SE526643C2 - Method to avoid bleaching in the application of fluorescence correlation spectroscopy - Google Patents

Abstract

A method for fluorescence correlation spectroscopy in connection with studying a time-varying course, wherein molecules are exposed to energy for the purpose of exciting said molecules, wherein the subsequent emission, from said molecules, of fluorescence of a predetermined wavelength is detected, and wherein said energy is applied in pulses with a pulse frequency which at least amounts to two pulses per period of the time-varying course being studied.

Description

526 643 overlap in fl uorescence will, however, give a signal in both detection areas and thus a high but false correlation because the signal originates fi than the same dye.

Thanks to its excellent sensitivity and the fact that the measurement method does not permanently destroy the system being studied, FCS is particularly well suited in a number of biophysical contexts. The method has been used to study the degree of activity of enzymes, the functions of specific signaling proteins in living cells, protein aggregation and for DNA sequencing, to approach some applications.

Prior Art Fluorine Correlation Spectroscopy (F CS) is described in the prior art, for example in the publication Fluorescence Correlation Spectroscopy - An Introduction to its Concepts and Applications, Peta Schwille and Elke Haustein, Experimental Biophysics Group, Max ~ Planck-Institute for Biophysical Chemistry, Göttingen, Germany , 2002. This publication contains the on-line publication "Fluorescence oorrelation spectroscopy - an introduction to its concepts and applications", Petra Scwille and Elke Haustein, Biophysics Textbook Online (BTOL), 2002. This publication is hereby incorporated by reference as a description of the per se known part of the practice of the invention. The publication describes both theoretical background and experimental realizations. In FCS measurements according to this publication and other prior art, excitation of molecules with a continuous light source takes place alternatively with a pulsed light source with a high repetition frequency in the range 40 MHz (MegaHertz) and upwards, which gives a so-called quasi-continuous light source. In this way, the molecules are illuminated all the way in and out of the detection volume, which gives the side effect bleaching, that is, the molecules lose their ores uorescent properties. Quasi-continuous light sources typically have repetition rates of about 75 IVII-Iz (megahertz), and electronics for performing correlation have a sampling frequency of at least 10 MHz (megahertz).

Generally speaking, known techniques work with quasi-continuous pulse frequencies and sampling frequencies of the order of MHz (MegaHertz).

Another publication describing prior art fluorescence correlation spectroscopy (FCS) is "Fluorescence correlation spectroscopy: molecular recognition at the single molecular level", E Van Craenenbroeck and Y Engelborghs, J. Mol. Recognize. 13, pp. 93-100, 2000. This publication is hereby incorporated by reference as a description of the per se known part of the practice of the invention. 10 15 20 25 30 526 643 Another publication describing Fluorescence Correlation Spectroscopy (F CS) according to the prior art is “Fluorescence correlation spectroscopy: the technique and its applications”, Oleg Krichevsky and Grégoire Bonnet, Rep. Prog. Phys. 65 (2002) 251-297.

Patent publication WO03 / 021240 describes a method for detecting luminescent molecules by optical excitation in a confocal measuring volume, wherein different types of luminescent are excited at different times in a sample and wherein emitted radiation from the different types of molecules is detected by a detector.

Prior art methods of FCS are limited by the need to modify biomolecules to give them good uorescence properties, and these molecules nevertheless fade and lose their uorescence properties too quickly.

Object of the Invention The invention aims to solve the general problem of unsatisfactory fluorescence properties of molecules in connection with Fluorescence Correlation Spectroscopy (F CS).

Aspects of the problem that the invention intends to solve are to: - Eliminate the effect of bleaching of fluorescent molecules. In order to be able to study molecules of interest, for example proteins, these are modified according to known telmics with different dyes. Although these color sleeves have excellent orescence properties, they still fade relatively quickly and this is a problem in many applications, for example in studies of slow processes. Analysis of data is also made more difficult; bleaching involves the potential risk of underestimating the diffusion time and the number of particles.

- Eliminate the need to modify biomolecules to give them good orescence properties. An even more serious problem with bleaching is that it is not possible to use the inherent ores uorescence properties of proteins for FCS. The amino acids tryptophan and tyrosine, which are represented almost all proteins, fade too quickly to be used for FCS according to the prior art. However, in many contexts it is highly desirable to be able to study proteins in their natural form, without having to modify them, as this can change the property one wants to study. occur that the protein ceases to form aggregates when it is modified.

Summary of the Invention According to the invention, which is an improvement of the FCS method, the above-mentioned problem and limitations are eliminated, and thus a more useful FCS method is provided.

The heat solves the problem by minimizing the number of excitations of the molecules to the minimum number required to achieve observability. The invention is based on the fact stated by the sampling theorem, namely that a sufficient requirement to be able to correctly study a time course is that the sampling frequency is at least twice as high as the frequency being studied. According to the invention, it is essentially sufficient to excite the molecules on average twice per passage through the detection volume. This minimization of excitations virtually eliminates the problem of bleaching, since bleaching occurs to a negligible extent at this low number of excitations. The invention has, among other things, the following technical effects and advantages. 1) FCS on unmodified proteins can be made with the invention. By pulsing the excitation energy, the bleaching of molecules is minimized to such a low level that it is possible to use the bleach-sensitive fl orophores of proteins tryptophan and tyrosine, instead of dye molecules. This makes it possible to study proteins in their native form without disturbing the system beyond excitation. With the invention, it is therefore also possible to study processes and proteins in their natural environment without having to modify DNA for the expression of color-coded proteins. Overall, the invention provides an FCS method that is more useful in medical applications and enables new diagnostic methods. In addition to proteins and biomolecules, there are also other bleach-sensitive molecules of interest.

The invention is of course applicable and useful even in the analysis of such molecules. 2) With FCS according to the invention, there is no limit for slow processes. In traditional prior art FCS with continuous illumination in the detection volume, dyes fade sooner or later due to their inherent photostability. This means that slow processes where the molecules spend a large part in focus are impossible to study with FCS according to known technology. Since the technique according to the invention with pulsed excitation is adapted to the time cycle one wishes to study, only two excitations are needed per passage, regardless of how long a passage takes. 3) FCS according to the invention allows milder requirements for the performance of dyes. There are a number of dyes which have insufficient properties of FCS according to the prior art in terms of quantum exchange for emission and photostability but which are available by means of pulsed excitation according to the invention. 4) Improved opportunities for two-color FCS through the invention. By separating the excitation pulse rate for the two dyes, it is possible to minimize the overlap in ores uorescence between dyes. This will lead to greatly improved signal-to-noise ratio in correlation analysis and it will make it easier to choose in two or more color sleeves suitable for FCS. 5) Better signal-to-noise ratio. In traditional FCS according to the prior art, bleaching is limited by keeping the excitation at a relatively low level. With the improved F CS method according to the invention, pulsed excitation with higher laser power and thus higher signal is possible. In general terms, the reliability of the FCS method will be improved by the invention of low frequency pulsed excitation energy, even if one chooses to modify the studied molecules with dyes. The risk of diffusion time and particle number being underestimated is minimized; freer choice of color name allows you to choose a system that suits the experiment you want to perform rather than primarily making sure that you meet the requirements for FCS, as has been necessary with FCS according to known technology.

One aspect of the invention includes a method of fluorescence correlation spectroscopy in which molecules are exposed to energy to effect excitation of said molecules and in which subsequent fluorescence is detected from said molecules. In applications of the invention, the main aim is to minimize energy application to the molecules, and preferably said excitation energy is applied in pulses with a pulse frequency 10 64 64 substantially corresponding to twice the diffusion frequency of said molecules. Thus, said molecules with said energy are exposed on average substantially twice per passage of said molecules through a detection volume of a solution containing said molecules.

The diffusion time of studied molecules is generally in the range of 0.01 to 10 ms (millisecond). In applications according to the invention, therefore, a pulse frequency is used for exposing energy, preferably in the range of 200 to 0.2 kHz (kiloHertz).

According to the invention, in the analysis of diffusion processes, pulse sequences in the order of kHz (kiloHertz) are preferably used, i.e. in the limited range of 0.01 to 999 kI-lz (kiloHertz). In applications of the invention that analyze faster processes, for example chemical reactions, pulse frequencies in the MHz range (megaHerz) may occur.

The common denominator for different applications of the invention is to pulsate the exciting light with at least two pulses per period of the time variation of the studied process. Since the period and sequence of the time variation of the studied process are not always fully known in advance, the period and frequency mentioned in practical application of the invention are estimated, and the pulse frequency of the energy exposure is set in the range between 2 and approximately 10 pulses per period. According to other aspects of the invention, said molecules are exposed to pulses of energy of a first and a second wavelength. This is typically used in FCS analysis according to the invention of molecules that can be stained with two dyes, and with two dyes it is usually advantageous and in some cases necessary with two excitation wavelengths. In further aspects of the invention, the V molecules are exposed with energy in pulses by a first and a second predetermined pulse frequencies and possibly with a predetermined time delay between the pulses of said first and second pulse frequencies. With the time delay, the pulses originating from each color are separated by a time interval. Emission is detected from said molecules of florescence with a first and a second feed-determined wavelengths.

Said molecules are in one embodiment of the invention modified with a dye.

Said molecules may comprise a first kind of molecules modified with a first dye and a second kind of molecules dyed with a second dye.

In various applications of the invention, two-color FCS according to the invention can be used in the analysis of a molecule modified with two dyes, a process splitting the molecule and thereby separating the two dyes. In other applications, one has two molecules down each of a different colored dye, and when these molecules are joined together, cross-correlation can be detected.

Further aspects of the invention include an apparatus for uorescence correlation spectroscopy in which molecules are exposed to energy to effect excitation of said molecules and in which subsequent emission is detected from said molecules of oresorescence with a predetermined wavelength and functions. Furthermore, the invention may comprise a computer program product for controlling an apparatus for fluorescence correlation spectroscopy, molecules being exposed to energy to effect excitation of said molecules and detecting subsequent emission from said molecules by determining the frequency of perform the steps and functions as above.

In general terms, the UP can be used to study time-varying processes and processes where the said time variation can be measured with ores uorescence. Diffusion, chemical processes and population of long-lived energy levels in studied systems are examples of this.

Correlation analysis is used according to the invention to determine the number of units, e.g. the number of molecules, which contribute to ores uorescence and the frequency of the time variation of the process. For the sake of clarity, the invention is explained in more detail by exemplary embodiments which are carried out on diffusion as a process.

Brief Description of the Drawings The invention will be explained below with reference to the accompanying drawings, in which: Fig. 1 shows graphs of absorption and emission of two common dyes during excitation according to the prior art; Fig. 2 shows graphs of the two dyes according to Fig. I during excitation according to the invention; Figures 3-6 show graphs illustrating various experiments with the invention; Fig. 7 shows an overview block diagram of steps included in an embodiment of the method according to the invention.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION Fluorescence Correlation Spectroscopy (FCS1 10 15 20 25 The core of FCS is to measure ores uorescence and calculate autocorrelation on the detected signal. very good ores uorescence properties so that the signal can be detected at all.The presently most common approach is to focus laser light with a diffraction limited lens to a volume with length measurements of the same size as the wavelength, giving a volume of the order of fifty liters. . 1 1 GMT? 4D: 4D: 1 + 2 1+ 2 mo zo where D is the definition constant, m0 is the diameter of the detection volume and zo is the length. In order to obtain a good correlation amplitude, the particle number N must be low, which at the same time means lower intensity and poorer signal-to-noise retention.

Usually, the geometry of the detection volume is increased, which leads to the particle number N and the lateral partition time (må / 4D) being determined. The number of particles makes it possible to determine concentration and study chemical reactions, while the separation time gives a measure of size and can be used to study, for example, aggregation.

Frequency relationship The acquisition is based on the surveyors' insight into the following relationship. 10 15 20 25 30 526 643 In time-discrete observations of time-continuous processes by sampling at a certain sampling frequency, the well-known sampling theorem applies. Sampling can be considered as pulses of the observation event or detections with cm certain pulse fi- equator of a time-dependent quantity that varies with a certain studied frequency. This view is used in the present invention.

Fluorescence correlation spectroscopy (PCS) has an observed volume of any substance that contains molecules. These molecules diffuse into and out of the observation volume due to thermal motion. A time-dependent quantity in this context is the number of molecules which preferably average on average in the observed volume. The number of molecules in the observation volume at any given time follows a basically time-continuous process.

Purely mathematically, the process itself is time-discrete because a number, ie the size of a set, is indicated by natural numbers, between which there are discrete steps. For practical purposes, however, this number of molecules can be considered to have a time-discrete course.

The molecules settle in the observation volume for a certain approximately average time, which in the FCS context is called the diffusion time. In a fi sequence description, the time-continuous process varies with a frequency corresponding to a relationship where the diffusion frequency = l / diffusion time.

The observed volume of the molecules is ordered with energy that has a certain wavelength.

Molecules in the observed volume are excited by the energy and then emit a fluorescent light (fl uorescence) with a certain wavelength.

By means of a light intensity detector, the orescence emission is observed as a parameter which depends on the number of molecules the observed volume. The observation of the ores uorescenser nission, ie. the detection of the intensity of the fl uorescence emission fi- of the excited molecules occurring in the observed volume is ongoing during FCS. This can be compared to a camera with a constantly open shutter. the intensity of the orescence initiation depends on the number of excited molecules that settle in the observed volume. The fluorescence from a molecule after a pulse of excitation energy contributes to the intensity and is thus observable / detectable during the time that the excited molecule settles in the observed volume until it has diffused out. Thus, a molecule is observable / detectable during the average diffusion time.

To detect a fluorescence intensity that correctly corresponds to the number of molecules in the observed volume, all molecules in the volume must be excited. According to the present invention, irradiation of the observation volume takes place with exciting energy in pulses with a certain pulse sequence. By pulsing the excitation energy, the ores uorescence emission from the molecules in the observation volume acquires a time-discrete character with a frequency that is dependent on the diffusion frequency. Thus, the sampling theorem becomes applicable, and thus in order to correctly observe the time course for a frequency description of the number of molecules in the observation volume via the parameter ores uorescence emission, it is necessary and sufficient that the pulse frequency of the excitation energy is double the diffusion frequency. That is, during the time each molecule settles in the observation volume, it has time to be irradiated by an excitation pulse on average twice. - As the excitation energy is time discrete through the pulsed radiation, the ores uorescence emission fi from the observation volume is also pulsed, ie time discrete. The output signal from the detector for detecting the uorescence emission then also becomes discrete, that is to say the output signal comprises pulses of intensity peaks after each excitation pulse. In order for the output signal from the detector to deliver a signal corresponding to a correct number of molecules in the observation volume, it is thus sufficient in accordance with the invention to apply excitation energy in pulses with a pulse sequence substantially corresponding to the double diffusion equation of the currently studied molecules. The resolution can be compared to a camera with an open shutter and pulsed lighting with the possibility of a minimal or minimum pulse fi-sequence equal to double the diffusion fi-sequence.

This provides a time-discrete detection or sampling of a pair with a time-discrete character which is dependent on an at least in principle time-continuous process. This is in contrast to the prior art, which can be compared to a carnation with an open shutter and continuous lighting or quasi-continuous lighting. Which thus according to the prior art provides a time-continuous detection of an at least in principle time-continuous pairing which is dependent on a substantially time-continuous course.

Fluorescence Correlation Spectroscopy (FCS) with Zero Excitation Fig. 7 shows in a schematic block diagram the principal steps for performing FCS with pulsed excitation according to an embodiment of the invention, comprising the following steps: 7_02: Arranging an observation volume according to FCS with molecules to be studied. 103: Apply to the observation volume excitation energy in pulses with pulse frequency in the range substantially corresponding to twice the diffiction frequency of the molecules.

WE: Record emitted ores uorescence from the observation volume. ] _O_8_: Analyze said recorded ores uorescence which depends on the number of molecules in the observation volume.

Pulsed excitation according to the invention will generally not entail any major change in what an FCS instrument looks like, but it will be possible to use existing equipment with modified control of the excitation energy. According to a preferred embodiment of the invention, means are required for pulsing the emission of energy from the excitation source, that the repetition sequence must be controllable or adjustable and that in correlation analysis it takes place with the same time resolution as excitation.

The pulsation and control of the repetition frequency when the pulsation is achieved in different embodiments of the invention, for example by means of an aperture with controllable permeability for excitation energy generated by the excitation source.

In addition, in an embodiment of the invention adapted for experiments with two dyes, the excitation pulses for the different dyes are controllably separable over time. By time separation of the excitation pulses, the biggest problem in these types of measurements is minimized, namely false correlation from dyes that emit in both wavelength ranges. As an example, Fig. 1 shows graphs of absorption and emission for two common dyes, Alexa Fluor 532 (above in the figure) and Alexa Fluor 633 (below in fi guren) from Molecular Probes, Eugene, OR. If the excitation occurs simultaneously, it is required that the detector that is to measure the signal from Alexa Fluor 633 has an somlter that does not transmit a signal below 650 nm to avoid that it should also detect signal from Alexa Fluor 532. As shown in Fig. 1, Alexa also has Fluorine 532 has a significant emission up to 650 nm. This means that a large part of the emission from the Alexa Fluor 633 is filtered out only to avoid duplication of signal, and this means that the signal-to-noise ratio deteriorates considerably. It is a major problem for two-color F CS according to the prior art to find two dyes which do not overlap in emission while the excitation wavelength must not be too different. Too much difference leads to different geometries for excitation.

When instead the excitation pulses are separated, as in embodiments of the invention, preferably at least so much so that the signal has time to fade, given the lifetime of the excited state, one knows exactly which tern is excited at a given time and consequently in which detector the desired signal is registered. .

Fig. 2 shows graphs generated in an experiment in which a green light detector is equipped with an alter that transmits light with wavelengths shorter than 600 nm and a detector dries red light with a wavelength longer than 600 nm. When a green laser (532 nm) illuminates the sample, Alexa Fluor 532 is excited, but also Alexa Fluor 633 to some extent. At a red laser pulse (633 nm), only the Alexa Fluor 633 will be excited and the red detector will receive light. Correlation is now calculated between green ores uorescence when the green laser illuminates the sample and red fl uorescence at red excitation. In this way, the so-called cross-talk or crosstalk that would otherwise have arisen disappears at the same time as it is possible to take advantage of the maximum of emitted fl uorescence. This principle makes it easier to choose dyes but also improves the correlation analysis. It should also be possible to extend this to more than two dyes and thus open up opportunities to study reactions in which your reagents participate and more than one bond is created or broken.

Model description The effect of pulsing the excitation source can be shown with a simple computer model. This model takes into account the diffusion of molecules in and out of the detection volume (which is the same as the excitation volume), excitation and relaxation as well as bleaching. The model is O-dimensional, i.e. the geometry is assumed to be syrmenic in all respects.

The environment outside the detection volume is assumed to be unchanged with respect to the number of molecules. The flux of molecules into the volume is assumed to be equal to the fl ux that is aligned, which gives the probability ßr that a molecule diffuses in or out is given by the equation At P. = - dig Tdw 10 15 20 25 526 643 where At is the time interval considered and 14.37 the average diffusion time. At each time step in the calculations, this is tested for each molecule. This gives the number of molecules that settle in the volume at each individual occasion and are thus exposed to excitation. The next step is to determine how much ores uorescence the molecules that settle in the volume give rise to. This is done by calculating how energy levels are populated via excitation and depopulated through relaxation to lower energy levels. The system used is assumed to have three levels, a ground state N 1 (lowest energy) and two excited states, N; and N 3. Transition speed (number of transitions fi- from one level to one arman per unit of time) is denoted kf, - where i represents the initial level and j the final level. Fig. 3 shows an energy level diagram with transition speeds accordingly.

Population of the different energy levels is determined by the system of equations: N1 = "k12N1 + k21N2 + ks1Ns Nz = + kx2N1'k21Nz 'kzsNz I Na = + k23N2' ksiNa N = N, + N, + N, N 1: 2 = 3 = 0 where N means the time derivative of N. Also included is the assumption of iron weight, Relaxation of N; with transition to the ground state can take place either without radiation via collisions (k2 ° f ”) or via emission of photos (kffd). The number of emitted photons per unit time is given by N 2 kf and assuming that the emission is isotropic, that light-collecting optics cover the space angle Q and that the efficiency of the detector is then the detected intensity of: 1 I 71- '10 15 20 25 30 526 643 14 Transition speeds can be obtained from the literature and some simple calculations. Excitation is given by the intensity of the excitation source (hm), the cross-sectional area of the detection volume and the absorbency of the molecule (extinction coefficient 6): 1 ,,,,, _ mio 81, ”i nwå aoouv, w: ka: = 5 lens, which means that the diameter (2 mo) of the volume is approximately equal to the wavelength of the laser light. Relaxation rates are given by lifetime r; and the quantum yield 45 for N23 q, _ kr _ a. "kzrid + kzciu + kzs k21 + k23 l" = k21 + k23 1. ' The molecules in the model may have one or fl era tryptophan that stands for the fl uoresccns that are reported. Tryptophan has an extinction coefficient of 5500 M1 cm * (at 280 nm) and from the literature it is found that t; is 3 ns, d: is 10-30% and kg; 20-80 106 S4. The transition speed went N; estimated at 106 S4.

The big problem for F CS is bleaching and then arises N; populeras. This state is a triplet state with spin equal to 1 (while N 1 and N; are singlet states and have spin 0) and long-lived because relaxation to the basic state requires change of spin. Molecules in this long-lived state (lifetime about 1 us) have plenty of time to collide with O 2 (triplet) and thus relax to the basic state at the same time as 0; (singlet) is formed. This in turn is very reactive and risks destroying the molecule's uorescent properties. To take this into account, all tryptophans that end up in N are considered; as bleached and still incapable.

Results The graphs in Fig. 4 - Fig. 6 describe the effect of pulsed excitation. They correspond to three different “experiments” and each is described by the fl uoresccns recorded and the result of the calculation of autocorrelation. In calculations, the worst conditions have been assumed, i.e. the quantum yield is 10% and kg; is 80 106 s ". Furthermore, the intensity of the laser pulses is assumed to vary by 10%, pulse to pulse, and in addition there is 20% noise. The diffusion time is 100 ps, the time interval in the calculations is 1 us and the total measurement time is 100 ms.

Figure 4 shows continuous excitation of molecules with a tryptophan. Without bleaching, the autocorrelation curve (solid blue, barely visible) would follow the red line describing diffusion. This is the effect of bleaching, the molecules bleach as soon as they enter the volume, the intensity becomes minimal and the autocorrelation curve is determined solely by the bleaching process.

The graphs of Fig. 5 are obtained in another experiment in which the excitation is pulsed with the Nyqvist sequence, the excitation time is 100 p.s which means an excitation pulse every 50 ps.

The result is striking, the autocorrelation curve is visible and is well described by the combined effect of diffusion and bleaching. Bleaching can also be considered a kind of diffusion, when a molecule ceases to emit ores uorescence, it becomes synonymous with it having fed the volume.

In the third experiment, Fig. 6, the molecules have five tryptophars. The autocorrelation curve follows the diffusion closely and bleaching has become a slow and not very important process.

It is also noteworthy that the correlation amplitude at r = 0 is 0.1, which is in good agreement with the fact that there should be an average of 10 molecules in the volume (G (1: = 0) = 1 / IV).

In summary, the effect of pulsed excitation is obvious, that is, the negative effect of bleaching is eliminated. It is also clear that FCS is possible on proteins that contain fl your tryptophans. It is currently unclear whether with the invention it will be possible to perform measurements on proteins with only one tryptophan, but this is not of decisive importance. Pulsed excitation according to the invention improves the situation with regard to bleaching, in addition, most proteins contain more than one tryptophan.

The invention can be varied and implemented in various ways within the scope of the appended claims.

Claims (17)

526 643 nu n 4 u n f .. n u n n z:. ; Q '1' '' '' '' o "n n n u u u 0 I uu n v n 0 I, u o o o l 16 | n z: II ',' q u con uu n s u Patent claim A method of fluorescence correlation spectroscopy in the study of a time-varying process in which molecules are exposed to energy to effect excitation of said molecules and in which corresponding emission is detected from said molecules of fluorescence with a certain determined wavelength. a pulse frequency of at least two pulses per period of the time-varying course studied. The method according to the preceding claim, wherein said energy is applied in pulses with a pulse frequency in the range of two to 10 pulses per period of the time-varying course studied. The method of claim 1, wherein said energy is applied in pulses with a pulse frequency in the range of 0.01 to 999 kHz. The method of claim 1, wherein the applied energy is applied in pulses having a pulse frequency in the range of 0.2 to 200 kiloHertz. A method according to any one of the preceding claims, wherein the number of fluorescence emitting units is determined by means of a correlation analysis of said detected emission. A method according to any one of the preceding claims, wherein the frequency of the time variation of the orescence emitting process is determined by means of a correlation analysis of said detected emission. A method according to any one of the preceding claims, wherein said energy is applied in pulses with a pulse frequency substantially corresponding to twice the diffusion frequency of said molecules. The method of the preceding claim, wherein said molecules are exposed to pulses of energy of a first and a second wavelength. k) t !! 0: 0, ,,. ° .. '... Now. .. .abb Og Q-'l OOIO O nu no 0000 oo ooo 0 OI 0 oo 0 00 o I .U o oc u 0 000001 oc 00 o nu 0 526 643 -uno o u. .uuv, 'pnonvgo 0 0 0 0 0 an 0 onon .z _: z, '. . p n oo- o u l 0 '° I,. . o 0 0 17. u 2 o I ~ _ '_ ._ u .. nu n e n e The method of any of the preceding claims, wherein said molecules are exposed with energy in pulses of a first and a second predetermined pulse frequencies and with a predetermined time delay between the pulses of said first and second pulse frequencies. The method according to any of the preceding claims, wherein emission fi is detected from said molecules of fl uorescence with a first and a second feed-determined wavelengths. The method of any one of the preceding claims, wherein said molecules are modified with a dye. The method of any preceding claim, wherein said molecules comprise a first kind of molecules modified with a first dye and a second kind of molecules dyed with a second dye. The method of any of the preceding claims, wherein said molecules comprise more than two kinds of molecules modified with different dyes. The method according to any one of the preceding claims, wherein said molecules are of a first kind modified with two or more different dyes. The method of any of the preceding claims, wherein said molecules are exposed to said energy substantially on average twice per passage of said molecules through a detection volume of a solution containing said molecules. An apparatus for oresorescence correlation spectroscopy wherein molecules are exposed to energy to effect excitation of said molecules and wherein subsequent emission is detected from adjacent molecules of oresorescence having a predetermined wavelength, characterized by means arranged to perform any function of 1-1. 5. A computer program product for controlling an apparatus for oresorescence correlation spectroscopy wherein molecules are exposed to energy to effect excitation of said molecules and wherein subsequent emission is detected from said molecules of oresorescence at a predetermined frequency 0, o! N. 0 0 0 t 0 A I u I o n 0 000 I O I I 0 u q 00 I o n: in o u an: ull o o 0 a 000 I ln i oo n 526 643 .u: “.: z OI o o g n u o u u o 0 I f. u n u en o a o n n n o c z z _ ', q o 18 z z z 0.0 I .U g., ,,,. now characterized by a program code arranged to control a computer processor to perform the steps and functions according to any one of claims 1-15.