Diffraction-unlimited optical microscopy Optical microscopy, and fluorescence microscopy in particular, has emerged as one of the most powerful and convenient microscopic tools available today. This power does come at a price, however, in terms of a limited spatial resolution: traditionally fluorescence microscopy has been limited by diffraction to a resolution of a few hundred nanometers, far too large to discern nanostructuring in biological or material samples. Recent conceptual advances have emerged that challenge this once-thought ‘unbreakable’ barrier, and fluorescence microscopy with nanometer resolution is now within reach. In this review we highlight some of the approaches that have made this paradigm shift possible. Peter Dedecker, Johan Hofkens, and Jun-ichi Hotta Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, B-3001 Heverlee, Belgium E-mail:
[email protected]
One of the great paradoxes of life is that in order to truly
strengths and weaknesses, but one of the most convenient is optical
understand the everyday issues and concepts that affect and
microscopy, which, in an ideal case, requires only the illumination of
fascinate us, one invariably has to focus on the microscopic world
the sample. Because of this, optical microscopy tends to be relatively
around us. This idea, while no longer particularly surprising,
noninvasive, nondestructive, and convenient to use. Fluorescence
was realized as soon as the first lenses and microscopes were
microscopy in particular has proven to be one of the most powerful
developed, and has only become stronger as our ability to observe
microscopy tools available today, by combining the advantages of
smaller and smaller details improved. While this idea is usually
optical microscopy with the selectivity and extreme sensitivity of
brought up in the context of the life sciences, it is no different for
fluorescence emission. Indeed, fluorescence microscopy allows for
the materials of today and tomorrow. Designing new materials
measurements down to the level of individual molecules1–3, and
tailored to specific needs and properties requires both study
has been successfully applied in the study of, for example, polymer
and manipulation of their micro- and nanoscale structuring, and
dynamics4–9, catalysts10–15, dendrimers16.
microscopes are invaluable tools in modern-day research. Not all approaches to visualize small details are equally useful for
12
Nevertheless, revealing nanoscale structuring and ordering remains one of the great challenges of science. Indeed, one aspect that is true
a given sample or problem, however, and a wide range of microscopy
for every microscopy technique is that there is a lower limit to the
techniques have been developed. Each of these has its share of
sizes of the details that can discerned, that is, the spatial resolution is
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Fig. 1 Point spreading: after imaging, an object of negligible size (such as a single molecule) will show up as a distribution of much bigger dimensions. The centroids of these distributions are indicated by the green and blue markers. If the emitters are spaced close together, as is the case for the molecules in the blue circle, then they cannot be distinguished and the structural information is lost. (Adapted from 83. © Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission.)
limited. Intuitively, if the sample structure becomes too small, then
microscopes and objectives have advanced to the point where the level
it cannot be revealed. There are several possible reasons why this is
of aberrations is so low that their contribution is negligible. Therefore
so, ranging from technical aspects, such as instrument imperfections,
little gain in resolution can be expected from further improvements
vibrations, and measurement noise, to limitations imposed by the
in the quality of the microscope optics. While it is possible to modify
fundamental laws of nature. While the former limitations are, to some
the optical system in such a way that the point spreading is strongly
extent, specific to a particular instrument or experiment, the latter
reduced, e.g. by including a second objective20,21, these approaches
present limiting barriers that are hard to overcome.
tend to be limited by the fact that the instrument design and
In the case of optical microscopy, this fundamental limitation
alignment become increasingly complex.
stems directly from the wave-like character of light: no matter what
It thus appears that the spatial resolution in optical microscopy
kind of optical components we use, we cannot focus the light waves
is limited to a few hundred nanometers, and that details at smaller
to an infinitely small point, only to a finite region. This might seem
distances are fundamentally out of reach. Fortunately it turns out that
surprising at first: when we collect the fluorescence emission from a
it is possible to obtain an imaging resolution that is fundamentally
single, isolated molecule and image it through a lens system (such
unlimited by diffraction, and a variety of techniques that aim to
as a microscope), the resulting image will not be a point but rather
achieve this have recently appeared. Intuitively these techniques
a three-dimensional shape of much larger dimensions (hundreds of
work by making fluorescence emission into a rare event: if neither the
nanometers; see Fig. 1). The same holds true for the excitation light:
excitation illumination nor the fluorescence emission can be confined
even if the light source is infinitely small, we can only focus the light to
further, then we have to make sure that only a small fraction of the
a diffraction-limited region and cannot confine it further. This is known
molecules emits fluorescence even though all of them may experience
as point spreading, and is an immutable law of nature.
a similar intensity excitation. Alternatively, if we process the images
Point spreading is problematical if we want to image a sample
mathematically, then we can invert the problem, and try to minimize
that contains fluorescent molecules in close proximity, such as in a
the number of molecules that are not emitting beyond what is
finely structured sample. In this case the emission distributions from
normally allowed by diffraction. It turns out that the answer to this
closely spaced fluorophores overlap in the resulting fluorescence
problem lies in the properties of the fluorescent molecules themselves,
image, making it impossible to distinguish between molecules that are
and in the fact that fluorescence is a dynamic phenomenon22–24.
close together (Fig. 1). Hence point spreading imposes a fundamental limitation on the spatial resolution that can be attained. (We will not
Nonlinearity
go into further detail here; more extended, yet accessible, discussions
When we think about fluorescence emission, we tend to think about
have been published
elsewhere17–19.)
While imperfections in the imaging system worsen the point spreading beyond the theoretical minimum, the optics in today’s
it as a fairly well-behaved, constant phenomenon. If we take a fluorescent molecule and irradiate it with excitation light, we expect to see a continuous, and predictable, fluorescence emission. Moreover,
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Fig. 2 The emission of a single molecule of a fluorescent protein, clearly showing complex fluorescence dynamics, and the resulting nonlinearity in the fluorescence emission. The inset shows an expansion of the emission trace.
if we double the excitation intensity then we expect to see a twofold
makes use of this fact in some way. It is possible to roughly divide
increase in fluorescence emission, and so on. This seems intuitive,
these approaches based on whether they make use of nonlinearities
because it agrees with the observation of emission from a dye-loaded
in space (that is, try to exploit the nonlinearity to increase the spatial
solution in a fluorescence cuvette.
confinement of the fluorescence emission) or time (that is, try to
This apparent proportionality, however, only holds at the ensemble
separate the emission of the different labels in time). In what follows
(averaged) level. One of the most fascinating findings from single-
we will discuss each of these approaches, and mention some possible
molecule experiments is that fluorescence emission is in fact a very
advantages and disadvantages of each.
dynamic phenomenon: individual molecules are seen to rapidly cycle between emissive and nonemissive states, displaying a highly
Nonlinearity in space
complex and fascinating dynamic behavior25–29 (Fig. 2). The origin
For a more detailed example of how nonlinearities can be put to
of this on–off ‘blinking’ behavior is not always clear, though it can
work, let us look at the process of fluorescence emission itself. The
sometimes be attributed to, for example, the formation of triplet
initial absorption of an excitation photon causes the molecule to
states, photoisomerization, or electron transfer. It is also known to
enter an electronically excited state, where it remains for (usually)
be virtually ubiquitous, being observed in wide variety of systems,
a few nanoseconds, before returning to the ground state through
including organic molecules25–27,29, semiconductor nanocrystals28, and
the emission of a fluorescence photon. The central issue here is
fluorescent proteins30.
that there is always some nonzero delay between the absorption
Apart from transient nonfluorescent or ‘dark state’ formation,
increase up to the point where the molecule is constantly in the
well, including saturation (the fact that there is an upper limit for the
excited state, when it reaches a plateau. At this point, increasing the
emission rate due to the finite excited-state lifetime), and phenomena
excitation intensity does not increase the emission rate any further,
such as photoswitching or
photoactivation31–41.
The net result is that the fluorescence emission is no longer strictly proportional to the excitation intensity. For a given sample and
and the fluorescence emission is said to be saturated (and strongly nonlinear). Fig. 3 shows an example of this. In this figure a cross-section of the
experimental setup the emission can depend on time, on the excitation
fluorescence emission rate along the focus of a confocal fluorescence
intensity or intensities, or, by applying a spatial intensity distribution,
microscope is shown, at different excitation powers. The shape of the
on the position of the molecule with respect to the microscope’s focus.
diffraction-limited excitation intensity distribution is shown by the red
Every approach to achieve a diffraction-unlimited resolution
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and emission events. In other words, the emission rate can only
interruptions in the fluorescence emission can arise for other reasons as
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Fig. 3 The effect of saturation: he relative emission rate at the focus of a confocal fluorescence microscope and different excitation powers. The effect of saturation of the fluorescence emission is clearly visible. The relative emission powers, from red to purple, are 1, 5, 20, 100, and 1000, respectively.
However, as the excitation power is increased, the saturation of the
input laser beam. Especially interesting are ‘donut modes’, which are
emission drastically alters the resulting emission distribution.
characterized by a sharp intensity zero at the maximum, surrounded
The most crucial aspect of Fig. 3 is that it demonstrates that saturation
by a region of intense irradiation42–44 (Fig. 4). Only at the exact center
enables us to create emission distributions with sharp edges and
of the microscope focus is the intensity truly – mathematically – zero,
strongly confined nonemissive regions, even though these were not
while there is some irradiation intensity everywhere else in the focal
allowed in linear systems by diffraction.
volume.
This simple example shows how using fluorescence nonlinearities can allow us to bypass the limitations of diffraction. Saturation of the emission rate is not the only possibility, however,
Let us now assume that we have shaped the output from a laser into a donut mode, and that the wavelength and duration of the light is chosen so that it can deactivate the dye molecules. This
as any transition to a state with different emissive properties can
deactivation could be because the light induces the formation of a
be used as long as it is saturable (that is, its light-induced formation
nonfluorescent state (e.g. triplet or charge-transfer states, fluorescence
is much faster than its recovery) [22]. Approaches that make use of
photoswitching), or because it induces stimulated emission. Moreover,
this principle to create ‘sharp’ distributions in emission are known as
due to the intensity distribution of the donut shape, this deactivation
RESOLFT microscopy, standing for ‘reversible, saturable optically linear
occurs everywhere in the focal volume except at the center of the
fluorescence transitions’ [23].
focus. This approach becomes even more interesting when it is
By itself, the example shown in Fig. 3 is not directly useful
combined with saturation (Fig. 5). In this case, it is clear that the
for attaining a higher imaging resolution, since the fluorescence
confinement of the molecules in the fluorescent state can be made
confinement is not improved but, rather, worsened compared to the
arbitrarily small, limited only by the power and duration of the donut-
‘standard’ diffraction-limited case. Hence we must either modify the
mode beam.
imaging to use the saturation in a different way or process the images
At present the most successful technique that uses this approach
mathematically. A number of techniques have been developed that
is ‘stimulated-emission depletion’ (STED) microscopy45–50. The
attempt both approaches, and we will expand on these in what follows.
principle underlying this approach is shown in Fig. 6, and operates as follows: in the first step of the acquisition, the sample molecules in
STED microscopy
the diffraction-limited spot are excited by a normal mode (Airy disk)
While the focusing of the excitation light will always lead to some
laser pulse with a duration of picoseconds or less. This is followed
spatial distribution, the exact shape of this distribution can be modified
immediately (within a few hundred picoseconds) by a second donut-
into different ‘modes’ by manipulating the relative phases of the
mode laser pulse irradiating the same region, which induces stimulated
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Fig. 4 Different intensity distributions along the focal plane of a confocal microscope: a ‘standard’ Airy disk shape and a ‘donut mode’.
emission in the majority of the dye molecules, except those at the
state can be used, including triplet-state formation51 and fluorescence
center of the focus (due to the intensity distribution of the donut
photoswitching52–55. the sequence of the pulses likely has to be
mode).
interchanged, and an additional laser pulse may be necessary.
The sample molecules that are still in the excited state are then
The most attractive features of donut-based microscopy are
left to fluoresce normally, which is detected. Because of the depletion,
that the effective resolution increase is completely dictated by the
however, the fluorescence collection is effectively confined to a spot
experimental setup and the irradiation powers used. Moreover, the
with sub-diffraction dimensions (Fig. 5b), leading to an increased
imaging is altered directly, and obtaining a subdiffraction resolution
resolution. This measurement cycle is then repeated at the next point
does not require any additional processing. In addition, if stimulated
on the sample, until a complete image is obtained. In this way, a spatial
emission is used, then the image acquisition times can be as fast as any
resolution down to a few tens of nanometers has been
reported48,49.
As mentioned above, any saturable transition to a nonfluorescent
laser-scanning microscope56. As indicated, the effective resolution increase that can be obtained
Fig. 5 The effect of saturation using a donut-mode beam. (a) By saturating the transition to a nonfluorescent state, the spot of molecules that remain in the bright state can be made arbitrarily small (the relative excitation powers are 1, 5, 20, 100, and 1000, respectively). (b) The resulting apparent spot size.
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Fig. 6 A diagram of an experimental setup for STED microscopy, showing both the activation and depletion pulse, and the resulting reduction of the fluorescent spot size. (Reproduced with permission from 18.)
depends on the number of irradiation photons in the dump beam:
structure. One way to do this is by nonlinear structured illumination
more irradiation leads to a greater depletion and tighter confinement,
microscopy60.
and hence an improvement in resolution. While this is a conceptual
Structured illumination relies on projecting the excitation light
advantage, as the diffraction-limit is effectively broken, it also means
as a standing wave along the focal plane of the microscope, with
that rather high levels of irradiation are required, especially when
a sufficiently high intensity to saturate the emission (though other
the depletion has to occur within a very short time window (as is
saturable transitions can also be used). A cross-section of the
the case when using stimulated emission). This can be mitigated
fluorescence emission along the standing wave pattern looks similar
somewhat by making use of longer-lived nonfluorescent
states51–55,
to Fig. 5a, with ‘fluorescence deactivation’ replaced by ‘fluorescence
but photobleaching or photodestruction is likely to remain a limiting
emission’. By recording a set of images of the same sample, but with
factor in techniques that rely on saturation to create sharp emission
shifted orientations of the pattern, a diffraction-unlimited picture
distributions. Nevertheless, a wide range of fluorescent dyes, including
can be reconstructed (Fig. 7). Moreover, it is possible to to extend
fluorescent
proteins57,58
and ‘atto’
dyes58,
have been found to be stable
the underlying principle to three dimensions, as has recently been
enough to produce impressive results49,59.
demonstrated61,62.
Nonlinear structured illumination
microscopy in that the measurement time is theoretically independent
As already discussed, saturation of the emission rate can be used
of the labeling density and the photostability of the sample determines
directly to achieve a diffraction-unlimited resolution, though it
the performance. They differ, however, in that STED is based on
requires processing of the raw images to discern the underlying sample
confocal microscopy instead of widefield microscopy, and that image
Nonlinear structured illumination is fairly similar to STED
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Fig. 7 Examples of STED and nonlinear structured illumination. (Left) A comparison between a standard confocal image and a STED image of 20 nm fluorescent beads. (Right) A widefield image of 50 nm fluorescent beads. (a, b) Conventional and filtered image; (c) linear structured illumination; (d) nonlinear structured illumination. (Structured illumination data reproduced with permission from60. © National Academy of Sciences, USA.)
processing is essential for structured illumination but not required for
make use of steady-state saturation: the high- resolution information is
STED.
only obtained after the system has reached saturated conditions. While
The exact mechanism by which the resolution is increased, and
this makes for an intuitively clear picture, it is not essential. We will
the required imaging processing is usually expressed in terms of
not consider ‘dynamic saturation optical microscopy’ in detail here, but
Fourier components and spatial frequencies. A detailed discussion
two possible approaches have been discussed elsewhere63,64.
of this is beyond the scope of this paper, but has been published
Nonlinearity in time
elsewhere60. In a sense, the two approaches that we have introduced here both
The problem of limited spatial resolution can be approached in a
Fig. 8 The principle behind PALM imaging: by repeatedly activating, localizing, and bleaching, individual emitters a high-resolution picture of the sample can be reconstructed. (a) A simulated conventional image of the sample. (b) The PALM imaging. (c) The reconstructed picture.
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different way: let us assume that we somehow know that only a single emitting molecule is present in a given diffraction-limited spot. We can easily pinpoint the exact position of this molecule with nanometer precision, simply by looking at the centroid of the emission distribution65,66 (indicated by the markers in Fig. 1). If the density of the labeling molecules is sufficiently low, then this approximation is likely valid, though in that case it would be impossible to obtain any detailed structural information on the sample. If we are interested in subdiffraction microscopy, then our sample should contain many fluorophores within nanometers of each other. Hence, the only way to apply this method would be to separate the emission of the molecules in time, so that at any given moment only a single molecule within a given diffraction-limited spot would emit. Then, by locating and mapping out the positions of all of the molecules in time, a high-resolution image can be constructed, effectively bypassing the diffraction limit. Such a separation would be possible if one could precisely control the distribution of the molecules between the fluorescent and nonfluorescent states over an extended period. Until recently
Fig. 9 An example of localization microscopy. (a) A conventional image of clathrin-coated pits in a fixed cell; (b) a STORM/PALM image of the same region. (Reprinted with permission from 71. © AAAS.)
this task would have been considered virtually impossible, especially for large numbers of dye molecules. Now, however, a numbe rof
a new set of molecules is activated, and the cycle is repeated until
new fluorescent dyes have become available that display highly
the position of (nearly) all molecules has been determined. Because
efficient fluorescence photoswitching or photoactivation31–41.
the activation of the fluorescence is a stochastic process, different
Photoactivation means that the molecule starts out in a thermally
molecules are activated in each cycle, and the probability that two
stable nonfluorescent state but can be converted to a fluorescent
activated molecules will be present within a given diffraction-limited
state through irradiation with light of an appropriate wavelength,
spot can be neglected. Hence, by mapping out all the localized
where it remains until photobleaching. Photoswitching, on the other
positions, a high-resolution image of the sample can be reconstructed
hand, means that the molecule can exist in both a thermally stable
(Fig. 9).
fluorescent state and a nonfluorescent state, and can reversibly
PALM is a fascinating technique in that it does not attempt to
interconvert between these states through irradiation with light
modify the optical capabilities of the instrument in any way. Instead,
of an appropriate wavelength. By using these properties, precise
it sidesteps the issue completely by ensuring that situations where
control over emission of individual fluorescent labels is now possible.
there are two emitters within the same diffraction-limited spot are
However, other processes can be used as well, such as diffusion and
avoided. By this technique, localization precision to within a few tens
docking67.
of nanometers has been demonstrated68,71. The advantages of this
Several related techniques have appeared that make use of
technique are that it is relatively simple, as no special equipment
photoswitching or photoactivation in combination with localization,
is required, and fairly gentle, in the sense that only low irradiation
and are commonly known as ‘photoactivation localization microscopy’
intensities are required. Also, as some of the most promising
(PALM)68,
photoswitchable and photoactivable fluorescent markers are genetically
‘fluorescence photoactivation localization microscopy’
(FPALM)69, or ‘stochastic optical reconstruction microscopy’
encodable fluorescent proteins, this approach is especially useful for
(STORM)70. For convenience, we will refer to these techniques
biological samples.
as PALM microscopy. Briefly, the principle behind PALM, shown
However, the technique is not without its disadvantages: at present,
in Fig. 8, is as follows: a given sample is densely labeled with
the resolution along the optical axis is still fairly limited, and the
photoactivatable or photoswitchable dyes, which are initially in the
requirement for a large number of activation–localization–bleaching
nonfluorescent state. The sample is then weakly irradiated with a
cycles means that a fairly long measurement time (several minutes
small amount of activation light, so that only a very small fraction
or more) is required. Nevertheless, attempts to extend the imaging
of the molecules are activated. These activated molecules are then
to three dimensions71,72 and to use different spectrally shifted
imaged until they deactivate again, either through photoswitching or
labels simultaneously73,74, as well as to increase the speed of the
photobleaching, and their locations are precisely determined. Next
acquisition75–77, are currently underway.
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Outlook and conclusions It does not happen often that a barrier that has stood for over a hundred years is suddenly broken, but that is precisely what is happening today with fluorescence microscopy. Over the course of just a few years, several techniques have appeared that tackle or avoid this limitation in different ways, thereby allowing fluorescence microscopy to cross a familiar frontier into a previously inaccessible world. While the field of diffraction-unlimited fluorescence microscopy is still very young, it has evolved and continues to evolve at a very rapid pace. The validity of the different approaches presented here has been demonstrated, and the experimental focus has started to shift away from the ‘proof-of-concept’ stage to that of a valid and useful research tool. That is not to say, however, that there is a lack of challenges for subdiffraction imaging. For example, until recently, most experiments focused entirely on the imaging resolution along the focal plane of the microscope, using very thin samples, while effectively neglecting the resolution along the optical axis for the most part. By itself, this is not unreasonable, especially considering the recent nature of these approaches. However, in many experiments the ability to image ‘thick’ samples or to construct optical sections is essential. While considerable progress is being made in this direction71,72,78,79, no conclusive approach has emerged so far. A second complication is the time resolution of the acquisition,
This discussion leads to another issue. While diffraction-unlimited
though this is currently mostly a limitation for the PALM-based
fluorescence microscopy has, to some extent, been made possible
approaches. As we have tried to highlight in the paper, the approaches
through technical advances, a perhaps even more crucial aspect has
that aim to exploit nonlinearities to increase the confinement
been the fluorescent label itself. As we have emphasized here, all of the
(RESOLFT, nonlinear structured illumination) have the advantage that
techniques available today rely on special properties of the fluorescent
the duration to acquire an image is always the same, irrespective of the
label. This ranges from exceptional photostability to the presence
labeling density of the sample, and that it is possible to collect fewer
of long-lived nonfluorescent states, or ‘special’ properties such as
photons per molecule, as they do not need to be individually localized.
photoactivation or photoswitching. These processes are not yet fully
In contrast, in PALM-based microscopy, the rate at which molecules
understood, and the dyes that are available today all have their own
can be localized is essentially fixed, and is limited by the requirement
drawbacks, directly limiting the imaging. As such, there is a strong
that each activated molecule should emit enough photons to be
requirement for new dyes with improved properties80.
accurately localized. As such, it remains to be seen whether localization
As for the applicability of these techniques to practical
approaches will prove capable of resolving fast dynamics (minutes or
problems, in the experiments demonstrated so far the focus has
less) on meaningful samples.
mostly been on biological samples, and comparatively little has
But what exactly is a ‘meaningful sample’? To answer this question
been done with respect to the material sciences81,82 (Fig. 10). In
we have to look in closer detail at the imaging itself. Fluorescence
principle, however, these techniques can be applied to any sample
microscopy is interesting in that it never observes the sample structure
that can be studied by conventional fluorescence microscopy. One
directly, but observes the labeling molecules that have been introduced
possible limitation here might be the requirement for special probe
artificially. The ‘structure’ that is observed thus depends on two factors:
molecules, though, with careful experimentation and the knowledge
(i) which part of the sample has been labeled; and (ii) the density of
and labels available today, we feel that it should be possible to
the labeling. Intuitively: if we have an optical resolution of 50 nm but
obtain a significant improvement in resolution for most kinds of
the average distance between the labels is 200 nm, then the resulting
samples.
fluorescence image contains no meaningful information on features
20
Fig. 10 Applications in material science: a composite image showing the three-dimensional colloidal packing of 200 nm fluorescent latex spheres, acquired using STED microscopy. Both well organized areas and defects are clearly visible. (Reprinted with permission from 82. © 2007 American Chemical Society.)
In particular, research areas where these techniques are set
smaller than 200 nm. This is an aspect that has become very relevant
to have a significant impact include nanometer-sized particles or
with the development of high-resolution imaging.
structures such as plasmonic particles, phase transitions in colloidal
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systems, single active-site catalysis, laser-induced lithography and
the techniques that we have presented here have their own strengths
micropolymerization. Similar possibilities exist in polymer dynamics,
and weaknesses, and hence will be more or less suitable for a particular
where the material properties depend strongly on the conformation
experiment. At the moment it is impossible to say which, if any, of
of individual polymer chains, which is hard to obtain directly using
these techniques will eventually turn out to be superior, or if the way
diffraction-limited techniques. With time it should be possible to
forward will be to use them in a complimentary fashion. Perhaps an as-
extend these techniques to most fields where nanoscopic particles or
yet undiscovered approach will take over – it is too early to tell. What
features are important.
can be said, however, is that subdiffraction fluorescence microscopy
There is no doubt that diffraction-unlimited fluorescence microscopy is here to stay, and will continue to evolve rapidly. All of
has opened up an entirely new playing field, and that many untold possibilities await.
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