EP1386139A2

EP1386139A2 - Fluorescence fluctuation microscope analytical module or scanning module, method for measurement of fluorescence fluctuation and method and device for adjustment of a fluorescence fluctuation microscope

Info

Publication number: EP1386139A2
Application number: EP02724136A
Authority: EP
Inventors: Jörg LANGOWSKI; Malte Wachsmuth
Original assignee: Deutsches Krebsforschungszentrum DKFZ
Current assignee: Deutsches Krebsforschungszentrum DKFZ
Priority date: 2001-05-07
Filing date: 2002-05-07
Publication date: 2004-02-04
Also published as: US20040238730A1; WO2002090947A2; DE10291983D2; WO2002090947A8; WO2002090947A3

Abstract

A fluorescence fluctuation microscope, in which excitation light and detection light are coupled into or out of a microscope by means of a common beam path, comprises a closed loop scanning unit (22, 23).

Description

Fluorescence fluctuation microscope, measuring module or scanning module and method for fluorescence fluctuation measurement as well as method and device for adjusting a fluorescence fluctuation microscope

The invention relates to a fluorescence fluctuation microscope, a fluorescence fluctuation measurement module or a fluorescence fluctuation scanning module and a method for fluorescence fluctuation measurement. The invention also relates to a method and a device for adjusting a fluorescence fluctuation microscope.

A fluorescence fluctuation measurement module is known from WO 98/23944 A1, with which fluorescence fluctuation measurements or fluorescence correlation measurements and or fluorescence cross-correlation measurements can also be carried out with commercially available microscopes, in particular with inverted microscopes. In the solution shown there, for example, the corresponding measurement module is connected to an optical connection of a microscope, the paths of the excitation light and the measurement light being confocally aligned by the fluorescence fluctuation measurement module. This arrangement is particularly designed for the measurement of mobile particles or molecules which pass through the focus due to their own movement, in particular due to the Brown 'see molecular movement or molecular flows. On the other hand, KM Berland et. al. "Scanning Two-Photon Fluctuation Correlation Spectroscopy: Particle Counting Measurements for Detection of Molecular Aggregation", Biophysical Journal Volume 71, July 1996, pages 410 to 420 presents a scanning fluorescence fluctuation measurement in which immobilized particles or molecules periodically with respect to the excitation light The periodic movement can be achieved on the one hand by periodically moving the slide or on the other hand by periodically moving the excitation light. However, the scanning method shown there is not suitable for detecting mobile molecules or particles because the necessary periodicity cannot be achieved with these, in particular if these molecules or particles leave the periodic path of the slide or of the excitation light.

It turns out that these methods, which are known and very successful, are not able to detect or track non-immobilized molecules or particles to a sufficient extent. In this respect, the great advantages of fluorescence fluctuation spectroscopy with its high sensitivity and selectivity with regard to fluorescence, which extends to the recognition of individual molecules, cannot be used with non-immobilized molecules.

It is an object of the present invention to provide a fluorescence fluctuation microscope or a method for fluorescence fluctuation measurement which eliminate the disadvantage described above. As a solution, the invention proposes a fluorescence fluctuation microscope in which excitation light and fluorescent light are coupled into or out of a microscope via a common beam path, preferably confocal, and which is characterized in that a closed-loop scanning unit is provided.

In contrast to the resonant scanning units used in the known scanning fluorescence fluctuation measurement, a closed-loop scanning unit enables a specific location to be approached and the corresponding position to be held over a long period of time. This makes it possible to measure a sample in a targeted manner at different positions, these measurements being able to be carried out at a specific location, for example both according to WO 98/23944 A1 or according to the known scanning fluorescence fluctuation measurement.

While a corresponding closed-loop scanning unit can be provided directly on the specimen slide, it proves to be particularly advantageous if the corresponding closed-loop scanning unit is provided in the common beam path of detection light and excitation light, so that excitation light and detection light are moved over the sample. In this way, it is possible for the first time to record fluorescence fluctuation measurements even with moving molecules or particles or with corresponding flows, and these flows can be followed in a targeted manner. In addition, for the first time, the invention enables a spatially extended sample in its entirety with regard to fluorescence measure fluctuations, so that even complex relationships, such as those that occur in a biological cell, can be fully recorded.

Laser scanning microscopes are known from DE 198 29 953 AI, DE 197 33 195 AI and DE 43 23 129 AI, which can also be used for fluorescence measurements. Fluorescence fluctuation measurements are not mentioned in these publications. A glance at the embodiments shown in these publications shows the person skilled in the art that these arrangements, owing to their complexity, the relatively long optical and thus also mechanical paths between the beam splitter separating excitation light and detection light and the detectors, and also the pinhole arrangements provided for each detector, of the many other mechanical components and the integrated laser are not suitable for fluorescence fluctuation measurements, since all of these components on the one hand lead to background noise during fluorescence fluctuation measurements and to correlated vibrations and thus to sham measurement results. In this respect, these arrangements are indeed not suitable for time-critical fluorescence fluctuation measurements for fluorescence measurements designed to be integrative over time.

Accordingly, the invention also proposes a method for fluorescence fluctuation measurement, in which a closed-loop scanning unit focuses excitation light on a sample at a specific location and a fluorescence fluctuation measurement is carried out at this location and in which the closed-loop scanning unit then focuses the excitation light on the sample at one location and a fluorescence fluctuation measurement is also carried out at this other location.

Regardless of the other features of the present invention, a fluorescence fluctuation microscope is also advantageous, which comprises a microscope, a fluorescence fluctuation measurement module, for example according to WO 98/23944 A1, and a fluorescence fluctuation cam module. In this way, a device operating according to the method according to the invention can be provided at relatively low cost, since only the corresponding scan module needs to be provided. With such an arrangement, especially if the microscope-side connection of the fluorescence fluctuation scan module is configured identically to the microscope-side connection of the fluorescence fluctuation measurement module, the fluorescence fluctuation measurement module can also be used without problems for classic fluorescence fluctuation measurements. Accordingly, regardless of the other features of the present invention, a fluorescence fluctuation scan module with two connections, a first connection on the microscope side and a second connection on the measurement module side, in which the two connections are designed to be complementary, is advantageous.

A descan lens is preferably provided between the scanning unit and a beam splitter for excitation light and detection light. In the present context, under the term “descan lens” each optically see arrangement through which focused rays can be converted into parallel rays. In particular, such a descan lens can also be composed of several individual lenses. Such a descan lens, independent of the other features of the present invention, makes it easier to adjust the overall arrangement. This makes it possible to initially adjust excitation light and detection light, in particular confocal. This can be done in a manner known per se, in particular using a microscope. Such adjustment is considerably simplified, in particular, by the beam guidance divergent without the descan lens.

Following this, the descan lens can be used and adjusted without any problems, whereby it is only necessary to ensure that the beam path is sufficiently parallelized due to the descan lens. For this purpose, in particular a detachable adjustment holder connected to the descan lens and having a marker that can be displaced parallel to the optical axis can be used.

It goes without saying that the descan lens can be arranged on the one hand on the microscope-side connection of the fluorescence fluctuation measurement module or on the connection of the fluorescence fluctuation scan module that faces away from the microscope. The descan lens can preferably be adjusted with respect to an optical arrangement of the fluorescence fluctuation measurement module, in particular with respect to a pinhole. The scanning unit is preferably adjustable perpendicular to its optical axis with respect to the optical axis of a detector arrangement and / or with respect to the optical axis of an excitation light source. This can be ensured in particular by designing at least one of the corresponding connections to be adjustable with respect to the rest of the module, in particular adjustable perpendicular to the optical axis. In this way, after a parallel light beam has been generated in the above-described adjustment by the descan lens, it can be ensured that it passes through the scanning unit in an optimal manner or that its optical axis coincides with the optical axis of the arrangement consisting of the descan lens and stim - clearing or detection light path matches.

As already described above, the arrangement according to the invention makes it possible for the first time to use fluorescence fluctuation measurements for imaging. While the measurements themselves can easily be carried out in a reasonable time window, the associated calculations, which are necessary according to the prior art, prove to be so complex that it is difficult to speak of a “real-time” recording In the present case, also independently of the other features of the present invention, it is proposed to first statistically evaluate the pure measurement results and thus to significantly reduce the number of data to be processed before correlation analysis actually takes place. Such a procedure can be carried out in particular with a fluorescence fluctuation microscope which Means for spatially resolved detection of the measured intensity and means for spatially resolved detection of a correlation function integrated over time. These values can be determined on the one hand in a computer and, on the other hand, relatively promptly and without considerable effort by means provided locally on the corresponding detectors. A correlation time can then be determined immediately from the measured intensity and the integrated correlation function and displayed accordingly. In this way, a user can be provided with a spatially resolved representation of a fluorescence fluctuation measurement almost in “real time”, which includes the integrated correlation function on the one hand and the correlation time on the other hand.

Further advantages, goals and properties of the present invention are explained with the aid of the description of the attached drawing, in which a fluorescence fluctuation microscope according to the invention and an adjustment holder are shown schematically.

The drawing shows:

1 shows a part of a fluorescence fluctuation scan module and a

Fluorescence fluctuation measuring module in a schematic view,

Figure 2 shows the other part of the fluorescence fluctuation scan module and a corresponding microscope in a schematic view and

Figure 3 is a schematic representation of an adjustment bracket according to the invention. The fluorescence fluctuation microscope shown in FIGS. 1 and 2 comprises a commercially available microscope 1, in the tube 10 of which a scanning unit 2 is arranged, to which, on the other hand, a fluorescence fluctuation measurement module 3 is connected. The fluorescence fluctuation measurement module 3 used as an example in this exemplary embodiment comprises a laser light coupling via a fiber 30, the light emanating from the fiber being focused into an intermediate image plane 33 by means of a collimator 31 and a lens 32 with an adapted aperture. For this purpose, lens 32 in particular can be shifted accordingly. The beam waist of the excitation light beam serves as the light source for the arrangement described below, the corresponding light first being passed through an excitation filter 34 to a beam splitter 35. This beam splitter 35 reflects the excitation light and allows longer-wave fluorescence or detection light to pass through, so that two conjugate intermediate image planes 33 are generated accordingly. Starting from the beam splitter 35, the detection light is passed through a pinhole 36, which produces the confocality, or a pinhole 36 in the plane 33 and is spectrally divided into two detectors 38 and 39 by means of a beam splitter 37. The short-wave component is reflected on the beam splitter 37 and imaged on the detector 38 by an emission filter 40 and a detection lens 41. Accordingly, the long-wave portion that passes through the beam splitter 37 is imaged by a lens 44 onto the second detector 39 via a mirror 42 and a filter 43. In this embodiment, the pinhole 36 acts as a reference point for adjustment. Here, the lenses 41 and 44 are shifted until the pinhole 36 is imaged on the detectors 38 and 39. Furthermore, the lens 32 and — if appropriate — the collimator 31 are also adjusted such that the laser beam waist and pinhole 36 are arranged in a conjugate confocal manner.

In the exemplary embodiment shown in FIGS. 1 and 2, an inverted microscope 1 with an optical output 11 on the side is used, which in itself can also be used for connecting CCD cameras or dissection devices. In the microscope 1 used in the present case, a beam splitter cube 12 conducts approximately 80% of the light coming from a sample arranged on a sample carrier 14 and coming from an objective 13 via a tube lens 15 to the side exit 11 and 20% via a mirror 16 to an eyepiece 17 It goes without saying that other microscopes and other microscope outputs can also be used for an implementation according to the invention.

An intermediate plane 18 is provided in the tube 10 of the microscope 1 and is used as the focal plane for the fluorescence fluctuation measurement module 3 or for the fluorescence fluctuation scan module 2. It goes without saying that an intermediate level of the microscope 1 which is present at another location and can be used in a suitable manner can also be used for this purpose. In addition, the arrangement according to the invention can also be implemented without the use of such an intermediate image plane, the use an intermediate image plane, on the other hand, enables a relatively simple implementation of the invention, in particular also with other microscopes, since it only has to be ensured by suitable connections that a correspondingly provided intermediate image plane can be used as the focal plane.

The fluorescence fluctuation scanning module 2 comprises a scanning lens 20 at its microscope-side connection, the focal plane of which coincides with the intermediate image plane 18 when the scanning module 2 is connected to the microscope 1. On the image side, a telecentric plane 21 of the scanning lens 20 lies between two mirrors 22, 23 of a galvanometer scanner. In the present exemplary embodiment, rotary magnet galvanometer scanners in particular have proven to be advantageous. Here, the axis of rotation of the mirror 23 is tilted 15 ° from the horizontal in order to achieve the smallest possible distance between the mirrors, which is approximately 23.5 mm on the optical axis. These galvanometer scanners are designed as closed-loop scanners so that they can maintain a deflection once they have been reached. In this way, a position can be approached in a targeted manner and the fluorescence fluctuation can be measured. It goes without saying that such closed-loop scanners can also be used to move the sample holder 14. However, the use of a scanning unit in the light path has the advantage that much smaller masses have to be moved. By suitable choice of the masses of the mirrors, the effects of the scanning unit which distort the measurement can be prevented or minimized. As well the control loops available with closed-loop scanning units can be optimally designed.

When the scanner mirror rotates, the focus moves in the intermediate image plane 18 and in the object plane of the microscope. A collimation or descan lens 24 focuses the light accordingly on the intermediate image plane 33, the aperture angle being chosen to be identical to the aperture angle of the light emerging from the microscope 1 at the output 11. This aperture adjustment allows the fluorescence fluctuation scan module 2, consisting of tube 10, scan lens 20, scanner 22, 23 and descan lens 24, to be removed from the beam path between the output 11 and the fluorescence fluctuation measurement module 3 and to connect the fluorescence fluctuation module 3 directly to the output 11 or instead of the fluorescence fluctuation - Measuring module 3 to mount another confocal optics on the scan module 2.

For adjustment, the descan lens 24 can be displaced, in particular, laterally or perpendicularly to the optical axis. An auxiliary frame 50 is preferably provided for the adjustment of the descan lens 24, which can be attached to the descan lens 24 in order to align it appropriately. This auxiliary frame is designed in such a way that it can be used to check the parallelism of the light beam leaving the descan lens 24 by means of suitable markers 51 (exemplarily numbered in FIG. 3), and the descan lens 24 can be suitably readjusted. For this purpose, the adjustment bracket in the present exemplary embodiment has two guides 52, on which a screen 53 is connected in parallel. is arranged, which carries the corresponding marker 51. The parallelism of the corresponding light beam can be checked easily by a parallel displacement of the slot 53.

In addition, a cross table 25 is arranged on the fluorescence fluctuation scan module 2, by means of which the optical axes of the fluorescence fluctuation measurement module 3 and of the fluorescence fluctuation scan module 2 can be made to coincide.

The outputs of the detectors 38 and 39 are connected to a corresponding evaluation device, in particular a corresponding computer. This can in particular have separate inputs or cards with which individual functions can be easily controlled. This can be, for example, a correlator card for recording the correlation functions and a counter card, the counter card providing the sampling rates which are unusually high for conventional correlator cards for scanning microscopy. The necessary calculation can also be carried out to a sufficient extent in these cards so that the computer does not have to intervene directly. It goes without saying that the computer can, however, also be used for these calculations, in particular as a support.

In the measuring mode, the detector signal of the detectors 38, 39 is then first integrated in the photon counting mode, as is also common for the known fluorescence fluctuation spectroscopy, over time intervals Δτ of, for example, 10 μs for a defined time of, for example, 50 ms (that is 5000 in tervalle) added. Then the auto- or cross-correlation fluctuation, depending on single-channel or two-channel operation, G (τ) for τ> 0 is calculated from this data. The calculation of the correlation function from the measured photon pulses k i = I ... N, in each case summed up over a time interval Δτ in N successive intervals, can be carried out quickly as follows:

In addition, the amplitude G (0) is calculated from this data, which in the case of diffusion is inversely proportional to the number of particles in focus and thus inversely proportional to the concentration. The calculation of the amplitude G (0) from the counted photon pulses can be carried out as follows:

For diffusion-induced signal fluctuations, the integration via the correlation function \ dτ G (τ) = c ^• xj ^■ G (0), where τ ^ is the correlation or decay time of the signal correlations (in the case of diffusion, the mean length of stay of the Molecules in focus and inversely proportional to the diffusion coefficient), and the constant c number rically or analytically can be determined. The constant c can also be determined with a single conventional FCS measurement in the same sample.

Due to the numerical integration of G (τ), τ _< / is also known. For example, it can be done as follows:

In this respect, the values xj summed or integrated above over time can be used to represent the correlation time by appropriate means, for example by means of a screen visualization, in particular also almost in "real time". The calculations are very fast on today's computers and can be carried out in parallel with the point-by-point data acquisition With the existing structure, points or locations can be approached in succession for short times (eg 50 ms), so that data can then be taken point by point.

In this way, images with the intensity distribution, with the concentration, as a correlation function integrated over time, and with the correlation time as a contrast-giving signal can be created very quickly, without additional effort in terms of data analysis or the like being necessary.

Claims

claims:

1. Fluorescence fluctuation microscope, in which excitation light and detection light are coupled into or out of a microscope (1) via a common beam path, preferably confocal, characterized in that a closed-loop

Scanning unit (22, 23) is provided.

2. Fluorescence fluctuation microscope according to claim 1, characterized in that the scanning inlet (22, 23) is provided in the common beam path.

3. Fluorescence fluctuation microscope according to claim 1 or 2, comprise a microscope (1), a fluorescence fluctuation measurement module (3) and a fluorescence fluctuation scan module (2).

4. Fluorescence fluctuation microscope according to one of claims 1 to 3, characterized by a descan lens (24) between the scanning unit (22, 23) and a beam splitter (35) for the excitation light and the detection light.

5. Fluorescence fluctuation microscope according to one of claims 1 to 4, characterized in that the scanning inlet (22, 23) perpendicular to its optical axis with respect to the optical axis of a detector arrangement (38, 41, 36; 39, 44, 36) and / or is adjustable with respect to the optical axis of an excitation light source (31, 32).

6. Fluorescence fluctuation microscope according to one of claims 1 to 5, characterized by means for spatially resolved detection of the measured fluorescence intensity, with means for spatially resolved detection of a correlation function integrated over time and with means for displaying a correlation time.

7. Fluorescence fluctuation microscope according to claim 6, characterized in that the means for spatially resolved detection of the measured fluorescence intensity and / or the means for spatially resolved detection of the correlation function integrated via the time axis are provided in a separate unit from an evaluation computer.

8. Fluorescence fluctuation measurement module (3) with a microscope-side connection, characterized by a descan lens (24) on the connection.

9. Fluorescence fluctuation scan module (2) with a connection facing away from a microscope, characterized by a descan lens (24) on the connection.

10. Fluorescence fluctuation scan module according to claim 9, characterized by two connections, a first for a microscope (1) and a second for a measuring module (3), the two connections being complementary to one another.

11. Fluorescence fluctuation measuring module according to claim 8 or fluorescence fluctuation scanning module according to claim 9, characterized in that the descan lens is adjustable with respect to an optical arrangement, in particular with respect to a pinhole (36).

12. Fluorescence fluctuation measurement module or fluorescence fluctuation scan module according to one of claims 8 to 11, characterized in that the connection is adjustable with respect to the rest of the module, in particular adjustable perpendicular to the optical axis.

13. For fluorescence fluctuation measurement, in which a closed-loop scanning unit (22, 23) focuses excitation light onto a sample at a specific location and a fluorescence fluctuation measurement is carried out at this location, and in which the closed-loop scanning then takes place Unit (22, 23) focuses the excitation light on the sample at another location and a fluorescence fluctuation measurement is also carried out at this other location.

14. The procedure for adjusting a fluorescence fluctuation microscope according to one of claims 1 to 8, characterized in that first an excitation light path and a detection light path and then a descan lens (24) are adjusted.

15. Verfaliren according to claim 14, characterized in that after the adjustment of the descan lens (24), the scanning inlet (22, 23) is adjusted.

6. Device for adjusting a fluorescence fluctuation microscope (24), characterized by an adjustable holder (50) connected to the descan lens (24) and removable with a marker (51) that can be displaced parallel to the optical axis.