluorescence microscopy.
FPs can be fused to a protein of interest by genetic
manipulation of cells to create a fluorescent chimeric
version that can be expressed and imaged in living
organisms.
With the ability to visualize molecules in living cells,
the challenge then is to derive from the acquired fluores-
cence images information about molecular mobility, such
as diffusion and transport, and molecular state such as
interaction, conformation or post-translational modifica-
tions, to understand how biological patterns arise. Interac-
tions, for example, could be investigated by simultaneous
imaging of two or more labeled species. However, co-
localization of different molecular species in the same
spatially resolvable volume does not mean that interaction
is occurring. A standard microscope is an optical low-pass
filter that removes the fine details of an object, preventing
the observation of sub-wavelength structures (Abbe
diffraction limit). A point-like light source in the sample is
reconstructed by a microscope into a larger image called
point spread function (PSF), and its dimensions are related
to the optics of the microscope and the wavelength of the
light. For standard wide-field microscopy, the PSF is 250
nm in the lateral dimension (perpendicular to the beam
propagation), and in the longitudinal dimension spans the
height of a typical cell, resulting in a volume of about 5 fL.
Such dimensions are much larger than the size of a typical
protein (80 kDA equivalent to 10 e 7 fL), and therefore two
apparently perfect co-localizing objects in an image can be
far apart on the molecular scale.
Using a pinhole to reject out-of-focus light, confocal
microscopy reduces the longitudinal dimension to
m,
enabling optical section of a typical cell into several slices.
In spite of this achievement, the size of a typical confocal
volume (1 fL) is still seven orders of magnitude larger than
the size of a typical protein. Novel super-resolution tech-
niques such as PALM/STORM or STED can routinely go
down to a resolution of 20 nm. In PALM/STORM the
ability to localize single fluorophores with nanometer
accuracy is exploited to reconstruct a high-density super-
resolution picture from a series of images of sparsely
excited fluorophores [11,12] . In STED, the PSF of
a confocal laser scanning microscope is sharpened by
stimulated emission depletion of the fluorophores far away
from the center of the PSF [13,14] . To identify and localize
single molecules, both imaging methods benefit if the
density of molecules to be detected is low. Longer acqui-
sition times are required to attain the resolution needed to
distinguish between interacting and nearby molecules (or
a protein conformational change), thereby precluding its
use to monitor fast cellular dynamics.
Although most of the aforementioned techniques have
pushed the resolution well below the diffraction limit,
a different subfamily of methods has been developed to
directly assess functional observables such as diffusion,
conformation and interacting populations of molecules. In
a first group of functional techniques, the macroscopic
fluorescent steady state is locally perturbed by photo-
chemical means while monitoring its re-equilibration over
the cell. Such recirculation of fluorescent species is
a macroscopic reporter of the mediating intracellular
processes: transport, binding and diffusion. The oldest and
most common of these techniques is fluorescence recovery
after photobleaching (FRAP), in which a region of the cell
is first bleached using a strong laser beam for a short time;
the recovery of the fluorescence intensity is monitored
afterwards [15,16] .
In a second group of functional techniques the macro-
scopic fluorescent steady state is dissected in space and
time into the microscopic fluctuating parts that are aver-
aged out on a larger scale. Such fluctuations are a product
of the discrete molecular composition of the observed
system and can be harvested for information about the
underlying physicochemical processes that give rise to
them. In an ergodic system, the observation in a limited
region of space of a sufficient large number of events is
equivalent to observing a statistical sample of all the
ensemble's possible events. This principle is used in fluo-
rescence correlation spectroscopy (FCS) to obtain absolute
concentrations and diffusion times by measuring the
motion of individual fluorescent species through a small
(~ 0.5 fL) confocal volume [17,18] . With two-color FCS,
the co-diffusion of proteins through the confocal volume
will become coincidences in the intensity time traces [19] .
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