DE102015015497A1 - Various applications of high-resolution fluorescence-based microscopy methods (RESOLFT / STED et al.) Based methods, such as fluorescence-based nanostructuring - Google Patents

Abstract

A device and method is presented for controlling, controlling and precisely performing atomic resolution nanostructuring / nanomodification and other processes. This is realized by the temporally targeted excitation of spatially precisely located individual fluorophore molecules or atoms, so that they emit fluorescence photons when depleted, these emitted fluorescence photons strike other lying in the immediate vicinity of individual atoms or molecules, thus adjacent to the fluorophore or surrounding atoms or molecules certain processes in atomic dimension (eg their (energetic) excitation or modification, destruction, defragmentation, degradation of their physico-chemical structure or transport / movement of at least individual molecule sections or ablation / removal / removal of individual Atoms / molecules or the like) in order to enable precisely these processes, such as, for example, nanostructuring, nanomodification or information storage / information processing. In this case, this method and the corresponding device based on RESOLFT / STED or other high-resolution microscopy methods, but the photons of the emitted fluorescent light not for the detection or analysis, but for other applications, such as the above-mentioned processes such as nanostructuring / nanomodification, etc. used become.

Description

Introduction:

A method and apparatus, respectively, based on high-resolution fluorescence-based microscopy methods (such as RESOLFT / STED) in which a fluorophore is excited to fluoresce and then emit a fluorescence photon, is used. The emitted fluorescence photon is then not detected as usual by a detector, but is directed to the material surrounding the fluorophore, there to control individual atoms or molecules temporally and spatially precisely and precisely individually to a) whose phys.-chem. Properties with high resolution can be changed at any time and with pinpoint accuracy and / or b) prepared for further steps that are common in micro and nanotechnology.

Various applications are discussed.

State of the art:

Below is an overview of the current high-resolution or super-resolution microscopy methods. Many of them are based on fluorescence techniques.

RESOLFT:

RESOLFT (Reversible Saturable Optical Fluorescence Transitions) comprises the two super-resolution fluorescence microscopy techniques STED and GSD. However, RESOLFT and STED are sometimes equated in the literature.

STED:

The STED (stimulated emission depletion microscopy) method is based on transitions between energetically different electronic states, which occur consecutively [1] - [5].

In order to be able to perform the STED method, it is important that three different (electronic) states or energy levels exist in the sample or in the fluorescent dye labeling the sample ( 1 ):

- The lowest or lowest energy condition Z1:

The lowest energy state is a non-fluorescent or non-fluorescent state. He is also called dark state.

- The mean or energetic middle state Z2:

The energetic mean state is a fluorescent or fluorescent state. He is also called Hell State.

The transition from the non-fluorescent state to the fluorescent state is also called switching on or activation or switching. The transition from the fluorescent state to the non-fluorescent state is also called switching off or switching off or deactivation or switching.

- The highest or highest energy state Z3:

The highest energy state is a fluorescent state, since the atom / molecule can emit a fluorescence photon in this state.

The transition from the fluorescent state to the fluorescent state is called excitation, the transition from the fluorescent state to the fluorescent state is called depletion and can be done by emission of fluorescent light. Radiation-free transitions are also possible (eg quenching).

The activation, excitation, de-excitation and deactivation light may have different wavelengths.

In the STED process, in the initial state, the fluorescent dye molecules (fluorophores) are present on the sample surface in the circular region B1 to be detected in the energetically lowest state Z1 (non-fluorescent state). In a first exposure step, all fluorophores are raised from the non-fluorescent state Z1 to the energetically middle state Z2 (fluorescent state) by incident activating or switch-on light S1 with a corresponding wavelength. In a second exposure step, all fluorophores in the region to be detected are brought from the fluorescent state Z2 into the highest-energy fluorescent state Z3 by incident excitation light S 2 having the appropriate wavelength. In a third exposure step, with a depletion light beam S3, which has a center zero in the center and a nonzero intensity at the edges, the edge region RB1 of the region B1 is stimulated by stimulated emission, so that the fluorophores located in this edge region RB1 are deflected by the fluorescent state Z3 return to the fluorescent state Z2 again. Since the applied Abregungslichtstrahl S3 centrally has a zero, that is so centrally has no intensity, there remain the few or even a single fluorophore in the fluorescent state Z3. These can now themselves spontaneously by fluorescence, ie by emission of a fluorescence photon, return to the fluorescent state Z2. However, it is now also possible by means of a further, additional excitation beam S4 without a central zero point to deplete the fluorophores remaining in the central zero in the fluorescent state Z3 by stimulated emission, so that the time of fluorescence emission can be selected or determined.

The few fluorophores thus remaining in the central zero point in the fluorescent state Z3 then emit spontaneously or stimulate the fluorescence photons, which then reach the detector surface. Since the emission of the fluorescence photons from the fluorophores takes place successively within and outside the central zero, one can thus obtain an image of the surface which has a resolution far below the optical diffraction limit according to Abbé.

The de-excitation process itself, however, is also diffraction-limited from a purely optical point of view: if the depletion were linear with the intensity of the depletion beam, no significant improvement or increase in the resolution according to Abbé would be achieved [5]. However, only by a material effect, namely the saturation of the de-excitation with the Abregungslichtstrahl the resolution limit can be overcome [5]: as stated above, the intensity of the Abregungsstrahls S2 has a central zero point, which is not formed stepwise, but within the cross section Beam profile of Abregungsstrahls the transition from the edge region RB1 with maximum intensity to the center of the centrally stored zero steadily, so that actually only in a very, very small centrally located zero range NB1 of the zero point, the intensity is really equal to zero. However, even the lowest intensities of the Abregungsstrahls sufficient to completely depopulate the fluorescent state Z3, d. H. to transfer all the excited electrons from the excited state Z3 to the excited state Z2. Thus, only in a very, very small subarea, namely in the region of the zero point NB1, the fluorophores remain in the fluorescent state Z3, which corresponds to the center of the zero point of the Abregungslichtstrahls. The diameter of this subregion, namely the region of the zero point NB1, is far below the classical diffraction limit. The falling below the classical optical resolution limit according to Abbé does not take place in a purely wave-optical way, but is achieved by means of a material's own effect, namely saturation.

The depletion beam S2 does not necessarily have to be circular or rotationally symmetric with a central zero. Also conceivable are all other intensity distributions, which, however, must have at least one zero point or at least one local intensity minimum. Also conceivable are several zeros arranged in an array (grid), other non-point geometric figures or a linear zero. The latter would be particularly useful if you have to scan large sample areas and if only one room dimension is interesting for a super-resolved representation (line scan).

If the substances to be tested are autofluorescent, d. H. they fluoresce on their own, without having to add a fluorescent dye (fluorophore) and immobilize, then you can perform the STED and the other processes just without fluorophore, whereby a complex process step (immobilizing the fluorophores) can be omitted.

The optical properties of fluorescence radiation (wavelength, intensity, half-life, polarization, etc.) also depend on the physico-chemical properties of the atomic / molecular environment, in particular the wavelength of the emitted fluorescence radiation is influenced by the atomic / molecular environment (chemical-shift). , Thus, conclusions and information can be drawn or obtained from the detected fluorescence radiation, in particular the wavelength, but also from the remaining optical properties of the emitted fluorescence radiation, via the physicochemical properties and the atomic / molecular structure of the immediate environment of the fluorophore.

In principle, anything that is applicable to normal fluorescence (time-resolved fluorescence measurement, mapping, lifetime imaging nanoscopy (FLIN), etc.) is also applicable to STED.

GSD:

In the case of the GSD (Ground State Depletion) method, the de-excitation beam S3 does not cause any de-excitation from the fluorescent state Z3 to the fluorescent state Z2, but instead a transition from the fluorescent state Z2 to the non-fluorescent dark state Z1 is induced (Case 1), or Transition from the fluorescent state Z3 in the non-fluorescent dark state Z1 induced (Case 2), either before the excitation light S2 excite the electrons from the fluorescent state Z2 in the fluorescent state Z3 (Case 1) or after the excitation light S2, the electrons from the fluorescent Stimulate state Z2 in the fluorescent state Z3 [6] - [8].

The dark state is often a long lasting triple state.

In principle, the STED method is not limited to electronic stimuli and transitions between electron electronic levels, but other types of excitement such as between individual vibrational or rotational levels of the fluorophore molecule appear possible. For example, if the de-excitation process from Z3 to Z2 occurs between two levels of vibration in the same electronically excited state, then instead of a UV fluorescence photon, an IR photon may be emitted; or when the fluorophore is in state Z3, in the form of a high state of vibration in an electronically excited state, then an IR depletion beam S3 or S4 can bring the fluorophore to a lower vibrational state in the same electronically excited state, from which it then Emission of a fluorescence photon goes into the electronic ground state.

Instead of the normal fluorescence transitions in the STED process, X-ray fluorescence can also be used, especially as X-ray radiation has a very small wavelength, which is advantageous for the resolution. In addition, in the context of the fluorescence-based nanostructuring, which will be discussed in detail below, the already known and proven photoresist layers can be used.

Also, other optical or non-optical activation or excitation, z. B. by Raman processes, elastic or inelastic collision or scattering processes with electrons, protons or other particles, conceivable to get from an energetic lower state Z1 to an energetic middle state Z2 or from an energetic middle state Z2 to an energetically high state Z3 , For example, you can u by means of an applied voltage or opto-acoustic processes. a. The fluorophores can be brought from state Z1 to state Z2 or from state Z2 to state Z3, or by electroluminescence one can transport the fluorophores from state Z2 to state Z1 or from Z3 to Z2. However, in these alternative modes of activation and deactivation, by means of which the fluorophore in particular passes from state Z3 to state Z2, one would lose the positional resolution simply because of the missing zero position; Unless one dominates a process in which particles could deliver a part of the energy by inelastic collision or scattering processes to the fluorophore (electronically) on or off, especially from state Z3 to state Z2, because highly accelerated particles such For example, electrons, de Broglie have a wavelength that are far below the optical wavelengths, namely in the lower nanometer range. Instead of zeros, one could use the minima or maxima of diffraction and interference patterns of interfering electrons or the focus of an electron beam for localized excitation or depletion of the fluorophores, since the diameter of the interference fringes or focal diameters due to the de Broglie relationship at high particle energy may be much smaller than the zero of the de-energizing light S3.

If the electrons are accelerated to high velocities to obtain a very low de Broglie wavelength, but have too much energy for the resonant excitation or depletion of the fluorophore, then the electrons can also graze to only a fraction the energy to the fluorophore. Another advantage would be that a grazing incidence would not easily destroy the fluorophore.

Due to the larger mass compared to electrons, the de Broglie relationship with protons and neutrons accelerated to high velocities would allow much smaller wavelengths to be achieved, and thus much smaller focal diameters and narrower interference fringes. however, the danger of destroying the sample or the fluorophore by the incidence of these massive particles also increases. Thus, only dead matter such as semiconductor materials but no sensitive organic samples could be investigated. Particle beams with electrons, protons or neutrons would also be of interest for the later-presented fluorescence-based nanostructuring.

PALM:

Also PALM (Photoactivated localization microscopy) or STORM (Stochastic optical reconstruction microscopy) is based on an activation and deactivation of the fluorescent molecules (fluorophores) [9] - [12]: In the initial state, the fluorophores are present on the sample surface to be examined in the deactivated dark state Z1 , Due to the large-area exposure of the sample surface with activation light of low intensity, a small proportion of the fluorophores is converted into a fluoresceable bright state Z2. It is important to keep the intensity of the activation light so low that only very few fluorophores reach the bright state. For stochastic reasons, these few activated fluorophores are generally relatively evenly distributed on the surface, so that on average there is a distance between two individual activated fluorophores which is a multiple of the optical resolution limit according to Abbé. An increase in the intensity of the activation light would result in a larger number of Convert fluorophores to the activated light state. This would reduce the mean distance between two activated fluorophores. However, this average distance must under no circumstances fall below the optical resolution limit according to Abbé. Upon initial activation of a minor portion of the fluorophore on the sample top, these few relatively spaced apart activated fluorophores are excited to fluoresce by further excitation light excitation. In this case, the excitation light has such a sufficient intensity that as far as possible all activated fluorophores are excited to fluorescence. The excited fluorophores emit the fluorescent light. Since the individual activated fluorophores have a large distance to one another, which is a multiple of the resolution limit according to Abbé, the fluorescent fluorophores appear to the observer or the receiving local resolution camera (CCD / CMOS) individually and isolated and diffraction-dependent as a luminous circular or spherical disk with a circular or spherical radiation distribution (diffraction disc). In an isotropic medium, for reasons of principle and simple symmetry considerations, the fluorophore molecule emitting the fluorescent light must be in the center or center of the luminous spherical disk. Due to the emission of the fluorescent light, these fluorophores are de-energized, in order then to be converted again by deactivation into the deactivated dark state. The emitted fluorescent light is recorded spatially resolved by means of the CCD / CMOS camera: since the fluorescence radiation characteristic of the emitting fluorophore is circular or spherical, the fluorescent light is arranged in a circle or spherically symmetric about the emission site (fluorophore) or in other words, the fluorophore is located precisely in the Center of the luminous circle or Kugelscheibchens, so that the local position of the center of the luminous circle or Kugelscheibchens, which stands for the emission location of the fluorescent light and thus for the local position of the fluorophore, simply by calculating the geometric center of the luminous circular or Kugelscheibchens can be determined. The fluorescence image recorded in this way is first revised computer-aided by further narrowing the actual location of the fluorescent fluorophore molecule by determining the center of the luminous spherical disc for reasons already outlined above, with an accuracy that is far below the optical resolution of Abbé , This is only possible if, as a prerequisite, the distance of the individual fluorescent fluorophores is large enough so that two adjacent luminous spherical disks do not overlap, but appear isolated and circular. The thus revised image is then finally saved. This procedure is repeated and, for stochastic reasons, each time this procedure is repeated, most other fluorophores on the sample surface are usually activated and excited to fluoresce. At each repetition, a different fluorescence frame is taken with the CCD / CMOS camera. After a sufficiently large number of repetitions, the revised, different individual images are then superimposed; and there is (almost) complete imaging of the fluorescence-marked structure on the sample surface to be examined, with a resolution below the resolution limit according to Abbé.

It is obvious that this method requires complex computer-aided calculations which either take a very long time and / or correspondingly powerful computer hardware and software are necessary.

SIM:

A third high- or super-resolution microscopy method is the so-called SIM (Structured Illumination Microscopy) method [13] - [18]. In this case, the surface to be microscopically illuminated with a periodic stripe pattern with a corresponding period with the wave vector k _S (structured illumination), whereby the resolution can be significantly increased. This can be explained clearly in the reciprocal space (k-space) with the aid of the Fourier transform: Without the fringe pattern, the surface of the object to be microscopically has a Fourier spectrum whose wave vectors are all within a zone, for example a sphere, in reciprocal space. which is located in the reciprocal space around the origin of the wave vector coordinate system. Due to the (sinusoidal) structured illumination with the fringe pattern, this sphere is displaced in the reciprocal space around the wave vector k _{S of} the structured illumination, so that it is no longer arranged concentrically around the origin of the coordinate system but in its first quadrant. As a result, all k-vectors of the sphere are vectorially added to the wave vector k _{S of} the structured illumination, so that as a result of this vector addition all k-vectors have a greater length or higher magnitude and thus a smaller wavelength λ because of k = 2π / λ. However, a lower wavelength means, according to Abbé, a higher resolution because of d = λ / NA = λ / nsinα (with d = minimum observable distance, NA = numerical aperture), so that the optical resolution limit is shifted downwards by the structured illumination.

You can also easily observe this effect experimentally (moiré effect).

Further high-resolution or super-resolution microscopy methods are SPDM (Spectral Precision Distance Microscopy), SPDMphymod with "flashing dyes", SOFI (Superresolution optical fluctuation imaging), SALM (Spectrally Assigned Localization Microscopy), 4Pi, SNOM or NSOM (near-field scanning optical microscope) and / or technical derivations and / or combinations thereof or with other microscopy methods [19] - [23].

Nanostructuring:

There are already many processes or processes only structuring of different materials (semiconductors, metals, polymers, ceramics, etc.) on the nanometer scale (nanostructuring). By way of example, the following method is now pointed out: UV-sensitive molecules have been immobilized on the surface of a nanometer-scale material to be patterned. By exposure to UV light whose wavelength is within the absorption range of the UV-sensitive molecules, the UV-sensitive molecules can be activated and excited to initiate a photoreaction and thereby a nanostructuring process. A disadvantage is the precise immobilization of UV-sensitive molecules on the material surface, and precisely at the point where the nanostructuring is to take place. In the fluorescence-based nanostructuring, presented below, the surface of the material to be structured can be covered extensively with fluorophores, in order to produce z. B. optically to control individual fluorophores by means of the STED method, d. H. to activate and stimulate, so targeted targeted the nanostructuring process can start.

Task:

In many application areas of micro- and nanotechnology, the performance of the micro- or nano-components is very much dependent on the spatial density as well as the spatially local resolution of the micro- and / or nanostructures in the components. For example, in microelectronics, integrated circuits (chips) such as processors and memory chips are all the more powerful the greater their density of transistors or switching elements is (see degree of integration in integrated circuits such as VLSI to GLSI). d. H. the closer the transistors are packed, the higher the performance of the integrated circuit. As the integrated circuits are most often fabricated by optical lithography, in the fabrication of such microelectronic devices, Abbé optical resolution is a limit that limits the transistor density of the integrated circuits and thus their performance. It is an object of this invention to provide a method or device that falls below this limit of optical resolution according to Abbé, for. B. to greatly increase the integration density of nanostructures beyond the limit imposed by the optical resolution.

Solution:

The method and the corresponding device is based on a high-resolution fluorescence-based microscopy method, such as RESOLFT / STED. In the method / device according to the invention, precisely this same microscopy method is used to generate a fluorescence photon in a highly localized manner by exciting a fluorophore in a highly resolved manner and thereby emitting a fluorescence photon. However, the emitted photons of the fluorescent light are not used for detection or analysis, i. H. the fluorescence photon emitted by the fluorophore does not pass into a detector, but the fluorescence photon emitted by the fluorophore is branched off in advance, before it would strike a detector surface, and then, for another application, such as e.g. Nano-structuring / nanomodification or information storage / information processing [24]; z. For example, the fluorescence photon emitted by the fluorophore is deflected to modify a molecule in the surrounding material, with a spatial resolution below the resolution limit of Abbé.

Instead of RESOLFT / STED, it is also possible to use other high-resolution fluorescence-based microscopy methods, such as PALM or SOFI, in which a fluorophore emits super-resolved superimposed resolved fluorescence photon on which the inventive method / device is based.

Detailed description of the invention and exemplary embodiments and application examples

As already described in detail in the prior art, in the RESOLFT / STED method, among other things, a small number of fluorescence photons are emitted from a fluoresceable molecule (fluorophore) in a spatially highly resolved and precisely localized manner; and with a spatial resolution down to the atomic scale of a few nm, which is far below the classical optical resolution limit according to Abbé for light. In the RESOLFT / STED method, the emitted fluorescence photons are used for optical detection and subsequent analysis by falling on an optical detector and triggering a corresponding measurement signal; however, these emitted fluorescence photons can also be used for other applications as follows [24]: All applications have in common that they go through a STED process as described in detail under the prior art. As a result, the fluorophore present in state Z3 emits a few high-resolution fluorescence photons by going from state Z3 to state Z2. These are referred to below as the fluorescence photons emitted by the fluorophore. All applications now have in common that they use the fluorescence photons emitted by the fluorophore otherwise than for pure imaging detection. Thus, the STED / RESOLFT process can be used for fluorescence-based nanostructuring and for the realization of actuators, sensors or other devices or methods, as shown below:

I.) Fluorescence-based nanostructuring

Nanostructuring of material is either a modification or ablation of nanoscale material.

By a photon-based modification of a material is meant the photon-induced change of the physico-chemical structure of the fluorophore locally surrounding and irradiated or treated material by the incident photons photolytically and / or pyrolytically stimulate the material and thus a photon-induced Reaction (photochemical or photothermal process) trigger. This leads to a change in the molecular structure such as molecular arrangement or generation of new molecular structures by cleavage or formation of new compounds, eg. B. networking. This goes hand in hand with the change in physicochemical properties. For this purpose, the optical properties (refractive index, transmission, reflection, absorption coefficient, optical activity, polarization, birefringence, non-linear optical properties, etc.), the electrical properties (resistance, conductance, dielectric, electrical polarizability, etc.), the magnetic properties (susceptibility , Magnetization, magnetic polarizability, etc.), mechanical properties (density, strength, stability, etc.), thermal properties (thermal conductivity, coefficient of thermal expansion, heat capacity, etc.), acoustic properties (propagation velocity and acoustic wave attenuation, etc.), physico-chemical properties ( Enthalpy of vaporization, sublimation enthalpy, enthalpy of condensation, wettability, surface tension, adsorption properties and other surface or volume properties, etc.) or chemical properties (reaction enthalpy, reactivity, selective etchability, eg For example, to generate photonic crystals or semiconductor heterostructures, local reaction inhibition, local increase in reactivity or local pretreatment, etc.).

Ablation is the defined removal or removal of material by means of (laser) light without visible thermal damage to the surrounding material. The abraded site is characterized by sharp edges and Abtragsflächen with minimal thermal damage such as debris, so that in micro or nanotechnology, the properties of the surrounding material are not changed.

The fluorescence-based nanostructuring is realized as follows: Here, a high-resolution fluorescence-based microscopy method such as RESOLFT / STED / GSD ( 1 ) (the method is not limited to RESOLFT / STED, but may include all other high-resolution, fluorescence-based microscopy methods, but for simplicity and clarity, the STED or GSD method is chosen here for demonstration): First, by an S1 turn on activation of the fluorophore from the non-fluorescent dark state Z1 into the fluorescent bright state Z2. Thereafter, the fluorophore is converted by incident excitation light S2 in a fluorescent state Z3. Subsequently, the fluorophore and its surroundings are supplied with an excitation light S3, which has a zero point centrally. Thus, and because of the saturation effect already explained above, only the one fluorophore remains in the excited, fluorescent state Z3, in a range well below the classical optical resolution, ie the still excited and fluorescent region is actually much smaller than after the optical resolution limit of Abbe actually possible, while all the surrounding fluorophores have either (as in the case of STED) been degenerated into the fluorescent bright state Z2 but which is not fluorescent, or the same (as in the case of GSD) into the non-fluorescent dark state Z1. The remaining in the fluorescent state Z3 fluorophore now emits spontaneously or stimulated by another Abregungsstrahl S4 a few fluorescent photons by going from state Z3 to state Z2. These fluorescence photons obtained by passing through the STED / GSD process are not used for purely imaging detection by falling into a detector, but the fluorescence photons produced by the STED or GSD process are used to apply them to the photon To direct surrounding material in order to apply this highly disrupted to the fluorescence photons emitted by the fluorophore, so that it is structured highorts resolved by either locally modified or locally removed, controlled, targeted and controllable with a Atomic scale resolution far below the optical resolution limit of Abbé.

The following nanostructuring and / or nanomodification proceeds as follows:
Through the previous STED process (see above and described in detail in the prior art), the fluorophore emits a small number of fluorescence photons. These are not used in this case for the detection, but they spread and hit the atoms or molecules of the surrounding, adjacent material from which they are absorbed and energetically excited, that are raised to a higher energy state. The excitation can be either photolytic or pyrolytic in nature, ie the excitation can be either electronic, vibratory or rotational. This initiates a photo-reaction that alters the physicochemical structure of the surrounding material. The photo-reaction can either be a photochemical or a photothermal reaction or a mixed form or hybrid form of both (photochemical and / or photothermal) or a multi-stage reaction composed of several individual photochemical and / or photothermal reaction stages is. This can be done for example by a direct bond breaking (photolytic) or by photon-induced vibrational or rotational movements of individual molecule groups (pyrolytic) or by modification of the steric structure or by cis-trans isomerism, [25] in which the emitted fluorescence photon the target Transferred from a cis state to a trans state or vice versa. It will be described later that this process can be used for information storage where the cis state can be interpreted as binary 1 and the trans state as binary 0 or vice versa. Other possible photo-reactions are Norrish I or II, inter alia on adjacent polymethacrylate molecules.

Also, a total defragmentation or degradation appears theoretically possible, so that there is a well-defined removal of the material on the nanometer scale at a certain point in this area. This leaves empty, material-free areas of a precisely defined size, shape and peripheral areas without thermal damage, which due to quantum effects change the nanophysical and / or nanochemical properties of the material thus treated, such as the absorption spectrum and thus the color impression. For example, photonic crystals with certain meta-optical properties such as negative refractive index can be produced thereby; or the electronic bands of the crystal structure may be modified to produce semiconductor heterostructures such as quantum dots, quantum wells or quantum wells. Also, the modification of the physicochemical properties of crystal structures seems possible.

For the excitation of the fluorophore different mechanisms come into question:
down-conversion: The photons of the excitation light have a higher quantum energy than the photons of the emitted fluorescence light: thus, the fluorophore can be excited by high-energy UV radiation, the wavelength of the emitted fluorescence light in the UV / Vis, visible or (near) IR spectrum lie (down-conversion, Stokes). This may be important for pyrolytic excitation of photothermal processes.
up-conversion: The photons of the excitation light have a lower quantum energy than the photons of the emitted fluorescence light; ie it is also conceivable that the excitation light has a lower quantum energy than the emitted fluorescent light (up-conversion, anti-Stokes), so that, for example, the fluorophore is excited by visible or IR light and thus emits high-energy UV light as fluorescent light is whose quantum energy is sufficiently large to stimulate electronic bonds photolytically and break directly and / or trigger another photochemical reaction, and thus the surrounding material in the impinged sites atomically accurate and targeted to modify and / or remove.

A multi-photon excitation of the fluorophores is conceivable, for example, from several photons from the near IR spectrum by a fs laser, so that the emitted fluorescent light is in the UV range and whose photons have sufficiently high photon energies for nanostructuring. In contrast, multi-photon nanostructuring, in which several emitted photons of the fluorophore together and at the same time cause a modification process of the surrounding material, is unlikely.

Multilevel processes are also conceivable in which structuring takes place indirectly or indirectly. For this purpose, photoresist layers can be used with or without photoinitiators, which serve for example as a protective layer for selective etching or as a reaction layer or as a sacrificial layer or as a functional layer. Other typical micro-or nanotechnology operations, such as sputtering or molding, may also join or be combined with it. In addition to photoresist layers of novolaks especially photoresist layers of UV-modifiable polymethacrylates appear particularly suitable at which Norrish reactions to refractive index change at atomic sites can be carried out [26]. For this purpose, the photo-layers with the fluorophores should then be doped or provided accordingly.

Thus, photo-modifiable / photoablative materials stored around the fluorophore, such as photoresists with or without photoinitiators, can be acted upon by the emitted fluorescence photon and locally modified or ablated locally in atomic resolution or otherwise altered by a photo-reaction triggered thereby.

In addition to the targeted triggering of certain chemical reactions on individual molecules (eg biomolecules), fluorescence-based nanostructuring can also be used to prepare simple and complex nanostructures, such as nanostructures. B. thin layers, voids, grid u. a. generate, which can be used inter alia for the further construction of nanomachines, or it can already existing nanosystems, such as biological systems, eg. Cells, modified; Thus, a genetic modification also appears possible if the fluorophore is specifically attached to a specific site on the DNA molecule.

II.) Fluorescence-based actuator

• – zur allgemeinen örtlich hochaufgelösten Beleuchtung eines anderen Objektes, um dieses mit wenigen Photonen zu beaufschlagen, damit z. B. die Photonen am anderen Objekt kontrolliert und gezielt gestreut werden, damit z. B. das andere Objekt „hell erscheint und so gemessen werden kann; auch eine Raman-Streuung oder eine weitere Fluoreszenz an anderen Objekten erscheint möglich
• – zum örtlich hochaufgelösten Lichteintrag/Lichteinkopplung in das Umgebungsmaterial: durch die gezielte Beaufschlagung der Umgebung (z. B. von benachbarten Molekülen) mittels der vom Fluorophor emittierten Fluoreszenzphotonen werden diese durch die benachbarten Atome/Moleküle absorbiert, um dort eine photonen-induzierte Reaktion, insbesondere eine photochemische/photolytische Reaktion oder Prozess, auszulösen; dabei kann es beispielsweise um die bereits weiter oben ausführlich diskutierte fluoreszenz-basierte Nanostrukturierung handeln, bei denen ganze Bereiche im Nanometermaßstab bearbeitet, modifiziert oder abgetragen werden, oder es kann um einzelne Moleküle handeln, die durch die Absorption der emittierten Fluoreszenzphotonen gezielt zu einer photochemischen Reaktion oder zu einem weiteren RESOLFT/STED-Prozess angeregt werden
• – zur örtlich hochaufgelösten Energieübertragung von einem Fluorophor/Molekül auf ein benachbartes Fluorophor/Molekül und von diesem Fluorophor/Molekül wiederum auf ein weiter benachbartes Fluorophor/Molekül oder von einem Fluorophor oder Nanomaschine auf eine andere benachbarte Nanomaschine; so kann ein minimaler Energiefluss in der Größenordnung von wenigen Energiequanten hinsichtlich Energiemenge und -richtung kontrolliert gesteuert werden
• – zum örtlich hochaufgelösten Energieeintrag oder -abfluss, beispielsweise in ein molekulares System oder in eine Nanomaschine, um diese mit Energie zu versorgen, damit diese arbeiten und ihre Funktion verrichten kann
• – zur örtlich hochaufgelösten Informationsübertragung (dies wird später ausführlich diskutiert)
• – zur örtlich hochaufgelösten punktuellen Erwärmung, beispielsweise um photothermische Prozesse auszulösen

The already extensively discussed fluorescence-based nanostructuring can already be interpreted as the first example, in which the RESOLFT / STED / GSD process is already used for the realization of an actuator, since the fluorophore in state Z3 is the fluorescence photon used for nanostructuring emitted, as an actuator can be construed. In addition to the fluorescence-based nanostructuring presented in detail above, the STED / RESOLFT process can also be used to implement further actuators:
As described in detail above, the STED process or a comparable process is run through. After the excitation radiation has been applied, only one or a few fluorophores remain in the fluorescent state Z3 in the region of the central zero point of the de-excitation beam S3. These fluorophore (s) now emit one or more fluorescence photons, which in this case are not used for detection, but serve as a point source of light

• - To the general local high-resolution lighting of another object to apply this with a few photons, thus z. B. the photons are controlled on the other object and scattered specifically, so z. B. the other object "appears bright and so can be measured; also Raman scattering or further fluorescence on other objects appears possible
• By localized exposure of the environment (eg of neighboring molecules) by means of the fluorescence photons emitted by the fluorophore, these are absorbed by the neighboring atoms / molecules in order to generate a photon-induced reaction there, in particular a photochemical / photolytic reaction or process trigger; This may be, for example, the fluorescence-based nanostructuring discussed in detail above in which entire regions are processed, modified or ablated on the nanometer scale, or they may be individual molecules that specifically target a photochemical reaction as a result of the absorption of the emitted fluorescence photons or to another RESOLFT / STED process
• For locally highly resolved energy transfer from one fluorophore / molecule to an adjacent fluorophore / molecule and from that fluorophore / molecule in turn to a further adjacent fluorophore / molecule or from one fluorophore or nanomachine to another adjacent nanomachine; Thus, a minimal energy flow in the order of a few energy quanta in terms of energy quantity and direction can be controlled controlled
• - To locally high-resolution energy input or outflow, for example, in a molecular system or in a nanomachine, to provide them with energy, so that they can work and perform their function
• For locally high-resolution information transmission (this will be discussed in detail later)
• - For local high-resolution point heating, for example, to initiate photothermal processes

To illustrate, a few selected application examples are described in detail below:

1.) Fluorescence-based data storage or data processing

Here, a method based on the STED process is proposed in order to enable digital building blocks in a very small space for a) switching of molecules (eg from a cis to a trans state and vice versa) and thus b) storage and / or storage possibly processing information. Here, the dimension or density is critical, because the smaller the dimension, the more powerful the data processing unit.

As described in detail above, the STED process or a comparable process is run through. After admission of the Abregungsstrahlung S3 remain only one or a few fluorophores in the fluorescent state Z3 in the region of the central zero point of the Abregungsstrahls S3. These fluorophore (s) now emit one or more fluorescence photons, which in this case are not used for detection, but which spread and strike neighboring molecules of the surrounding material. These neighboring molecules of the material surrounding the fluorophores have the properties of changing their state by incidence of a fluorescence photon, ie, being switched from one stable state to another stable conformation state or switching. These may be, for example, isomeric states, for example the cis-trans isomerism, for example of cyclohexane or derivatives. By applying the fluorescence photon emitted by the fluorophore to the neighboring molecule, it is precisely this acted upon molecule that changes from one isomeric state into the other, for example from the isomeric trans state to the isomeric cis state. This can be understood as storage of an information unit (bit), wherein, for example, the isomeric trans state stands for the digital information "0" and the isomeric cis state stands for the digital information "1". Of course, the isomeric states can also be interpreted inversely. If many cis-trans isomers are arranged and defined in the vicinity of the fluorophore or the fluorophores and are targeted and acted upon by the emitted fluorescence photon, then all of these cis-trans isomers assume the desired isomeric state, in each case for one Bit information is available. Thus, bit information can be stored, processed and read out again in the smallest space per isomer.

Also, information processing seems to be possible on this basis: if two fluorophores have access to the same cyclohexane molecule (or to another molecule that possesses the cis-trans isomerism), then the following procedure can be imagined: if both Do not emit fluorophores, then the cyclohexane molecule remains in its initial position, emits one of the two fluorophores, so switches the cyclohexane molecule, emit both fluorophores (almost) simultaneously, the cyclohexane molecule switches back to its original state; the switching behavior of the cyclohexane molecule under the influence of the two fluorophores corresponds to an exclusive OR truth table. The question remains as to what happens if both fluorophores act on the cyclohexane molecule exactly at the same time.

In addition to the cis-trans isomerism, other embodiments are possible and conceivable; Thus, for example, for the purpose of information storage on other Photoisomerie- or conformational isomerism states can be used. However, these would also have to be switchable and time-stable by means of an emitted fluorescence photon.

2.) Fluorescence-based nanoactuators / nanomachines

The STED method can also be used to operate or switch nanomachines or nanoactuators. Nanomachines or nanoactuators are machines with dimensions in the nanometer range that can perform mechanical work, in an area that is precisely localized to a few nanometers. The cis-trans isomerism can also be used in this application example: For switching or operating the nanomachines in atomic dimensions by the incident fluorescence photons emitted by the fluorophore or for carrying out actuator tasks, the cis-trans isomerism of the adjacent ones can also be used here Molecules can be exploited: by switching from the isomeric cis state to the isomeric trans state or vice versa, for example, work can be done in the smallest space. As already described in detail above, the STED or a comparable method is used for this purpose. After exposure to the Abregungsstrahlung S3 remain only one or a few fluorophores in the fluorescent state Z3. These fluorophore (s) now emit one or more fluorescence photons which propagate and strike adjacent molecules of the surrounding material. As a result, as already described above, the adjacent molecules are switched, for example, from the cis state into the trans state. As this moves mass in the form of molecular sections, mechanical work is thereby performed. This can be used to mechanically move other molecular entities or to supply energy to other molecular entities or nanomachines in mechanical form to perform mechanical work, to switch other molecular entities or other nanomachines, or to other molecular entities or nanomachines to submit or transmit essential information.

As a concrete example, it can be stated that the molecule in the cis state can then function as a nano-lever in the starting position, while the same molecule in the trans state serves as a nano-lever in the working state. Targeted switching is then accomplished by the STED method in that the emitted fluorescence photon transfers the molecule from the cis- to the trans-state and vice versa.

Not only is this process restricted to cis-trans isomerism, but any state or photoisomerism that alters the configuration of the molecule by the incidence or absorption of light quanta can be used.

3.) Fluorescence-based atomic or molecular light and / or energy source:

The fluorophore may serve as an atomic or molecular source of light or energy to illuminate surrounding material or to selectively deliver energy to an atomic or molecular entity to electronically excite, or to perform, or transmit information to.

For nanotechnology are also atomic or molecular light sources of interest, which operate according to the STED method and therefore can emit at a precisely defined location at a precisely defined time a well-defined number of photons with a well-defined quantum energy, for example one FRET process z. B. for a nanometer scale [27]. For this purpose, the fluorophore is activated and excited at the right time with the STED process in order to emit such a photon:
As already described in detail above, the STED or a comparable method is run through. After exposure to the Abregungsstrahlung S3 remain only one or a few fluorophores in the fluorescent state Z3. These fluorophore (s) now emit one or more fluorescence photons which propagate and strike adjacent molecules of the surrounding material.

In order to select the correct emission time of the fluorescence photon, the fluorophore remaining in the zero state in the fluorescent state Z3 can be acted upon by the stimulating emission light beam S4 after the de-excitation light S3, so that the fluorescence light emits the fluorescent light at the desired point in time by stimulated emission.

Such atomic light sources can also be used in combination to specifically stimulate optical resonance modes in adjacent microparticles or nanoparticles by the STED method (for example optical modes in the volume or in cavities of the particle, between its reflecting surfaces on the inner and / or outer surfaces). or outer sides, or modes in the particle-comprising spherical transparent layers can be excited or Whispering Gallery modes can be stimulated [28]). The micro- or nanoparticles can then be used, for example, as label-free (bio) sensors (for example, for the precise detection of individual target analytes or for the atomically accurate determination of the pH value) or as a detector with a spatially atomic resolution for external influences, the Change resonator conditions, e. G. Pressure or temperature). Since the STED method is also used in this application, a spatial resolution of the target analyte to be detected or of the parameters to be detected can take place on an atomic scale.

In addition to being used as a biosensor, the fluorophores can also use the STED method to mark the cantilever tips of an AFM or another scanning electron microscope or a field electron microscope atomically precisely in order to be able to calibrate them before the first measurement.

It can also be envisaged that two adjacent fluorophores emit the fluorescence radiation synchronized with the same phase by the stimulation Abregungslichtstrahl S4 are applied simultaneously after the de-excitation light S3 remaining in the Z3 in the state Z3 fluorophores, so that by stimulated emission both fluorophores simultaneously with a constant phase relationship emit the fluorescent light. Such a device can not yet be interpreted as a "nanolaser" because the resonator and thus a preferential propagation direction are missing. Maybe it is a "Nanosuperstrahler". However, if one integrates the two or more fluorophores within a quantum film or within a boundary layer of a semiconductor laser having reflective inner surfaces, then perhaps the emitted fluorescent light may be given a direction.

III.) Fluorescence-based sensor

As sensors, components or even individual molecules can be designated, which are suitable for measuring certain physical or chemical quantities that characterize an object or a process, by detecting them by means of an effect and generating and outputting a corresponding measurement signal.

The STED process can be used to realize high-sensitivity nanoscale sensors that have high or super-resolution resolution. The sensors are in the form of fluorescent molecules.

• – bei der ersten Funktionsweise beeinflusst die unmittelbare atomare oder molekulare Umgebung die physikalischen und/oder chemischen Eigenschaften des fluoreszierenden Sensormoleküls wie beispielsweise Fluoreszenzwellenlänge, Fluoreszenzintensität, Fluoreszenzdauer, Phase des emittierten Fluoreszenzlichts, so dass aus dem emittierten Fluoreszenzlicht des Fluorophors Rückschlüsse auf die unmittelbare physikalische und/oder chemische Umgebung geschlossen werden können. Dabei kann man sowohl kinetische als auch thermodynamische Vorgänge mit oder ohne Stoff- oder Phasenumwandlung untersuchen.
• – bei der zweiten Funktionsweise wird das fluoreszierende Sensormolekül entweder an ein mobiles oder bewegtes Medium oder an ein stationäres oder ruhendes Medium fixiert, so dass im ersten Fall der fluoreszierende Sensor mit dem mobilen Medium mitbewegt und im zweiten Falle der fluoreszierende Sensor im oder auf dem stationären Medium ruht. Dabei fungiert der fluoreszierende Fluorophor als Sender. Somit kann man im ersten Falle die Bewegung des Mediums nachverfolgen und somit Informationen über deren Kinematik und/oder Dynamik erhalten, oder man kann im zweiten Falle erkennen, wenn ein anderes Medium sich über dem stationären Medium ausbreitet oder sich darüber hinwegbewegt, um somit die Bewegung eines anderen Mediums anzuzeigen.

There are two types of sensors with different functions:

• In the first mode of operation, the immediate atomic or molecular environment influences the physical and / or chemical properties of the fluorescent sensor molecule, such as fluorescence wavelength, fluorescence intensity, fluorescence duration, phase of the emitted fluorescence light, so that conclusions can be drawn from the emitted fluorescent light of the fluorophore to the immediate physical and / or chemical properties. or chemical environment can be closed. It is possible to study both kinetic and thermodynamic processes with or without material or phase transformation.
• In the second mode of operation, the fluorescent sensor molecule is fixed either to a mobile or moving medium or to a stationary or stationary medium, so that in the first case the fluorescent sensor moves with the mobile medium and in the second case the fluorescent sensor in or on the stationary one Medium is resting. The fluorescent fluorophore acts as transmitter. Thus, in the first case, one can track the movement of the medium and thus obtain information about its kinematics and / or dynamics, or it can be seen in the second case, if another medium spreads over the stationary medium or moves over it, thus the movement of another medium.

Fluorescence-based sensor according to the first mode of operation

As already described in detail above, the STED or a comparable method is run through. After application of the depletion radiation S3, only one or a few fluorophores remain in the fluorescent state Z3 in the region of the central zero point. These fluorophore (s) now emit one or more fluorescence photons, which hit the detector surface and with which one can produce a spatially super-resolved image.

In the sensor according to the first mode of operation, the characteristics of the fluorescent light emitted by the fluorophore (wavelength, intensity, polarization, half-life, phase shift, etc.) also depend on the physico-chemical properties of the immediate atomic or molecular environment, such as the adjacent bonding ratios of the chemical environment since the physico-chemical properties of the molecular environment have an influence on the electronic structure or properties of the fluorophore molecule, which in turn influences the properties of the emitted fluorescence light (wavelength, intensity, half-life, phase shift, etc.), so that emitted fluorescent light, the physico-chemical environmental properties can be determined and thus z. B. conclusions on the bonding conditions in the immediate vicinity in atomic resolution are possible. A special example is the quenching.

This allows a variety of applications:
Since one can draw conclusions on the physical-chemical properties of the immediate atomic / molecular environment by means of the detected fluorescent light on the basis of their optical fluorescence properties such as fluorescence wavelength and duration, specifically with a spatial resolution on the nanometer scale, it seems possible, together with the pure spatial high-resolution location information for the purely imaging detection and representation to provide a spatial distribution of the physicochemical properties in atomic resolution (mapping).

The following application examples in the following subject areas seem conceivable:

without substance conversion:

It is possible to differentiate between materials in the field of kinetics and the field of thermodynamics with regard to the material examination without conversion of the substance by means of the sensor according to the first mode of operation:

kinetically:

• Kinetics, diffusion, osmosis, absorption, adsorption, physisorption, chemisorption, crystal growth, defect formation in the crystal lattice (semiconductor crystals: pitted defects, etc.), cracking, electrophoresis, mixing, quenching, convection, acoustic and / or thermal effects, corrosion, possibly also plasma possible

thermodynamically:

• Phase transformation, condensation, sublimation, phase transformation also in the superconductor, condensation, dissolution in solutions, mixing processes, crystallization, absorption, adsorption, transformation from one state of aggregation to another state of aggregation

technical or engineering scientific processes or processes:

The fluorophore is incorporated as a marker in a micro or nanodevice or is itself active as an active element, for example, for process monitoring during its own production or during operation, monitoring the Aging, changes in functional properties during operation, misalignment, wear, destruction

with material conversion:

It is possible to distinguish between material conversion applications using the sensor according to the first mode of operation between applications in the field of kinetics and from the field of thermodynamics:

kinetically:

Reaction kinetics, chemical reaction course between reaction partners: the fluorophore serves as a marker or is itself an active reaction partner, catalysis, nuclear chemical reactions

In general, the following fields of application can be distinguished from one another with respect to the sensor according to the first mode of operation:
Surface or volume effects, phenomena at the phase boundary or interface phenomena between the same or different phases and / or states of aggregation, investigations in the solid, liquid or gaseous state or in the plasma, or in not clearly defined physical states such as glass, sand, powder, etc.

The following concrete application examples are conceivable, but are only a limited selection:
It may be physical, chemical or biological properties, sizes and / or conditions or technical, natural and / or engineering parameters of static (temporally non-changing) or dynamic (time-varying) technical, physical, chemical or biological systems (natural or artificial), such as substances, materials, homogeneous or heterogeneous material systems, reaction systems, biological cells, technical devices and processes / processes in them (eg phase transformation, chemical reactions) non-time resolved or time resolved on an atomic or molecular scale in different Physical states / phases are determined.

After the RESOLFT / STED / GSD process has been run through several times as described above, a fluorescence photon emitted by the fluorophore is generated whose optical fluorescence properties, such as fluorescence wavelength or duration, and the like, are determined. a. Contains information about the chemical environment of the fluorophore and which are evaluated. One can equip a solid surface (crystalline, amorphous, semiconductor, metal, polymer, (bio-) chemical substances, etc.) with these fluorophores and thus the bonding conditions of z. B. by physisorption / chemisorption / Van der Waal forces deposited atoms or molecules.

It is thus possible to measure the surface states and also to follow the entire absorption or adsorption occupation or desorption process in time and space (sorption isotherm). It is also possible to indicate the change in the area above, z. B. when gas, steam or other fluid flows over the surface or within a flow channel, on whose inner surfaces the fluorophore has been immobilized. The particle or medium or fluid flowing past the fluorophore interacts with the fluorophore. This changes the chemical environment of the fluorophore and thus also the fluorescence properties of the fluorescence photons emitted by it, such as fluorescence wavelength or duration. In the case of transparent solids (glass, polymers), the fluorophores can also be bound in the material volume, or at least just below the surface, in order to find out about the bonding states in the material volume or just below the surface. It is also possible to attach the fluorophores to micro- or nanoparticles (microbeads) which are located in a fluid and dissolve in a liquid flow). Fluorophores do not necessarily have to be attached to solid surfaces, but can also be connected to liquid molecules or gas molecules. Optionally, the fluorophores can also be used in a plasma; or else the fluorophores may also be incorporated in the form of a ligand or a central particle into a complex compound or generally into a molecular segment of any (biochemical or synthetic or other) molecule to learn about the binding relationships within the molecule.

It is not only static situations that can be described, but also dynamic processes in which the physical-chemical properties of the environment change over time: for example, the fluorophores can be attached to a reaction partner (starting material) in the form of a label or marker undergoes chemical reaction. It is also possible that the fluorophore itself is a reaction partner. In any case, it is possible to draw in-situ conclusions on the temporal change in the bonding conditions in the course of the chemical reaction on the basis of the emitted fluorescence radiation. The chemical reactions can be any kind of chemical reactions; These include biochemical reactions (eg polymerase chain reaction) as well as technical reactions from technical chemistry (eg polymerisation). Nuclear reactions are also conceivable, if by radioactive radiation the Core number and thus the chemical element changes, this also has an influence on the fluorescence properties of the fluorescent radiation emitted by the fluorophore. Analogously, the temporal change of physical-chemical properties during the course of biological, physical, geophysical, geological, mineralogical processes or processes (phase transformation) can be tracked in-situ on a nanometer scale.

Another large area of application is engineering, in particular micro and nanotechnology. By a sensor according to the first mode of operation, errors in the production or production process of microstructures or nanostructures can also be found. The fluorophores can also be used for quality assurance or control during the production of microstructures or nanostructures; or fluorophores incorporated into micro- or nano-components are used for process monitoring to in-situ check the functionality and aging processes of operating micro- or nanostructures. Process monitoring also includes recognizing changes in functional properties during operation, misalignment, wear or destruction. The fluorophores themselves are incorporated as markers or themselves act as active components in the micro- or nanodevice.

If necessary, the fluorophores can also trigger a desired repair or self-destruct process of the micro- or nanostructure.

All of this also applies to biological, biochemical or chemical systems such as microreactors, nanoreactors or biological cells.

Fluorescence-based sensor according to the second mode of operation

In the sensor according to the second mode of operation, the fluorophore serves merely as a light source that emits fluorescent light with unchanged properties, uninfluenced by the local physical-chemical environment.

As has already been described in detail above, the STED or a comparable method is run through. After application of the depletion radiation S3, only one or a few fluorophores remain in the region of the zero point in the fluorescent state Z3. These fluorophore (s) now emit one or more fluorescence photons. Several different applications are possible, as discussed in more detail below:

cover sensor

The surface of a stationary object is equipped with fluorophores that emit super-resolved fluorescent light. This fluorescent light is detected super-resolved by a detector. A change in the detection signal occurs only when the fluorophore is partially or completely obscured by another mobile object (gas or liquid flow, moving solid particles of larger or smaller diameter such as sand, powder, etc.). From the detection signal, one can then determine the time of the vorbebeiflugs passing the passing particle or in the case of many fluorophores arranged in the form of a two-dimensional field on the surface of the stationary object, one can measure the exact location coordinates of the passing particle in atomic resolution. With partial obscuration of many smaller particles passing by, one can perform a particle density measurement, or in the case of passing liquids or gases, one can perform transmission or absorption or reflectance measurements super-resolved; or attach to the inside / face of μ-fluid devices or μ-fluid channels or biological cells to indicate the presence of fluids and to study their nanometer flow behavior.

Tracking of objects

The fluorophore is immobilized on a mobile object. Now, when this mobile object sets in motion and the fluorophore now emits fluorescence radiation, then the constantly changing location of the fluorophore and thus the mobile object, with which it is firmly connected, and thus track its trajectory in situ and currently, and although in atomic resolution far below the Abbé resolution limit (high-resolution tracking).

It is also possible to attach the fluorophores to micro- or nanoparticles (microbeads) and to introduce them into a liquid flow) or else the fluorophore itself is in liquid form and is thus itself part of the liquid flow. Thus, one can follow the course of the entire liquid on the nanometer scale. The flow may flow to Bernoulli and may be a stratified flow (Newtonian fluid), or it may be a turbulent flow. This can be used to determine the shear behavior, especially at the interface with other liquids or to the wall of the liquid-carrying microchannel, nanoscale turbulence, the internal friction (viscosity) or the Reynolds number (transition point from laminar to turbulent flow) and other rheological effects super-resolved. It must either the Abregungsstrahl S3 are moved to zero or the corresponding optics, or there are many fluorophores immobilized, and the Abregungsstrahl always hits only a certain place.

dosimeter

In this application, single UV photons can be detected at high resolution, which are used as excitation light in PALM or STED. Unlike the PALM or STED method, in this case the detectors that detect the emitted fluorescence photon are not used to obtain an image of the sample in super-resolution, but here the goal is that the fluorophore itself be used as a sensor, to display the existence and amount of incident UV excitation light spatially resolved. If even a small amount of UV excitation light is incident on the surface containing the fluorophore, this will be manifested by fluorescence appearance if the sites with activating light S1 have previously been turned on and optionally (in the case of STED) descent light S3 if necessary (with a zero) has been used to provide only single point sensitive to the incident UV light. Otherwise, the sample remains dark, because if no UV excitation light S2 is incident on the fluorophore-loaded sample surface or the corresponding sites have not been switched on with activation light, then the fluorophores will also emit no fluorescent light. The advantage with the STED method is that individual points of the sample can be switched on and off in an atomic dimension, so that these individual points can be activated or deactivated for detection by a previously unseen spatial resolution of the distribution of the excitation light S2 to reach. In practice, this is a spatially resolving dosimeter with a spatial resolution far below the optical resolution limit to u. a. also to study the intensity distribution within the excitation light beam.

The SIM method is also suitable for this type of application, since the exposure to periodically structured light can considerably increase the resolution.

IV.) Other applications

1.) Fluorescence-based labeling (identification / authentication / information carrier)

As already described in detail above embodiments, the STED or a similar method is run through. After application of the depletion radiation S3, only one or a few fluorophores remain in the region of the zero point in the fluorescent state Z3. These fluorophore (s) now emit one or more fluorescence photons.

Not only biological molecules, but also molecules of engineering materials / materials in particular of micro or nanomachines (or of other devices which have only a very, very limited surface of the order of a few atomic diameters, for example cantilever tips of an AFM or Field electron microscopes) can be atomically or locally marked using the STED method in order to store information about this material / material or machine (manufacturer, date and country of manufacture, product or serial number, production or process parameters, chemical composition, physico-chemical, functional and / or technical properties / features, etc.), in order later to permit identification and / or authentication and / or follow-up thereof by means of an atomic local retrieval of the information by means of the STED method.

3D printing / stereolithographic printing can also be used specifically for this application or in general for any other application, so that the fluorophores can be precisely positioned not only on the surface, but also in the bulk material itself at atomic scales.

2.) Optical trap

As already described in detail above, the STED or a comparable method is run through. After application of the depletion radiation S3, only one or a few fluorophores remain in the region of the zero point in the fluorescent state Z3. These fluorophore (s) now emit one or more fluorescence photons.

If the fluorophore has now been inserted in an optical trap, then by means of the STED method, the position of the optical trap (such as in DE 10 2014 005 219 A1 exactly localized). One can then closely track the laser-beam-based cooling of individual atoms or molecules accurately. This is important because the laser beam used to cool the fluorophore or the molecule labeled with the fluorophore must have just the right phase relationship in the right place so that the fluorophore / molecule is also cooled by depletion. Otherwise, if the STED measurement results in an inappropriate spatial position, the phase of the incident laser beam or the position of the atom to be cooled / molecule must be readjusted via a feedback signal.

Instead of STED, other technologies may also be used to locate below the diffraction limit.

3.) FRET pair (donor and acceptor)

One can also combine the STED process with the FRET (Förster resonance energy transfer) process. The fluorophore is the acceptor or donor in the FRET pair (consisting of donor and acceptor) or the FRET pair in its entirety serves as a single fluorophore. The FRET process allows conclusions to be drawn about the physical-chemical environment.

As already described in detail above, the STED or a comparable method is run through. After application of the depletion radiation S3, only one or a few fluorophores remain in the region of the zero point in the fluorescent state Z3. These fluorophore (s) now emit one or more fluorescence photons that strike a spatially resolving detector, resulting in a high resolution image of the sample.

The fluorophore can be present as a FRET pair; or the fluorophore may be present as an acceptor of the FRET pair, then the excitation of the acceptor by means of the STED process is highly resolved, d. H. the resonance energy transfer between the acceptor and the donor takes place after the depletion beam S3 has narrowed the position of the acceptor by means of the zero, and thus the emission of the fluorescent light of the donor is also very high resolution; or the fluorophore may be present as a donor of the FRET pair, then the emission of the donor by the STED process is highly resolved after the Abregungsstrahl S3 has de-energized the environment of the fluorophore or donor.

If the fluorophore is present as a FRET pair or as an acceptor or donor of the FRET pair, then the high-resolution location information can be combined with the corresponding information about the physicochemical environment at the same location. In this way, a super-resolved spatial distribution (mapping) of the physicochemical properties of the examined sample surface is obtained.

Actually, this represents a special case of a sensor according to the first mode of operation.

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Legend:

1 : Electronic Termscheme in the RESOLFT / STED process

2 : Principle of SIM illustrated in reciprocal space

QUOTES INCLUDE IN THE DESCRIPTION

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Cited patent literature

• DE 102014005219 A1 [0093]

Claims (1)

Use of an optically non-diffraction-limited, optically high- or super-resolution microscopy method and / or apparatus such as RESOLFT, STED, GSD, PALM, FPALM, PALMIRA, STORM, dSTORM, SIM, 3D-SIM, SMI, SPDM, SPDMphymod with "flashing dyes" , SOFI, SALM, 4Pi, 4Pi-STED, TIRF, 3DLIMON, LSI-TIRF, SNOM and / or engineering derivates and / or combinations thereof or other microscopy methods such as FCS or FCCS for fluorescence-based nanostructuring and other applications require a high or super-resolution.

Images