JP2006526798A - Scanning microscope with apparatus for fluorescence correlation spectroscopy - Google Patents

Abstract

A scanning microscope is provided that enables direction-dependent measurements of the diffusion rate and flow rate of sample elements such as molecules.
The present invention relates to a scanning microscope having an apparatus for fluorescence correlation spectroscopy. The scanning microscope is characterized in that the intensity and / or detection sensitivity of the illumination light is spatially adjusted within the measurement volume.

Description

  The present invention relates to a scanning microscope for fluorescence correlation spectroscopy.

  In scanning microscopy, the object to be tested is illuminated with a light beam in order to observe the reflected light or fluorescent light emitted by the sample. The focus of the illumination light beam is generally moved in the object plane by tilting two mirrors in the object plane using a controllable beam deflection device. The deflection axes are usually located perpendicular to each other so that one mirror deflects in the x direction and on the other in the y direction. For example, the mirror is tilted using a galvanometric positioner. The measurement of the power of light coming from the object depends on the scanning position of the illumination light beam.

  Particularly in confocal scanning microscopy, the object is scanned in three dimensions by the focus of the light beam. In general, a confocal scanning microscope has a light source, a focusing optical system in which light from the light source is focused on a pinhole aperture (so-called excitation aperture), a beam splitter, a beam deflector that controls the beam, a microscope optical system, It has a detection aperture and a detector for detecting detection light or fluorescent light. The illumination light becomes one through the beam splitter. Fluorescent light or reflected light coming from the object returns to the beam splitter via the beam deflector, passes through it, and finally focuses on the detection aperture. There is a detector behind this opening. Detection light not directly generated from the focal region takes another optical path and does not pass through the detection aperture. Thus, pixel information that yields a three-dimensional image is obtained as a result of continuous scanning of the object. In most cases, 3D images are performed by hierarchical data imaging. Theoretically, the path of the scanning light beam meanders on or within the object (scans a line in the x direction at a constant y position, then x scans and y repositions to the next line to be scanned) Is then interrupted, then scanning this line in the negative x direction at a constant y position, etc.). To enable hierarchical data imaging, after scanning a layer, the sample table or objective is repositioned and the next layer to be scanned is brought to the focal plane of the objective.

  For example, the increase in resolution in the optical axis direction is realized by using a double objective lens arrangement (4π arrangement) as described in Patent Document 1 entitled “Double Confocal Scanning Microscope”. The light coming from the illumination system is divided into two partial beams. These beams simultaneously irradiate the sample so as to extend in opposite directions through two objective lenses arranged in mirror image symmetry. The two objective lenses are arranged on different surfaces of the common object surface. As a result of this interferometer illumination, an interference pattern is formed at the object point that exhibits one main maximum value and several secondary maximum values during constructive interference. Here, the secondary maximum value is generally arranged along the optical axis. Using a double confocal scanning microscope compared to a conventional scanning microscope with interferometric illumination, an increased axial resolution is achieved.

  Confocal scanning microscopes are also used in fluorescence correlation spectroscopy (FCS). Here, the effective measurement volume (measurement volume) is set mainly by the confocal volume. FCS preferably allows analysis of molecular dynamics in the solution. Random molecular passages marked by one or more fluorescent dyes are detected through the measurement volume, for example to draw conclusions regarding the reaction rate or diffusion time.

  The FCS measurement with a confocal scanning microscope in which the diffusion rate is determined is, inter alia, a point spread function (PSF) —the intensity distribution of the illumination light in the focal volume—that is cylindrically symmetric and similar in both the z and lateral directions. The problem of having a distribution (profile) of. Therefore, the correlation signal does not give a clear result with respect to diffusion and flow velocity in various directions. The effect of the observed sample response on the correlation curve makes the analysis more difficult.

EP0491289

  Therefore, the object underlying the present invention is to propose a scanning microscope that allows each direction-dependent measurement of the diffusion rate and flow rate of sample elements such as molecules. Theoretically, this provides a way to easily distinguish between the response dynamics of the correlation curve and the effects of movement.

  This object is solved by a scanning microscope, and the intensity and / or detection sensitivity of the illumination light is spatially adjusted (modulated) within the measurement volume.

  The present invention has the advantage that with proper spatial adjustment, the movements in different directions are distinguished from each other and from the effects caused by different dynamics by the appropriate combination of such measurements. Eventually, in addition to motion along the x, y and z space axes, all possible motions are analyzed, eg diffusion is distinguished from direct transport.

  Preferably, the measurement volume is defined by at least one image point, so-called a person skilled in the art how to fluoresce a given fluorescent molecule within the measurement volume and have a suitable standard of combined probability. You can see how to make a spatial distribution and how to detect this fluorescence. The term “image point” does not mean a location without a mathematical dimension.

  The measurement volume may have a plurality of image points, and the detection light emitted at the image points is detected simultaneously—in parallel—preferably with a plurality of detectors.

  In a preferred embodiment, the intensity and / or detection sensitivity of the illumination light is adjusted spatially and periodically.

  Preferably, the adjustment of the intensity of the illumination light and / or the detection sensitivity may be switched on / off with a switch or spatially varied. This has the advantage that fluorescence correlation spectra of the same measurement volume imaged with different adjustments are analyzed by comparison with each other. Preferably, after correction (eg, background correction) is made, the fluorescence correlation spectrum of the same measurement volume imaged with adjustments on and off is then split by the other.

  In a variant, the adjustment of the intensity of the illumination light and / or the detection sensitivity is varied over time. In this case, the fluorescence correlation spectra of the same measurement volume imaged with different adjustments are analyzed by comparison with each other. By adjusting the intensity and detection sensitivity simultaneously (lock-in method), a spatial adjustment of the “effective” measurement volume is made.

  Preferably, the adjustment of the illumination light intensity and / or detection sensitivity is made by interference. In a preferred embodiment, the light waves that contribute to the interference exhibit a phase relationship that can be adjusted to each other, so that the spatial adjustment is changed by changing the phase relationship.

  In a particularly preferred embodiment, the adjustment of the intensity and / or detection sensitivity of the illumination light is made by the interference of two illumination light beams in opposite propagation directions. Preferably, the scanning microscope is in a 4π arrangement for two objective lenses facing each other, between which the sample is arranged. The illumination light is preferably split into two parts of the illumination beam bundle, each of which passes through the arm of the interferometer and reaches the sample via one of the objective lenses. Similarly, the detection light emitted by the sample passes through the objective lens, first enters the beam combiner with two detection light beam bundles, and then travels together to the detector. Preferably, the beam splitter acts as a beam combiner for the detected light beam bundle.

  This embodiment is particularly suitable for measuring axial flow velocities, since the phase relationship of the light passing through the interferometer arm varies constant over time. The rate of time change varies, for example, until it matches the flow rate.

  In a preferred modification, an amplitude filter that adjusts the intensity and / or detection sensitivity of illumination light is provided. Preferably several side lobes are created in the measuring volume. The side lobes are formed in the same way.

  In another variation, a phase filter or a combination having a phase filter and an amplitude filter may be provided to make adjustments to the intensity and / or detection sensitivity of the illumination light.

  In another variation, a lens arrangement (eg, a microlens arrangement) or a combination of multiple beam splitters is provided to make adjustments to the intensity and / or detection sensitivity of the illumination light.

  The scanning microscope is preferably designed as a confocal scanning microscope.

  The subject of the invention is schematically shown in the diagram and is described below with reference to the drawings. Elements that function similarly are given the same reference numbers.

  FIG. 1 shows a scanning microscope which is particularly suitable for measuring the indicated correlation function. The incident laser beam is split into two beams by a beam splitter (not shown). One of the partial beams goes directly to the 4π resonator. Here another beam splitter (BS) produces two beams. These beams pass through two objective lenses (OL) and are focused on the same position of the sample, where they create an axial pattern by interference.

  Instead, the other partial beam divided into a plurality of adjacent beams by a microlens scanner (LA) is also used. The position of the beam varies within the beam profile. Eventually, an additional movable lateral pattern is created in the sample.

  A pure lateral pattern is created by switching off one of the two sides of the 4π resonator.

  Here, a doped prism (DP) is shown as one of many possibilities for rotating the pattern around the optical axis.

  FIG. 2a shows the point spread function (PSF) of a one-photon 4π confocal microscope calculated as a detection pinhole aperture of 488 nm excitation light, a numerical aperture of 1.2, immersion and Airy disc size. The small figure shows the pattern change as the relative phase of the interference beam changes.

  FIG. 2b shows the temporal average intensity distribution of the movement pattern. It is consistent with the confocal PSF.

FIG. 3a shows the laterally adjusted PSF resulting from the coherent superposition of multiple excitation focal points located at a distance of 200 nm on the x-axis. The phases of adjacent focal points are shifted by π relative to each other. Their amplitude depends on the distance to the center of the Gaussian envelope and decreases to 1 / e ² times after 400 nm. PSF is expressed in the focal plane (xy) and xz directions. The small drawing below shows the pattern change as the focus moves in the envelope plane.

  FIG. 3b shows the average of the temporal movement pattern.

  FIGS. 4a and 4b show an effective PSF made by the lock-in detection method from the combination of the patterns of FIGS. 2a and 3b represented for two different phase shifts φ between the excitation adjustment and the detection adjustment.

FIG. 5a shows the PSF resulting from the combination of the horizontal and axial patterns. Axis pattern and horizontal pattern varies at the frequency ω _{2 =} 9ω _1/10, shown in a small figure in this example. A complete circle is determined by φ = 2π. The envelope resulting from the time average is shown in b.

FIG. 6a shows the effective PSF resulting from the combination of patterns shown in FIG. 5 using the lock-in detection method with a frequency difference of ω ₁ −ω ₂ . The diagonal structure is used to recognize movement along the bisector of the coordinate system, for example, to completely determine the diffusion tensor.

1 shows a scanning microscope suitable for measuring the indicated correlation function. The point spread function (PSF) of a one-photon 4π confocal microscope is shown. The average of the movement pattern in time is shown. FIG. 4 shows an effective PSF made by the lock-in detection method from the combination of the patterns of FIGS. 2a and 3b represented for two different phase shifts f between excitation adjustment and detection adjustment. The PSF resulting from the combination of the horizontal pattern and the axis pattern is shown. FIG. 6 shows the effective PSF resulting from the combination of patterns shown in FIG. 5 using a lock-in detection method with a frequency difference of ω ₁ −ω ₂ .

Claims (19)

  A scanning microscope with a device for fluorescence correlation spectroscopy, characterized in that the intensity and / or detection sensitivity of the illumination light is spatially adjusted within the measurement volume.   2. A scanning microscope according to claim 1, wherein the measuring volume is defined by at least one image point.   2. The scanning microscope according to claim 1, wherein the intensity and / or detection sensitivity of the illumination light is adjusted spatially and periodically.   The scanning microscope according to claim 1, wherein the adjustment of the intensity of the illumination light and / or the detection sensitivity changes with time.   5. Scanning microscope according to claim 4, characterized in that the fluorescence correlation spectra of the same measuring volume taken with different adjustments are analyzed by comparison with each other.   2. The scanning microscope according to claim 1, wherein the adjustment of the intensity of the illumination light and / or the detection sensitivity is turned on / off by a switch.   7. The fluorescence correlation spectrum of the same measurement volume taken when the adjustment is switched on and when the adjustment is switched off, is analyzed by comparison with each other. Scanning microscope.   The scanning microscope according to claim 6, characterized in that the fluorescence correlation spectrum of the same measuring volume taken when the adjustment is switched on and when the adjustment is switched off is split by the other. .   The scanning microscope according to claim 1, wherein the adjustment of the intensity of the illumination light and / or the detection sensitivity is made by interference.   The scanning microscope according to claim 9, wherein light waves contributing to interference exhibit a phase relationship that can be adjusted to each other.   The scanning microscope according to claim 9, wherein the adjustment of the intensity of the illumination light is made by interference by two illumination light beams in opposite propagation directions.   10. The scanning microscope according to claim 9, wherein the adjustment of the detection sensitivity is made by interference by two detection light beams in opposite propagation directions.   The scanning microscope according to claim 9, wherein the scanning microscope has a 4π arrangement.   The scanning microscope according to claim 1, further comprising an amplitude filter that adjusts the intensity of the illumination light and / or the detection sensitivity.   The scanning microscope according to claim 1, further comprising a phase filter that adjusts the intensity of the illumination light and / or the detection sensitivity.   2. The scanning microscope according to claim 1, further comprising a lens arrangement and / or a beam splitter for adjusting the intensity of the illumination light and / or the detection sensitivity.   The scanning microscope according to claim 1, wherein the sample in the measurement volume is optically excited by multiphoton excitation.   The scanning microscope according to claim 17, wherein the illumination light is a pulse.   The scanning microscope according to claim 1, wherein the scanning microscope is a confocal scanning microscope.

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