Single Molecule Detection (SMD)
Single Molecule Incentive
The ultimate limit to analytical sensitivity is the reliable detection of single
molecules. Recent technical advances in optical detection and manipulation have
made the detection of isolated, light emitting probe molecules a reality. Thus we
are witnessing a burgeoning interest in the imaging and spectroscopy of single molecules,
particularly within the fields of cell biology and drug discovery.
Of particular importance to biology is the possibility of direct, real-time visualization
of single biological macromolecules and their assemblies under native physiological
conditions, offering great promise for enhancing our understanding of the behaviour,
interactions and trafficking of individual biological macromolecules within the
living cell. Such studies have increased medical and pharmaceutical significance
within the developing post-genomic era of proteomics, providing the means to track
the behaviour and mechanistic involvement of biochemically relevant single proteins.
It is reasonable to ask why it is interesting to observe the emission from a single
fluorophore, since numerous fluorophore labels can be detected within a diffraction-limited
spot. There was a time when the scientific community was content with detecting
the combined signal of a vast number of fluorescent (or Raman active) molecules
from a point of interest within the sample. There is a rapidly growing realization
however, particularly within the life science and materials science fields, that
the information content afforded by imaging the single fluorophore markedly exceeds
that offered by the "bulk" ensemble measurement, yielding invaluable insight into
individual molecular properties and their micro-environment.
Experiments on single molecules have attracted vivid interest in many branches of
fundamental research because they allow for the study of molecular properties normally
disguised in inhomogeneous distributions of an ensemble. In contrast to observing
the position of several labelled molecules at a given position, single molecule
detection provides additional information, such as:
- Polarization or image spot shapes indicate orientation and reorientation.
- Time traces of intensity, emission spectrum, or fluorescence lifetime provide information
on local dynamics and diffusion.
- Fluorescence Resonance Energy Transfer (FRET) provides information on the proximity
of specific labelled sites less than 10nm apart.
- Position sensitivity allows an investigator to locate a molecule and follow simultaneously
the translational motion, re-orientational motion, and the internal dynamics of
the individual molecules.
Single molecule studies are uniquely poised to yield information about molecular
motion, behaviour, and fluctuations over time and space. There are many biological
molecules that can avail from examination at this level, typical subjects being
key members of a system that are receptive to specific cellular signals, environmental
perturbations or drug intervention. Cellular mechanisms that have been examined
include ion channel activity, protein folding, enzyme activity, membrane structure,
molecular motors/motility and vesicle transport. Single molecule detection is a
way to study detailed physical and chemical properties that allows for scrutiny
of fundamental principles and mechanisms, and may lead to technological and methodological
developments.
Single molecule techniques also have key potential in material development. The
single molecule is an excellent probe of local (nanoscale) properties since it is
a quantum light source with spectrum and lifetime that is sensitive to its chemical
and physical environment. The rotational and translational motion of single molecules
can be measured and used to understand the local mechanical properties of the material
in which the molecules are embedded. Thus groups are seeking to understand and catalogue
the great variety of behaviours of single molecules in technologically relevant
environments to better use these probes to study the small-scale structure.
Parallel Detection by Camera
One of the compelling aspects of single molecule studies is the ability to directly
observe distributions of individual properties and molecular behaviour. Obtaining
distributions of such parameters requires studying a large number of individual
molecules. Single molecule microscopy using camera detection has the capability
to effectively investigate many spatially separated individual molecules in a parallel
fashion.
Such an approach can make for more efficient experiments and ensure that the molecules
are imaged under identical conditions. Specific statistical distributions of properties
can be determined which contain more detailed information than the ensemble-averaged
mean values obtainable by bulk measurements.
From an analytical perspective, single molecule measurements have ultimate sensitivity,
opening a new era of femtomolar chemistry, by which biological reactions and high-throughput
screening can be carried out with minute samples, which are normally insufficient
for measurement purposes.
One area of particular commercial vibrancy is the drive towards extremely efficient
whole genome sequencing using single molecule approaches. The ultimate goal here
is to make available the capability to screen the entire 3 billion bases of the
human genome within 24 hours, for less than $1000!
Camera-Based SMD Techniques
The continually improving technologies of modern lasers, ultrasensitive detectors,
analysis software and novel optical configurations, permit detection and investigation
of single molecule fluorescence images, trajectories, spectra, and lifetimes.
Some of the camera-based techniques adaptable to single molecule detection are listed
below, their suitability depending more on the specific system under study:
- Confocal Raman Microscopy
- HyperSpectral Imaging
- Spinning Disk Confocal Laser Scanning Microscopy
- Total Internal Reflectance Fluorescence Microscopy (TIRFM)
- Widefield Epifluorescence Microscopy
Background Photon Noise - The Need to Minimize
Background photon noise level must be low compared to the signal level in order
to allow discrimination of single molecules. This is usually the most serious limitation
to sensitive detection. Just as stars can be seen at night but are obscured by sunlight
scattered from our atmosphere during the day, the key to SMD is to minimize background,
which includes fluorescence or Rayleigh/Raman scattering of the bulk medium containing
the molecule of interest.
This is normally realized by some combination of spectral, temporal or spatial filtering.
Spatial discrimination, has the effect of increasing the apparent concentration
of the molecule of interest - one molecule confined to a 1μL and a 1fL volume has
effective concentrations of 10-18 M and 10-9M respectively.
The proportion of the total signal that originates from the molecule can thus be
increased significantly by miniaturization. Spatial discrimination also helps to
isolate single molecules, as the probability of finding more than one molecule in
the same volume element (for a given bulk concentration) decreases with a decreasing
volume.
TIRFM
A relatively straightforward way to limit the observation volume is to excite the
molecules by Total Internal Reflection (TIR) illumination. TIRFM makes use of an
optical effect that can be adapted to observe fluorescent events occurring at the
interface between two optical media of different refractive indices. Excitation
light incident upon such a boundary, travelling at an angle greater than the critical
angle, undergoes total reflection.
The electromagnetic field of the total internal reflected light extends into the
sample beyond the interface, extending only a few hundred nanometres into the second
medium of lower refractive index - essentially in the z direction. Furthermore,
this Evanescent Field decreases exponentially in intensity along the z-axis of penetration.
Only the section of the specimen located within the evanescent field undergoes fluorescence
excitation.
TIRFM is limited to the area within a few hundred nanometres of the glass/sample
interface, where total internal reflection is occurring. By imaging the surface
onto a camera, individual molecules can be registered as each migrates to the proximity
of the surface. This imaging method allows the observation of many distinct molecules
simultaneously, yielding a high throughput and increased statistical viability.
TIRFM is an increasingly popular technique for visualizing, with high signal to
background ratio, single molecule processes that occur in and around the membrane
of living cells (partially due to availability of novel membrane-specific fluorophores).
In contrast to the spinning disk confocal technique, which can yield rapid optical
sections of approx. 1μm thickness, the excitation volume of a TIRF evanescence field
extends only approx. 100nm into the sample, the intensity of which decays exponentially
across this distance. Since only a very thin sliver of excitation is being produced,
we only detect photons that are created within that excitation volume, which has
the effect of significantly improving signal to background.
Whilst a thin excitation volume of the order of 100nm depth sounds very useful,
it must be remembered that due to the nature of the TIRF effect, we cannot step
this excitation plane through the volume of the cell. TIRFM is a surface interface
phenomenon, and as such is restricted to studying sample that is within 100nm or
so of the glass interface of the slide. For events within the bulk volume of the
cell, we must rely on widefield epifluorescent or a rapid confocal scanning approach.
Multiple Probes and FRET
It is often of interest to dynamically image multiple different biomolecules, and
their interactions within the sample volume. The TIRFM technique can readily be
extended to image multiple fluorophores labels, through integration of a multi-line
laser system, preferably a solid-state laser solution with Acousto-Optical Tunable
Filter (AOTF) modulation.
This technique can be readily adapted for FRET analysis, preferably through integration
of a suitable beam splitting device on the emission side.
Electron Multiplying CCD (EMCCD) - the Last word in Ultra-Sensitivity
EMCCD technology is the ideal detector for dynamic single molecule imaging. The
extraordinary Signal to Noise (S/N) offered is significantly greater than that afforded
by conventional CCD cameras operated at fast readout speeds. EMCCDs exhibit frame
rates that are ideally suited to dynamic acquisition of transient single molecules
and their interactions. Since EMCCDs are array detectors, multiple single molecules
can be illuminated and imaged in parallel, greatly improving the experimental throughput
and statistical viability of SM data.
Underlying all direct imaging studies of living cells or organisms, is the desire
to preserve the living subject for as long as possible, through minimization of
both phototoxic cell/tissue damage and also photobleaching of the incorporated fluorophores.
As such, techniques for studying single molecules benefit markedly from using EMCCD
detection technology, permitting the laser or lamp excitation light to be attenuated.
The spectral properties of single molecule systems can be accessed in a number of
ways. For example, simply through fast switching between multiple fluorophores excitation/emission,
or even splitting of the emission signal simultaneously onto different areas of
the sensor. Through minimization of excitation powers, the rates of dye photobleaching
and cell phototoxicity are significantly reduced.
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