WO2005080945A2

WO2005080945A2 - Method for avoiding bleaching when using fluorescence correlation spectroscopy

Info

Publication number: WO2005080945A2
Application number: PCT/SE2005/000223
Authority: WO
Inventors: Anders Hedqvist; Torleif HÄRD
Original assignee: Anders Hedqvist; Haerd Torleif
Priority date: 2004-02-20
Filing date: 2005-02-18
Publication date: 2005-09-01
Also published as: SE0400398D0; WO2005080945A3; SE0400398L; SE526643C2; WO2005080945A9

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

Method of avoiding bleaching during application of fluorescence correlation spectroscopy

Technical field The present invention relates to a method for avoiding biomolecule bleaching when using fluorescence correlation spectroscopy.

Background Fluorescence correlation spectroscopy (FCS) is a biophysical method of analysis which has many applications in bioscience and medicine. More specifically, FCS is an optical measuring method which involves molecules in solution being excited by laser light and the subsequent emission, what is termed fluorescence, being detected. If the concentration of the molecules is low, and the volume of the solution in which the molecules are excited is sufficiently small, the fluorescence signal will fluctuate with time as the molecules diffuse in and out of the volume due to thermal motion.

Correlation analysis of the detected signal gives the number of molecules which are on average located in the volume as well as their mean diffusion time. The method is extremely sensitive and it is perfectly possible to study individual molecules.

FCS was developed at the beginning of the 1970s as a variant of what is termed dynamic light scattering. Even at an early stage, the experiments were designed with confocal optical geometry for excitation and detection of emission. By means of placing an aperture in the image plane where the fluorescence is depicted, the background signal which originates from parts of the sample other than the excitation focus is suppressed. While FCS previously suffered from low signal levels and a great deal of noise, it has, with the introduction of new sensitive detectors, first and foremost APD (avalanche photo diode) , been developed so as to become a very useful and reliable measuring method .

In the middle of the 1990s, the first experiments involving the modification of molecules with two distinctly different dyes were carried out. Cross correlation of the emission from each respective dye gives a signal when the dyes are linked together, and their movement is thereby correlated, for example as a result of being attached each to its own end of a suitable molecule. If the link is broken, the correlation ceases and this makes the method excellent for studying all types of situation in which chemical or mechanical bonds are formed or broken. This variant is usually termed two-colour FCS and has become ever more widely accepted and used. A crucial problem associated with this type of measuring method is that of finding two suitable dyes which have distinctly different fluorescence properties. However, any spectral overlap in the fluorescence which might possibly exist will give a signal in both detection ranges and, as a result, a high but false correlation since the signal is originating from the same dye.

Due to its outstanding sensitivity and the fact that the measuring method does not permanently destroy the system being studied, FCS is particularly well suited for a number of biophysical contexts. The method has been used to study the degree of activity exhibited by enzymes, the functions of specific signal proteins in living cells and protein aggregation, as well as for DNA sequencing, to name some applications.

State of the art

Fluorescence correlation spectroscopy (FCS) is described in the prior art, for example in the publication Fluorescence Correlation Spectroscopy - An Introduction to its Concepts and Applications, Petra Schwille and Elke Haustein, Experimental Biophysics Group, Max-Planck Institute for Biophysical Chemistry, Gδttingen, Germany, 2002. This document is included in the on-line publication "Fluorescence correlation spectroscopy - an introduction to its concepts and applications", Petra Schwille and Elke Haustein, Biophysics Textbook Online (BTOL), 2002. This publication is hereby incorporated by reference as a description of that part of the implementation of the invention which is known per se. The publication describes both the theoretical background and experimental implementations. In FCS measurements as described in this publication and other prior art, the molecules are excited either with a continuous light source or, alternatively, with a pulsed light source having a high repetition frequency in the 40 MHz (megahertz) range and upwards, which provides what is termed a quasi-continuous light source. In this way, the molecules are illuminated for the whole of their path in and out of the detection volume, resulting in the side effect of bleaching, that is of the molecules losing their fluorescent properties. Quasi-continuous light sources typically have repetition frequencies of about 75 MHz (megahertz) and the electronics for performing the correlation frequently have a sampling frequency of at least 10 MHz (megahertz) . Expressed in a general manner, use is made, in the prior art, of quasi-continuous pulse frequencies and sampling frequencies which are in the MHz (megahertz) order of magnitude.

Another publication which describes fluorescence correlation spectroscopy (FCS) in accordance with the prior art is "Fluorescence correlation spectroscopy: molecular recognition at the single molecular level", E Van Craenenbroeck and Y Engelborghs, J. Mol. Recognit. 13, pp. 93-100, 2000. This publication is hereby incorporated by reference as a description of that part of the implementation of the invention which is known per se. Yet another publication which describes fluorescence correlation spectroscopy (FCS) in accordance with the prior art is "Fluorescence correlation spectroscopy: the technique and its applications", Oleg Krichevsky and Gregoire Bonnet, Rep. Prog. Phys. 65 (2002) 251- 297.

The patent publication WO03/021240 describes a method for detecting luminescent molecules by means of optical excitation in a confocal measuring volume, with different types of luminescents being excited at different times in a sample and with the emitted radiation from the different types of molecule being recorded by a detector.

Methods for FCS in accordance with the prior art are limited by the fact that it is necessary to modify the biomolecules in order to provide them with good fluorescence properties and by the fact that, despite this, these molecules become bleached and lose their fluorescent properties too quickly.

Object of the invention

The object of the invention is to solve the general problem of molecules having unsatisfactory fluorescence properties in connection with fluorescence correlation spectroscopy (FCS) .

Aspects of the problem which the invention is intended to solve are:

- to eliminate the effect of the bleaching of fluorescent molecules. In order to be able to study molecules of interest, for example proteins, the latter are modified, in accordance with the prior art, with different dyes. Even if these dyes have outstanding fluorescence properties, they are, nevertheless, bleached relatively rapidly, with this being a problem in many applications, for example in connection with studies of slow processes. It also makes it more difficult to analyse data; bleaching means there is a potential risk of underestimating the diffusion time and the number of particles.

- to eliminate the necessity of modifying biomolecules in order to provide them with good fluorescence properties. An even more serious problem associated with bleaching is that it is not possible to use the inherent fluorescence properties of proteins for FCS. The amino acids tryptophan and tyrosine, which are present in virtually all proteins, are bleached too rapidly to be used for FCS in accordance with the prior art. However, in many contexts, it is particularly desirable to be able to study proteins in their natural form without having to modify them, since doing so may alter the property which it is wished to study. This is important when determining whether proteins form aggregates since a situation can arise where 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 abovementioned problem, and the restrictions, are eliminated, thereby achieving an FCS method which is more utilizable.

The invention solves the problem by minimizing the number of excitations of the molecules to the minimum number which is required for procuring observability. The invention is based on the fact that is specified by the sampling theorem, namely that a sufficient requirement for being able to correctly study a time dependent course is for the sampling frequency to be at least twice as high as the frequency which is being studied. According to the invention, it is essentially sufficient to excite the molecules on average twice per passage through the detection volume. Minimizing excitations in this way in practice eliminates the problem of bleaching since bleaching occurs to a negligible extent in connection with this low number of excitations. The invention has the following technical effects and advantages, inter alia.

1) Using the invention, FCS can be carried out on unmodified proteins. By means of pulsing the excitation energy, the bleaching of molecules is minimized to a level which is so low that it is possible to use the bleaching-sensitive fluoro- phores of the proteins, i.e. tryptophan and tyrosine, instead of dye molecules. This makes it possible to study proteins in their native form without disturbing the system over and above exciting it. It is therefore also possible, when using the invention, to study processes and proteins in their natural environment without needing to modify DNA for expressing dye-labelled proteins. All in all, the invention achieves an FCS method which is more utilizable in medical applications and renders new diagnostic methods possible. In addition to proteins and bio- molecules, there are also other bleaching- sensitive molecules of interest. The invention is naturally also applicable and useful when analysing these latter molecules.

2) When using FCS according to the invention, there is no restriction on slow processes. In the case of traditional FCS in accordance with the prior art, where there is continuous illumination in the detection volume, dyes are bleached sooner or later depending on their inherent photostability . This results in slow processes, where the molecules spend a large part in focus, being impossible to study by FCS in accordance with the prior art. With the technique according to the invention, involving pulsed excitation, being adapted to the time dependent course which it is wished to study, only two excitations are required per passage, however long a passage takes.

3) FCS in accordance with the invention enables the demands placed on the performance of dyes to be more lenient. There are a number of dyes which, while having properties which are inadequate for FCS in accordance with the prior art in regard to quantum yield for emission and photostability, can be used when employing pulsed excitation in accordance with the invention.

4) The invention results in the possibilities of performing two-colour FCS being improved. By means of separating the excitation pulses for the two dyes, it is possible to minimize the overlap in fluorescence between dyes. This will lead to a greatly improved signal to noise ratio in connection with correlation analysis and will make it simpler to select two or more dyes which are suitable for FCS.

5) Better signal to noise ratio. In traditional FCS, in accordance with the prior art, bleaching is restricted by the excitation being kept at a relatively low level. The improved FCS method in accordance with the invention makes possible pulsed excitation with higher laser power and, as a result, a higher signal.

In general terms, the reliability of the FCS method will be improved by means of the invention, involving excitation energy which is pulsed at low frequency, even if the choice is made to modify the molecules under study with dyes. The risk of the diffusion time and the particle number being underestimated is minimized; a wider choice of dyes makes it possible to select systems which fit the experiment which it is wished to perform rather than first of all having to ensure that the requirements for FCS are satisfied, as has been necessary when using FCS in accordance with the prior art.

One aspect of the invention comprises a method for fluorescence correlation spectroscopy in which molecules are exposed to energy for the purpose of exciting said molecules and in which the ensuing fluorescence from said molecules is detected. When the invention is being used, it is endeavoured, substantially, to minimize the application of energy to the molecules and said excitation energy is preferably applied in pulses having a pulse frequency which principally corresponds to twice the diffusion frequency of said molecules. This thereby exposes said molecules to said energy substantially on average twice for every passage of said molecules through a detection volume of a solution containing said molecules.

The diffusion time for molecules under study is generally in the range of 0.01 to 10 ms (milliseconds). A pulse frequency for the exposure of energy which is preferably in the range from 200 to 0.2 kHz (kilohertz) is therefore used in applications in accordance with the invention. According to the invention, pulse frequencies of the kHz (kilohertz) order of magnitude, that is in the limited range of 0.01 to 999 kHz (kilohertz), are preferably used when analysing diffusion processes. In applications of the invention which analyse more rapid processes, for example chemical reactions, pulse frequencies in the MHz (megahertz) range may occur. The common feature of different applications of the invention is that of pulsing the excitatory light with at least two pulses per period of the time variation of the process being studied. Since the period and frequency of the time variation of the process being studied are not always entirely known in advance, said period and frequency are estimated when the invention is being applied in practice, and the pulse frequency for 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 the FCS analysis, in accordance with the invention, of molecules which may be coloured with two dyes and, when two dyes are being used, it is usually advantageous, and in some cases necessary, to have two excitation wavelengths. In yet other aspects of the invention, the molecules are exposed to energy in pulses of a first and a second predetermined pulse frequency and, possibly, with a predetermined time delay between the pulses of said first and second pulse frequencies. The time delay separates, with a time interval, the pulses which originate from each respective colour. Emission from said molecules of fluorescence of a first and a second predetermined wavelength is detected. In one embodiment of the invention, said molecules are modified with a dye. Said molecules can comprise a first type of molecule which is modified with a first dye and a second type of molecule which is coloured with a second dye.

In different applications of the invention, two-colour FCS in accordance with the invention can be used when analysing a molecule which is modified with two dyes, with a process cleaving the molecule and thereby separating the two dyes. In- other applications, two molecules are present, each of which has its own, differently coloured dye, and cross correlation can be detected when these two molecules are joined together.

Other aspects of the invention comprise an apparatus for fluorescence correlation spectroscopy, wherein molecules are exposed to energy for the purpose of exciting said molecules and wherein the ensuing emission of fluorescence of a predetermined wavelength from said molecules is detected, wherein means are arranged for effecting the steps and functions as described above. Furthermore, the invention can comprise a computer program product for controlling an apparatus for fluorescence correlation spectroscopy, wherein molecules are exposed to energy for the purpose of exciting said molecules and wherein the ensuing emission of fluorescence of a predetermined wavelength from said molecules is detected, with the computer program product comprising program code which is arranged to direct the computer processor to carry out the steps and functions as described above.

In general terms, the invention can be used to study time-varying courses and processes where said time variation can be measured by fluorescence. Diffusion, chemical processes and the population of long-lived energy levels in systems under study are examples of this. Correlation analysis is used, in accordance with the invention, for determining the number of units, for example the number of molecules, which contribute to the fluorescence, as well as the frequency of the time variation of the process. For the sake of clarity, the invention is explained in more detail by means of exemplifying embodiments which are performed on diffusion as the process.

Brief description of the drawings The invention will be explained below with reference to the attached drawings in which:

Fig. 1 shows graphs of absorption and emission for two common dyes while being excited in accordance with the prior art; Fig. 2 shows graphs of the two dyes shown in Fig. 1 while being excited in accordance with the invention;

Figs. 3-6 show graphs which illustrate different experiments using the invention;

Fig. 7 shows a synoptic block flow chart of steps which are included in an embodiment of the method in accordance with the invention.

Description of embodiments of the invention Fluorescence correlation spectroscopy (FCS)

The core in FCS is to measure the fluorescence and calculate the autocorrelation on the signal which has been detected. In order to obtain a measurable correlation amplitude, the number of molecules or particles which contribute to the fluorescence signal must be small. This requires these particles to have very good fluorescence properties so as to ensure that the signal can at all be detected. The most common approach at present is to use a diffraction-limited lens to focus laser light to a volume having a linear dimension which is the same size as the wavelength, thereby giving a volume of the femtolitre order of magnitude. The emitted fluorescence is collected using the same optics, i.e. what is termed epi-illumination, and the autocorrelation is calculated in accordance with the following formula:

where (I (t) ) is the time average of the intensity and τ is the separation in time between two groups of data which are to be correlated. Using a Gaussian intensity profile for the cross section of the laser beam, the autocorrelation is given by:

where D is the diffusion constant, ωø is the diameter of the detection volume and zo is the length. In order to obtain good correlation amplitude, the particle number IV must be low, something which at the same time means lower intensity and a worse signal-to-noise ratio.

The geometry of the detection volume is usually fixed, with this resulting in it being the particle number N and the lateral diffusion time (ω² ₀ /4D) which are determined. The particle number makes it possible to determine concentration and to study chemical reactions while the diffusion time provides a measure of size and can be used, for example, for studying aggregation.

Frequency relations

The invention is based on the inventors' insight regarding the following relations.

The well-known sampling theorem applies in connection with time-discrete observations of time-continuous processes by means of sampling at a certain sampling frequency. Sampling can be regarded as being pulses of occasions of observation, or detections, with a certain pulse frequency, of a time-dependent quantity which varies with a certain studied frequency. This way of looking at the matter is used in the present invention.

In fluorescence correlation spectroscopy (FCS) , there is an observed volume of some substance which contains molecules. These molecules diffuse into and out of the observation volume due to thermal motion. A time- dependent quantity in this connection is the number of molecules which are preferably on average located in the observed volume. The number of molecules in the observation volume at any given point in time follows a course which is in principle time-continuous. From the purely mathematical point of view, the course is per se time-discrete since a number, i.e. the magnitude of a quantity, is specified by natural numbers between which there are discrete steps. For practical purposes, however, this number of molecules can be regarded as exhibiting a time-discrete course.

The molecules are located in the observation volume for a certain, preferably average, time which, in connection with FCS, is termed the diffusion time. In a frequency description, the time-continuous course varies with a frequency which corresponds to a relation where the diffusion frequency = 1/the diffusion time.

The observed volume containing the molecules is irradiated with energy which has a certain wavelength.

Molecules in the observed volume are excited by the energy and subsequently emit a fluorescent light (fluorescence) of a certain wavelength. A light intensity detector is used to observe the fluorescence emission in its capacity as a parameter which depends on the number of molecules in the observed volume. The observation of the fluorescence emission, i.e. the detection of the intensity of the fluorescence emission from the excited molecules which are located in the observed volume, proceeds continuously during FCS. This can be compared to a camera whose shutter is continuously open.

The intensity of the fluorescence emission depends on the number of excited molecules which are located in the observed volume. The fluorescence from a molecule after a pulse of excitation energy contributes to the intensity and is therefore be observeable/detectable during the time the excited molecule is located in the observed volume and until it has diffused out. Consequently, a molecule can is observeable/detectable during the average diffusion time.

In order to detect a fluorescence intensity which correctly corresponds to the number of molecules in the observed volume, all the molecules in the volume must be excited. According to the present invention, the observation volume is irradiated with excitatory energy in pulses having a certain pulse frequency. As a result of the excitation energy being pulsed, the fluorescence emission from the molecules in the observation volume is given a time-discrete character having a frequency which depends on the diffusion frequency. This thereby makes it possible to apply the sampling theorem and, consequently, in order, by way of the parameter fluorescence emission, to correctly observe the time course for a frequency description of the number of molecules in the observation volume, it is necessary and sufficient for the pulse frequency of the excitation energy to be twice the diffusion frequency. That is to say that, during the period each molecule is present in the observation volume, it has on average time to be irradiated twice by an excitation pulse.

Since the excitation energy is time-discrete as a result of the pulsed irradiation, the fluorescence emission from the observation volume is also pulsed, that is time-discrete. The output signal from the detector for detecting the fluorescence emission will then also be discrete, that is to say the output signal will comprise pulses of intensity peaks after each excitation pulse. In order to ensure that the output signal from the detector supplies a signal which corresponds to a correct number of molecules in the observation volume, it is consequently sufficient to apply emitting energy, in accordance with the invention, in pulses having a pulse frequency which substantially corresponds to twice the diffusion frequency of the molecules which are currently being studied.

The invention can be compared to a camera having an open shutter and pulsing illumination with the possibility ^' of a minimal or smallest pulse frequency which is equal to twice the diffusion frequency. This gives a time-discrete detection or sampling of a parameter having a time-discrete character which depends on a course which is at least in principle time-continuous. This is in contrast to the prior art, which can be compared to a camera having an open shutter and continuous illumination or quasicontinuous illumination, with this consequently, according to the prior art, giving time-continuous detection of a parameter which is at least in principle ' time- continuous and which depends on a course which is in principle time-continuous.

Fluorescence correlation spectroscopy (FCS) with pulsed excitation

Fig. 7 shows a synoptic block flow chart of the principle steps for carrying out FCS with pulsed excitation in accordance with one embodiment of the invention, with this FCS comprising the following steps:

702 : Arrange an observation volume in accordance with

FCS containing molecules which are to be studied.

704 : Apply, to the observation volume, excitation energy in pulses having a pulse frequency in a range which substantially corresponds to twice the diffusion frequency of the molecules.

706 : Record the fluorescence which is emitted from the observation volume. 708 : Analyse said recorded fluorescence, which depends on the number of molecules in the observation volume.

Pulsed excitation according to the invention will in general not involve any major change in the appearance of an FCS instrument; instead, it will be possible to use existing equipment in which control of the excitation energy is modified. According to a preferred embodiment of the invention, the requirement is for means to be present for pulsing the emission of energy from the excitation source, for making the repetition frequency controllable or adjustable and for making the correlation analysis to take place with the same time resolution as the excitation. The pulsing and control of the repetition frequency for the pulsing is brought about in different embodiments of the invention by means, for example, of an aperture whose perviousness for excitation energy generated by the excitation source is controllable.

In one embodiment of the invention which is adapted for experiments using two dyes, the excitation pulses for the different dyes are separable in time in a controllable manner. Separating the excitation pulses in time minimizes the greatest problem of this type of measurement, namely false correlation from dyes which are emitting in both wavelength ranges. As an example, Fig. 1 shows graphs for the absorption and emission in the case of two common .dyes, i.e. Alexa Fluor 532 (above in the figure) and Alexa Fluor 633 (below in the figure) from Molecular Probes, Eugene, OR. If the excitation takes place simultaneously, it is necessary for the detector which is to measure the signal from Alexa Fluor 633 to have a filter which does not transmit a signal below 650 nm in order to avoid the detector also detecting a signal from Alexa Fluor ,532. As can be seen from Fig. 1, Alexa Fluor 532 also has significant emission up to 650 nm. This means that a large part of the emission from Alexa Fluor 633 is filtered off simply to avoid any signal overlap, and this means that the signal-to-noise ratio is significantly impaired. In the case of two-colour FCS in accordance with the prior art, it is a major problem to find two dyes which do not overlap in regard to their emissions but which, at the same time, must not have excitation wavelengths which are too different. This is because too great a difference leads to the excitations having different geometries.

When, as in embodiments of the invention, the excitation pulses are instead separated, preferably at least to such a degree that the signal has time to subside, given the lifetime of the excited state, it is then known precisely which dye is being excited at a certain time and, consequently, in which detector the desired signal is being recorded.

Fig. 2 shows graphs generated in connection with an experiment in which a detector for green light is provided with a filter which transmits light of wavelengths shorter than 600 nm and a detector for red light having a wavelength longer than 600 nm. When a green laser (532 nm) illuminates the sample, Alexa Fluor 532 is excited, on the one hand, but Alexa Fluor 633 is also excited to some degree. In the case of a red laser pulse (633 nm) , only Alexa Fluor 633 will be excited and the red detector receives . light. The correlation between the green fluorescence, when the green laser illuminates the sample, and the red fluorescence, in connection with red excitation, is now calculated. In this way, the crosstalk which would otherwise have arisen disappears at the same time as it is possible to make maximum use of the emitted fluorescence. While this principle makes it simpler to select dyes, it also improves the correlation analysis. It should also be possible to increase this to more than two dyes, with this opening up the possibility of studying reactions in which more reagents participate and more than one bond is created or broken.

Model description

The effect of pulsing the excitation source can be demonstrated using a simple computer model. This model takes account of the diffusion of molecules into and out of the detection volume (which is the same as the excitation volume) , excitation and relaxation as well as bleaching. The model is 0-dimensional, i.e. the geometry is assumed to be symmetrical in all respects.

The environment outside the detection volume is assumed to be unchanged in regard to the number of molecules. The flux of molecules into the volume is assumed to be equal to the flux which is directed outwards, with this resulting in the probability of a molecule diffusing in or out being given by the equation

At P — ' diff

Where At is the time interval which is under consideration and τ_d±_ff is the mean diffusion time. In each time step in the calculations, this is tested for each molecule. This gives the number of molecules which are present in the volume on each separate occasion and thereby subject to the excitation.

The next step is to determine how much fluorescence the molecules which are present in the volume give rise to.

This takes place by calculating how energy levels are populated by way of excitation and depopulated by means of relaxation to lower energy levels. The system which is used is assumed to have three levels, a basal state Ni (lowest energy) and two excited states, N_∑ and N₃.

The transition rate (number of transitions from one level to another per unit of time) is designated k±_j where i represents the initial level and j the final level. Fig. 3 shows an energy level diagram with transition rates in accordance with this.

The population of the different energy levels is determined by the equation system:

N_x = -k_nN +k_2lN₂ +k^N N₂ = +k_nN_x -k_2lN₂ -k_nN₂ N₃ = +k₂₃N₂ - £₃₁N₃

N = N_j +N₂+N₃ N₁₌ N₂ = N₃ = 0

where N denotes the time derivative of N. The assumption of equilibrium is also included.

Relaxation of N_2f with transition to the basal state, can take place either radiationlessly, by way of collisions (ic⁰"¹¹), or by way of photon emission {kH^d ) .

The number of photons emitted per unit time is given by N_∑k^ and, with the assumption that the emission is isotropic, that the light-collecting optics cover the solid angle Ω and that the efficiency of the detector is ξ, the detected intensity is given by:

4π ^{2 21}

Transition rates can be obtained from the literature and some simple calculations. The excitation is given by the intensity of the excitation source ( Jj_aSer) , the cross sectional area of the detection volume and the absorptive ability of the molecule (extinction coefficient ε) : nlO k_n = σ — l laassee -rr l -^■ — laser πωl 0.00 IN, πωl

As a rule, FCS experiments make use of diffraction- limited lenses which result in the diameter (2ω₀) of the volume being approximately equal to the wavelength of the laser light. The relaxation rates are given by the lifetime τ₂ and the quantum yield φ for N :

i=_fc "2,1 ₊ ' _fc '"2₃

The molecules in the model can possess one or more tryptophan (s) which is/are responsible for the fluorescence which is calculated. Tryptophan has an extinction coefficient of 5500 M^"1 cm^-1 (at 280 nm) while the literature states that τ₂ is 3 ns, φ is 10-30% and k₂₃ is 20-80 1 100⁶⁶ ss^""1. The transition rate for N₃ is estimated to be 10⁶ s^_1.

The major problem for FCS is bleaching, with this arising when N₃ is populated. This state is a triplet state having a spin of 1 (while Ni and N₂ are singlet states having a spin of 0) and long-lived since relaxation to the basal state requires a change of spin. Molecules in this long-lived state (lifetime about 1 μs) have plenty of time, to collide with 0₂ (triplet) and thereby relax to the basal state at the same time as 0₂ (singlet) is- formed. This latter is in turn very reactive and risks destroying the fluorescent properties of the molecule. In order to take this into account, all tryptophans which end up in N₃ are regarded as being bleached and henceforth incompetent.

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 fluorescence which is recorded and the result of calculating the autocorrelation. The worst conditions, i.e. the quantum yield being 10% and k₂₃ being 80 10⁶ s^-1, have been assumed in the calculations. Furthermore, the intensity of the laser pulses is assumed to vary by 10% from pulse to pulse and, over and above that, there is 20% noise. The number of molecules is 10, i.e. without bleaching there should on average be 10 molecules in the volume (but not at every individual time point) . The diffusion time is 100 μs, the time interval in the calculations is 1 μs and the total measurement time is 100 ms.

Figure 4 shows continuous excitation of molecules containing one tryptophan. Without bleaching, the autocorrelation curve (continuous blue line, scarcely visible) should follow the red line which describes the diffusion. This is the effect of bleaching; the molecules are bleached as soon as they enter the volume, the intensity becomes minimal and the autocorrelation curve is determined solely by the bleaching course.

The graphs in accordance with Fig. 5 were obtained in another experiment in which the excitation is pulsed with the Nyqvist frequency, and the diffusion .time is 100 μs, which means an excitation pulse every 50 μs . The result is striking; the autocorrelation curve is visible and is described well by the combined effect of diffusion and bleaching. Bleaching can also be regarded as being a type of diffusion; when a molecule ceases to emit fluorescence, this becomes tantamount to the molecule having left the volume.

In the third experiment, Fig. 6, the molecules contain five tryptophans. The autocorrelation curve closely follows the diffusion and bleaching has become a slow and not particularly important course. It is also notable that the correlation amplitude at τ = 0 is 0.1, which agrees well with the fact that there should on average be 10 molecules in the volume (G(τ=0) =1/N) .

To sum up, the effect of pulsed excitation is evident; that is to say, the negative effect of bleaching is removed. It is also clear that it is possible to use FCS on proteins which contain several tryptophans. It is at present unclear whether it will be possible, to use the invention to carry out measurements on proteins which only contain one tryptophan, but this is not of crucial importance. Pulsed excitation in accordance with 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 a variety of ways within the scope of the attached patent claims .

Claims

1. 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 and wherein the subsequent emission from said molecules of fluorescence of a predetermined wavelength is detected, characterized in that said energy is applied in pulses having a pulse frequency which at least amounts to two pulses per period of the time-varying course being studied.

2. 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 being studied.

3. The method according to Claim 1, wherein said energy is applied in pulses with a pulse frequency in the range of 0.01 to 999 kilohertz.

4. Method according to Claim 1, wherein said energy is applied in pulses with a pulse frequency in the range of 0.2 to 200 kilohertz.

5. Method according to any of the preceding claims, wherein the number of fluorescence-emitting units is determined by means of a correlation analysis of said detected emission.

6. Method according to any of the preceding claims, wherein the frequency of the time variation of the fluorescence-emitting process is determined by means of a correlation analysis of said detected emission.

7. Method according to any of the preceding claims, wherein said energy is applied in pulses with a pulse frequency that substantially corresponds to twice the diffusion frequency of said molecules.

8. The method according to any of the preceding claims, wherein said molecules are exposed to pulses of energy of a first and a second wavelength.

9. The method according to any of the preceding claims, wherein said molecules are exposed to energy in pulses of a first and a second predetermined pulse frequency and with a predetermined time delay between the pulses of said first and second pulse frequencies.

10. The method according to any of the preceding claims, wherein emission, from said molecules, of fluorescence of a first and a second predetermined wavelength is detected.

11. The method according to any of the preceding - claims, wherein said molecules are modified with a dye.

12. The method according to any of the preceding claims, wherein said molecules comprise a first type of molecule which is modified with a first dye and a second type of molecule which is coloured with a second dye.

13. The method according to any of the preceding claims, wherein said molecules comprise more than two types of molecule which are modified with different dyes.

14. The method according to any of the preceding claims, wherein said molecules are of a first type which are modified with two or more different dyes.

15. The method according to 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.

16. An apparatus for fluorescence, correlation spectroscopy, wherein molecules being exposed to energy for the purpose of exciting said molecules and wherein the subsequent emission, from said molecules, of fluorescence of a predetermined wavelength being detected, characterized by means that are arranged to perform steps and functions in accordance with any of the claims 1-15.

17. A computer program product for controlling an apparatus for fluorescence correlation spectroscopy, wherein molecules are exposed to energy for the purpose of exciting said molecules and wherein the subsequent emission from said molecules of fluorescence of a predetermined frequency is detected, characterized by program code that is arranged to direct a computer processor to carry out the steps and functions in accordance with any of the claims 1-15.