WO2003060610A1

WO2003060610A1 - Methods and systems for microscopic imaging

Info

Publication number: WO2003060610A1
Application number: PCT/EP2003/000335
Authority: WO
Inventors: Ralf Wolleschensky; Michael Kempe; Magued B. Nasr; Ayman F. Abouraddy; Mark C. Booth; Bahaa E. A. Saleh; Malvin C. Teich; Alexander V. Sergienko
Original assignee: Carl Zeiss Jena Gmbh; Trustees Of Boston University A Massachusetts Corporation
Priority date: 2002-01-16
Filing date: 2003-01-15
Publication date: 2003-07-24

Abstract

The invention relates to methods and systems that are based on multi-photon absorption with improved spatial resolution and which outstrip the lateral and also the axial resolution of known optical systems. The invention is inter alia used in lithography and for writing on and reading static optical memories. The aim of the invention is to provide an improved spatial resolution as compared to the prior art, especially for use in laser scan microscopy and spectroscopy. According to the invention, microscopic images are produced on the basis of the non-linear absorption of light. Correlated pairs of N-fold photons, N = 2, 3 n, such as pairs, groups of three, groups of four etc. are focused onto a point of a sample. The N-fold photons may be correlated in time and space.

Description

title

Methods and arrangements for microscopic imaging

FIELD OF THE INVENTION The invention presented here relates to methods and arrangements which are based on multiphoton absorption with improved spatial resolution and with which the lateral and also the axial resolution of known optical arrangements is exceeded. The invention can be used, inter alia, in lithography and for writing and reading optical memories.

State of the art

Laser scan microscopy and spectroscopy aim to obtain three-dimensional information about an object with high spatial resolution. Among the imaging techniques used, fluorescence imaging is the most common in life sciences. A classic tool for this is the confocal laser scanning microscope (CLSM) [publication: J.B. Pawley, Handbook of Biological Confocal Microscopy, Plenum Press, New York and London, 7995]. Either classic laser light or light from an incandescent light source can be used for the excitation.

In applications such as lithography, laser light is used to modify the chemical structure of a material (the photoresist). Due to the high spatial selectivity of this process, two-dimensional structures can be created in the photoresist. When using chemical etching techniques, the material can be found in the regions where the chemical structure of the photoresist changes was modified [Publication: T. Tanaka and S. Kawata, "Three-dimensional fabrication and three-dimensional observation by two-photon-absorption for micro-structure," Proc. SPIE, vol. 3937, pp. 92-96, 2000].

In another application, light is used to locally change the properties of a material that is used to store information in an optical memory [publication: Y. Kawata, H. Ishitobi, and S. Kawata, "Use of two-photon absorption in a photorefractive crystal for three-dimensional optical memory, "Opt. Lett., volume. 23, No. 10, pages 756-758, 1998].

The excitation of the endogenous or exogenous fluorophores within the object or the modification of the object by light (ie the chemical structure of the photoresist) usually takes place through the absorption of photons in a single step (one photon is required per excited molecule) - the linear sorption; or in a multi-stage process (the quasi-simultaneous absorption of at least two photons per excited molecule is required) - the non-linear absorption.

In order for non-linear absorption to take place in the object, a peak intensity of the excitation light is required at the site of the interaction in order to achieve a sufficient degree of absorption. Such high peak intensities can often only be achieved by using short-pulse laser radiation for excitation, combined with a strong focus of the light. Due to the high peak values, fading and damage to sensitive objects (e.g. due to photoelectric dissociation or plasma generation) are a common problem in multi-photon fluorescence microscopy. This disadvantage is ultimately due to the random nature of the impingement of photons from classic light sources (e.g. incandescent lamps or lasers), so that two or more photons are less likely to arrive simultaneously within the absorption cross-section of the medium that is to be excited.

For laser scan microscopy with single-photon excitation, three-dimensional imaging possibilities result from a confocal arrangement, the fluorescence emerging from a small point in the excitation volume being confocally imaged onto a point detector. Generally there is a pinhole in front of a standard photo receiver. In a multi-photon Fluorescence microscope (MPFM), the nonlinear character of the excitation (ie the nonlinear dependence of the degree of absorption on the immediate laser intensity) ensures a localization of the excitation that is comparable (for a two-photon fluorescence microscope - TPFM) or even better than the confocal resolution without Using a confocal receiver arrangement.

Description of the invention

The object of the invention is to further develop methods of the type described in the introduction, in particular methods in connection with laser scan microscopy and spectroscopy, in such a way that an improved spatial resolution compared to the prior art is achieved. Furthermore, it is an object of the invention to provide suitable arrangements for carrying out these methods.

Based on this, it is provided according to the invention to generate microscopic images on the basis of nonlinear absorption of light, with correlated pairs of N-fold photons, N = 2.3-n, such as pairs, groups of three, groups of four, etc., focused on a point on the sample become.

The N-fold photons can be correlated in time and space.

Light sources which generate entangled photons or squeezed photons are advantageously used.

The arrangement according to the invention can preferably be a microscopic arrangement consisting of a laser scanning microscope with object or beam scanning.

The invention further includes methods and arrangements in which a source of non-classically generated, collinearly emitted photons is located in or near a telecentric plane of the illumination beam path of the microscope.

The absorption can be a multiphoton absorption which comprises the absorption of at least two photons per molecule of the fluorophore to be excited. The invention also includes the use of the proposed new methods and arrangements in lithography, in which the multiphoton absorption takes place in a photoresist and changes the structure of the material.

In particular, this includes the use of multi-photon absorption for writing to an optical memory.

The invention also includes those methods and arrangements in which the light source consists of at least one nonlinear crystal in which a frequency conversion of pumped photons into entangled photons takes place.

The subject matter of the invention further includes configurations in which multiple passes through the crystal (s) are used, - an amplification of the pairs generated by the crystal is used, the amplification of the pairs generated by the first pass through the crystal by focusing the beam onto the second pass through the crystal, there is a source of non-classically generated, collinearly emitted photons in or near the pupil plane of the objective lens that images the sample, this plane coincides with the primary telecentric plane of the illuminating beam path, the light source is inside the housing of the Objective lenses, - the light source is in a different telecentric plane than the primary telecentric plane, the light source is in a telecentric plane in front of the optical elements that are responsible for scanning the sample by the beam, a spectroscopic Un tersuchung of samples with high spatial resolution on the basis of the fluorescence excitation is carried out by non-classical light and a source, not classically generated collinearly emitted photons is in or close to a telecentric plane of the BL LEVEL ^¬ tung beam path, not classical behind the source light within the Illumination beam path, an element for separating the paired photons, an element for optically delaying the photons of the pair to one another and one Element for re-combining the photons of the pair after the delay.

With the invention, a three-dimensional resolution is achieved which is better than that which has hitherto been achieved when using a laser scanning microscope with multi-photon absorption.

In an N-photon absorption process in which light of the wavelength λ is used, according to the invention presented here, the resolution in a confocal arrangement corresponds to a CLSM with linear absorption of light of the wavelength λ divided by N. In contrast to this, in the case of an N-photon absorption using known microscopic arrangements, the wavelength λ is decisive for the resolution. The advantages of the non-linear absorption of light of the wavelength λ compared to the linear absorption of light with a wavelength of λ / N when using the CLSM lie in a greater depth of penetration, less scattering and simplified optics.

In the absorption of non-classical light according to this invention, the degree of absorption depends linearly on the light intensity, so a continuous light source of medium power (radiation power) is sufficient for an effective multiphoton absorption. This fact reduces the complexity and cost of the light source required. In addition, the disadvantages associated with high peak intensities for the sample can be avoided. This extends the application of the MPFM to samples that are sensitive to damage and fading. Furthermore, the use of non-classical light, such as entangled photons, enables novel spectroscopic methods which, in conjunction with the optical arrangement described in this invention, allow detailed information about the sample to be obtained with high spatial resolution.

In the context of this invention, non-classical light means light with special photon statistics. Such a non-classical light source generates N photons that are correlated in time and space. One of the objects of this invention is to demonstrate methods and arrangements by which the path from N photons to the sample is designed to unite at one point in the sample and to act simultaneously through an N photon absorption process. Non-classical light can pass through the process of spontaneous parametric frequency conversion (SPDC) in a nonlinear crystal such as /? - barium borate (BBO) or lithium niobate (LN). When such a second order nonlinear crystal is pumped with a highly coherent pump laser, entangled photons (photon pair beams) are generated. The nonlinear material can also be poled periodically (eg PPLN) in order to achieve a higher efficiency in the generation of entangled photons. Non-classical light can also be generated by squeezing in an optical parametric amplifier (OPA) or oscillator (OPO). The principles of microscopy and spectroscopy with entangled photons have been described in US Pat. No. 5,796,477 [MC Teich and BEA Saleh, "Entangled-photon microscopy, spectroscopy, and imaging"]. An overview of the generation and behavior of squeezed light can be found in Ml Kolobov, "The spatial behavior of nonclassical light," Reviews of Modern Physics, Volume 71, No. 5, pages 1539-1589 (1999).

The following discussion of the invention relates to entangled photon fluorescence excitation generated in a nonlinear crystal by spontaneous parametric downconversion without limiting the scope of the invention thereto.

The key task of the optical arrangement for the excitation of the sample is to ensure that the N correlated photons of the non-classical light source arrive at a diffraction-limited point of the sample with a full numerical aperture of the objective lens. Often some or all of the N photons of the non-classical light source are emitted collinearly. In this case, and in one of the configuration variants for the optical configuration, the source of the non-classical light is close to the rear focal plane of the objective lens, which focuses in the sample. This level coincides with the primary telecentric level of the microscope's illumination beam path. The fluorescence emitted as a result of the absorption of photon pairs is imaged on a detector, the signal of which is processed electronically. The receiver device could be a confocal arrangement using a point detector. Alternatively, non-confocal measurement is possible using a large area detector (ie the receiver area is much larger than the point wash function in the receiver plane). In order to obtain an image, the sample is scanned by the excitation beam, then the processing and display of the receiver signal follows, as is known in laser scan microscopy. In another configuration presented in this invention, the entangled photon source is placed near a plane that is conjugated to the primary telecentric plane of the microscope's illumination beam path. In this case, more complex configurations of the source can be implemented. Such a source of entangled photons can, for example, be in a conjugate telecentric plane of a standard CLSM.

Another design variant contains elements for creating an optical delay between the photons that form a pair. Such an optical delay can be between the source and the objective lens and enables the investigation of spectroscopic properties of a sample with high spatial resolution.

Brief description of the drawings

The invention will be explained in more detail below on the basis of exemplary embodiments. In the accompanying drawings:

Fig.la the statistical properties over time, i.e. the number of

Photons that arrive in a time interval ΔT when using a non-classical light source with N times (in this case 4 times) photons, compared to a classic light source,

Fig. Lb the schematic representation of an N-photon absorption process by S real or virtual states from energy level E _ι to E ₂ ,

2 shows the schematic representation of the fluorescence excitation, which according to the invention is based on the classic two-photon absorption (left) or on the absorption of entangled photons (right), FIG. 3 shows a diagram of the basic optical focusing configuration according to the invention using the in Description of the notation used, Figure 4 is a diagram (not classical) stimulating the axial course of the excitation of the verschränk ^¬ th photon in comparison to the two-photon (TP) (classic) shows 5 shows a diagram which shows the lateral course of the excitation of the entangled photons (non-classical) in comparison to the two-photon (TP) excitation (classical),

6 shows a diagram showing the depth discrimination of the microscope of entangled photons with confocal reception compared to one

TPFM shows

7 shows a diagram in which a preferred configuration of the microscope of entangled photons is shown,

8 shows a diagram in which a further preferred configuration of the microscope of entangled photons is shown,

9 shows a diagram in which a preferred variant of the microscope of entangled photons with elements for the use of spectroscopic contrast information is shown.

Detailed description of the drawings

The present invention makes use of the difference between the absorption in a classic light source and that in a non-classic light source, such as e.g. entangled photons in a focused beam. 1a shows the temporal statistical properties of a non-classical light source that generates correlated photons compared to a classical light source. Within each time interval ΔT, a classic light source emits an arbitrary number of photons that follow a statistical distribution dependent on the light source, while a non-classical light source emits an N times the number at arbitrary times.

Fig.lb shows an N-photon absorption process between the energy levels E _ι and E. The intermediate states S can be virtual energy states or real states.

The effects of this difference on the dependence of the absorption rate and the sample interaction are known. The following explanations focus on the meaning for the resolution and describe configurations in which this difference is used for the high-resolution image. The later meaning is explained very simply in the case of a two-photon absorption, although it is clear that the invention shown here also applies to multi-photon processes. In addition, the discussion is for simplicity limited to one dimension in the transverse direction. The expansion to a two-dimensional system with rotational symmetry to the optical axis is easy and the conclusions are unchanged.

The axial and transverse course of the two-photon excitation of a sample, which is decisive for the resolution of the arrangement, should be determined for two cases: Classic two-photon excitation. In this case, a light source with a wavelength of 2λ is used to excite the sample. Excitation by entangled photons. Entangled photons are used which are generated to excite the sample in a non-linear optical crystal (NLC) using a frequency conversion process. In this case, the pump emits light of the wavelength λ and the degenerate frequency-converted photons each have the wavelength 2λ. The limitation to degenerate photons is for the sake of reasoning and in no way limits the scope of the invention.

The two cases are shown in Fig.2. G ²⁾ (x, z ,, t; x, z ₂ , t) is the probability of measuring a pair of photons at space-time points (x, z, t and (x ₂ , z ₂ , t_) In this context, fluorescence excitation via a two-photon process is of interest in the same arrival times f, = t ₂ = t, and since the function 0 ²⁾ (x, z, t; x ₂ , z ₂ , t) is time constant for a monochromatic light source, the time variable disappears and there remains Ö ²⁾ (x, z,; x ₂ , z _2> ).

The transfer function from object to image for a linear monochromatic optical system is given by

where all fields have the same polarization. Here E _o (xjω) and E _χ (x;, ω) are the scaled electrical fields in the object and image plane, respectively, and h (x _jt x _o ; ω) is the system's amplitude point blurring function between the image - and Ob / e / febene.

The optical system shown in FIG. 3 is used for focusing in both cases - the classic excitation and the excitation with entangled photons. The scheme according to the invention illustrates a simple lighting arrangement with a non-classical light source in a telecentric plane. For a plane at a distance z from the focal plane, the amplitude point blurring function is [publication: JW Goodman, Introduction to Fourier Optics, Chapter 6, McGraw-Hill, New York, 1968]

in which

Transformed the generalized πx 2 λ pupil function o (x) = (x) exp J JfV is [publication: B.E.A. Saleh and M.C. Pond, Fundamentals of Photonics, chap. 4, Wiley-Interscience, New York, 1991].

Here λ is the wavelength of the monochromatic, scalar electric field, / is the focal length of the lens and d is the distance between the telecentric (object) plane and the lens. In these calculations, the pupil function of the lens aperture p (x) is assumed to be rectangular and with the width A. It should be noted that this is the amplitude point blurring function for a one-dimensional optical system (in the transverse direction), therefore the proportionality constant of

changed in.

XdfV λjdfV

For the next discussion, the following assumption is made about the arrangement in Fig. 2a (classic light source): the pump light is a monochromatic plane wave,

Approximation of thin lenses,

Probe reacts to the arrival of a pair of photons on only one

Space-time point.

For classic two-photon excitation, the absorption rate of the sample (of the fluorophore) is proportional to G ^ ² ^ (x _!, Z; x _!, Z) oc / ² (χ _{j 3} z) The axial course of the excitation along the z axis, ie at x = 0, is given by

in which

and the transverse course in the focal plane (z = 0) is given by

J ² (x, z θ) oc sinc ⁴ (x). (5)

The quantity z '= is the standardized length in the axial direction, measured from the zAV z

Focal plane, where z _c = 2λ _p F _# and typically / ^ ^ and V ^' »1, so that z'z"

X

The set x is the normalized length x, in the image plane, so that x '= - -, where χ „x _c = 2λ _p Fχ (see Fig. 2).

f Here F is the number of lenses F, defined as F »= -, where / and A is the focal length

^# A or the aperture of the lens and 2λ is the wavelength of the light source. It should be noted that x _c and z _{c are} the characteristic lengths in the transverse and axial directions, respectively. The width of the excitation curve in the transverse and axial directions is proportional to these values.

For the discussion of the excitation by entangled photons, the following assumption is made of the experimental conditions of the arrangement in Fig. 2b (non-classical light source): the pump light is a monochromatic plane wave,

Approximation of thin lenses,

Probe reacts to the arrival of a pair of photons on only one

Space-time point a spectral filter of very narrow bandwidth, the center frequency of which coincides with the frequency of the degenerate photons, is placed behind the crystal, so that only degenerate photons contribute to the excitation of the sample.

For the excitation by entangled photons, G ⁽² (χ „z; xi, z), abbreviated as G ⁽²⁾ (x _u z) for the sake of simplicity, is calculated with

(6)

where g (a - /?)

J2πμ ₂ p ² ) exp (j2π (a - ß) p), (7)

^£ eq

_£ ι - ^{~ + d} ι ■ u, - - ^ ³ -, £ /, = -, N _f = is a Fresnel number,

¹ f N _f f N _f ^f λ

t- is an equivalent crystal thickness; £ is the crystal thickness and n (2λ)

the refractive index of the ordinary beam in the NLC for the degenerate wavelength. Since μ _ι «1 and μ« 1 for all practical values of £ and distances d, equation (7) is simplified to g (α-ß) = δ (-ß). As a result of equation (6), the axial course of the excitation is given by

which should be compared to the classic case in equation (3). The transverse course of the excitation is given by

G ⁽²⁾ (x;, z '= 0) oc sinc ² (2x;), (9)

which should be compared to equation (5). It should be noted that the arguments for the axial and for the transverse course of the excitation are twice and the potency of the functional dependency is half that of the classic results.

Fig. 4 shows the normalized axial course for the classic two-photon excitation (solid line) and excitation by entangled photons (dotted line). 5 shows the same curves for the transverse course.

It is shown that the focusing of degenerate photons, which were generated for example by the frequency conversion process in a nonlinear crystal, the size (both axially and transversely) of the course of the excitation by 31% compared to the classic two-photon excitation (i.e. 1 / e width of the entangled photon = 0.69 1 / e width of the classic) reduced.

Another important feature for three-dimensional imaging is the possibility of producing optical cuts, also known as depth discrimination. It can be described as the response of the system to the scanning of a thin, laterally homogeneous, fluorescent layer by the focus. In contrast to classic two-photon excitation, the excitation, which uses non-classic light, does not provide any optical cuts.

G ⁽²⁾ (z ') oc J dχ G ^{2) (x „' z ') = const. (1 0)

This is a consequence of the linear dependence of the degree of absorption on the excitation photon flux. The depth differentiation can be achieved by adding a confocal receiver unit analogous to single-photon excitation in a CLSM. The behavior is

where 2λ _p and λ are the wavelength of the excitation light and the fluorescence and f _{c is} the intensity point washing function for the confocal receiver arrangement. This behavior could be compared to the behavior in the case of two-photon excitation with a non-confocal receiver arrangement

GS W ∞) dx [I ² (x _l ', z') (12)

or with a confocal receiver arrangement

Fig. 6 shows these curves under the simplified assumption that λ _p = λ _f . It should be noted that the course of the non-classical excitation at wavelength 2λ _p with a confocal receiver arrangement is equal to the distribution of the single-photon excitation at wavelength λ _p n of a CLSM.

In summary, a system that uses non-classical light of wavelength 2λ as excitation via a focusing arrangement according to the invention described here behaves in terms of resolution like a system with single-photon fluorescence excitation of half the wavelength (i.e. λ). The former system provides a higher resolution than a non-confocal MPFM system that uses a classic light source with a wavelength of 2λ.

Preferred configurations of the invention are discussed below.

In a preferred configuration, as shown schematically in FIG. 7 a, the non-classical light source is part of the objective lens. Due to the condition of phase matching in the nonlinear crystal, a changed angle of incidence of the pump laser on the crystal has an undesirable effect on the properties of the photons generated. Therefore, such an arrangement can only be used in connection with object scanning.

In another version, shown in Fig. 7b, the non-linear crystal is outside the objective lens with the possibility of double passage of the pump light through the crystal to increase the performance of the non-classical light source. In both variants, the nonlinear crystal must be placed near the rear focal plane of the objective lens, which is also the primary telecentric plane.

In FIG. 7 a, the collimated light of the pump laser L illuminates the sample Sa and thereby passes through a dichroic beam splitter MDB, scanning optics SO, the microscope tube lens TL and a specially developed lens for entangled photons EPO, which is shown in detail on the right-hand side. The EPO consists of a light-linear crystal NLC in or near the pupil plane PP of the microscope objective O, which coincides with the primary telecentric plane PTP of the illumination. A filter HPF is located between the EPO and O, which filters the wavelength hides the pump laser, but allows longer wavelengths to pass through (frequency conversion arrangement, as described in the literature). The EPO elements can advantageously be installed in a lens housing of a conventional standard microscope. The fluorescence excited in the sample passes through EPO, TL and SO, is reflected by the beam splitter MDB and is imaged onto the detector DE through the pinhole optics PO through a pinhole PH.

In Fig. 7b, in contrast to the arrangement in Fig. 7a, the non-linear optics NLC is attached outside the housing of the objective lens, the incoming light is directed there by a polarization beam splitter PBS or a dichroic beam splitter. The back of the NLC is reflective, so that the pump light and the frequency-converted light are reflected back in the direction of the PBS. The PBS directs the pump light back towards the laser L and directs the frequency-converted light, which is polarized at right angles to the pump light, via the lens O onto the sample Sa. In this arrangement, the detector is on the right

Side, and the fluorescent light coming from the sample is reflected by the MDB. The arrangements shown in Figures 7a and 7b are for one

Determines sample that is moved in at least two directions by a scanning table (not shown) (object scanning). Furthermore, the optical system can include the scanning optics SO and the tube lens TL in both arrangements, as in FIG

Fig.7a shown, or not, as shown in Fig.7b.

In FIG. 7c, the pump light L and the frequency-converted light, after having once passed through the nonlinear crystal NLC, are focused back into the nonlinear crystal NLC by a spherical mirror M. After passing the PBS, the frequency-converted light is collimated by the lens L and directed to the sample, as described in Fig. 7b. This configuration has the advantage that the light intensity L within the crystal is much higher due to the focusing, so that the frequency-converted photons can be amplified to increase the radiation power of the pairs of entangled photons.

The disadvantage of the limitation to object scanning is avoided in a configuration according to FIG. 8, where the source of the entangled photons is located in a conjugated telecentric plane of the microscope. This arrangement of the source, again assuming that correlated photons are collinear generated ensures diffraction-limited focusing of the non-classical light. An advantage of this configuration is that more space is available for the source so that, for example, resonance-enhanced manufacturing of entangled photons can be used.

8 shows a standard confocal scanning arrangement with laser L, XY scanner for two mutually perpendicular scanning directions, optical transmission system RL, overview lens SO, tube lens TL, lens O, probe Sa, dichroic beam splitter MDB, pinhole optics PO, pinhole PH and detector DE , Behind the laser is a non-classical light source EPS (preferably based on frequency conversion in a non-linear crystal) with filter HPF for removing the pump light, as well as the telecentric optics CL1, CL2, in a telecentric plane of the illumination beam path. Thus, the light source of a standard LSM is replaced or combined with a non-classical light source in a telecentric plane of the illumination beam path, whereby the method for the multi-photon fluorescence excitation, which is described in this invention, is used.

If the non-classical light source (i.e. entangled photons) is placed in the pupil plane of the microscope, the pair-wise photons can be separated from one another and delayed from one another, as shown in FIG. 9, to enable spectroscopic examinations, as are already known. For example, the separation in the non-degenerate case, at different wavelengths of the photons of the pair, can be achieved by using a suitable dichroic beam splitter, the transmission edge being between the wavelengths of the entangled photons. If the photons of the pair differ in their polarization, polarization beam splitters can alternatively be used. Finally, even with indistinguishable photons of a pair, incomplete separation can be achieved by using a 50:50 beam splitter (half of the pairs per beam splitter are separated). The delay can be achieved by moving a delay unit in one strand of the arrangement.

FIG. 9 partially shows the arrangement described in FIG. 8, but between CL1 and CL2 are the lenses CL3 and CL4 and a dichroic mirror DC1 which directs the photons of the pair in the two different directions d1 and d2. The Both beams are then brought together again by the mirror DC2. Ml is a deflecting mirror for the light beam dl. The mirror M2 deflects the light beam d2 onto a reflector RR, which can be shifted in the path length between d1 and d2 to set the delay.

Claims

claims

1 . Methods and arrangements for microscopic imaging on the basis of non-linear absorption of light, wherein correlated pairs of N-fold photons, N = 2.3- n, such as pairs, groups of three, groups of four, etc., are focused on a point on the sample.

2. The method and arrangement for microscopic imaging according to claim 1, in which the N-fold photons are temporally and spatially correlated.

3. The method and arrangements according to one of the preceding claims, in which the light source generates entangled photons.

4. The method and arrangements according to one of the preceding claims, in which the light source generates squeezed photons.

5. The method and arrangements according to one of the preceding claims, in which the microscopic arrangement consists of a laser scanning microscope with object or beam scanning.

6. The method and arrangements according to one of the preceding claims, in which a source of non-classically generated, collinearly emitted photons in or near a telecentric plane of the illumination beam path of the

Microscope.

7. The method and arrangements according to one of the preceding claims, wherein the absorption is a multiphoton absorption, which comprises the absorption of at least two photons per molecule of the fluorophore to be excited.

8. The method and arrangements according to one of the preceding claims for use in lithography, in which the multiphoton absorption takes place in a photoresist and changes the structure of the material.

9. The method and arrangements according to one of the preceding claims, in which the multi-photon absorption is used for writing to an optical memory.

10. The method and arrangements according to one of the preceding claims, in which the light source consists of at least one nonlinear crystal in which a frequency conversion of pumped photons into entangled photons takes place.

1 1. Method and arrangements according to one of the preceding claims, in which multiple passes through at least one crystal are used.

1 2. Method and arrangements according to one of the preceding claims, in which an amplification of the pairs is used.

13. The method and arrangements according to one of the preceding claims, in which the amplification of the pairs generated in the first pass through the crystal is carried out by focusing the beam on the second pass through the crystal.

14. The method and arrangements according to one of the preceding claims, in which a source of non-classically generated, collinearly emitted photons is located in or near the pupil plane of the objective lens that images on the sample.

1 5. The method and arrangements according to claim 14, in which this plane coincides with the primary telecentric plane of the illuminating beam path.

16. The method and arrangements according to one of the preceding claims, in which the light source is located within the housing of the objective lenses.

1 7. Method and arrangements according to one of the preceding claims, in which the light source is in a different telecentric level than the primary telecentric level.

1 8. Method and arrangements according to one of the preceding claims, in which the light source is in a telecentric plane in front of the optical elements which are responsible for scanning the sample by the beam.

1 9. Methods and arrangements for the spectroscopic examination of samples with high spatial resolution, preferably according to one of the preceding claims, based on the fluorescence excitation by non-classical light, in which a source of non-classically generated, collinearly emitted photons in or near a telecentric Level of the illumination beam path is located.

20. The method and arrangements according to one of the preceding claims, in which behind the source of non-classical light, within the illuminating beam path, an element for separating the paired photons, an element for optically retarding the photons of the pair to one another and an element for reuniting the photons of the couple after the delay.