WO2004109360A2 - Microscope a balayage pourvu d'un dispositif de spectroscopie a correlation de fluorescence - Google Patents

Microscope a balayage pourvu d'un dispositif de spectroscopie a correlation de fluorescence Download PDF

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Publication number
WO2004109360A2
WO2004109360A2 PCT/EP2004/050984 EP2004050984W WO2004109360A2 WO 2004109360 A2 WO2004109360 A2 WO 2004109360A2 EP 2004050984 W EP2004050984 W EP 2004050984W WO 2004109360 A2 WO2004109360 A2 WO 2004109360A2
Authority
WO
WIPO (PCT)
Prior art keywords
scanning microscope
microscope according
modulation
illuminating light
intensity
Prior art date
Application number
PCT/EP2004/050984
Other languages
German (de)
English (en)
Other versions
WO2004109360A3 (fr
Inventor
Jörg Bewersdorf
Original Assignee
Leica Microsystems Heidelberg Gmbh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from DE10329756A external-priority patent/DE10329756B4/de
Application filed by Leica Microsystems Heidelberg Gmbh filed Critical Leica Microsystems Heidelberg Gmbh
Priority to JP2006508311A priority Critical patent/JP2006526798A/ja
Publication of WO2004109360A2 publication Critical patent/WO2004109360A2/fr
Publication of WO2004109360A3 publication Critical patent/WO2004109360A3/fr

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Classifications

    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders

Definitions

  • the invention relates to a scanning microscope with a device for fluorescence correlation spectroscopy.
  • a sample is illuminated with a light beam in order to observe the reflection or fluorescent light emitted by the sample.
  • the focus of an illuminating light beam is moved in a plane of the object with the aid of a controllable beam deflection device, generally by tilting two mirrors, the deflection axes usually being perpendicular to one another, so that one mirror deflects in the x direction and the other in the y direction.
  • the mirrors are tilted, for example, with the help of galvanometer control elements.
  • the power of the light coming from the object is measured depending on the position of the scanning beam.
  • a confocal scanning microscope generally comprises a light source, focusing optics with which the light of the source is focused on a pinhole - the so-called excitation diaphragm - a beam splitter, a beam deflection device for beam control, microscope optics, and one Detection aperture and the detectors for detecting the detection or fluorescent light.
  • the illuminating light is coupled in via a beam egg.
  • the fluorescent or reflection light coming from the object reaches the beam splitter via the beam deflection device, passes it, and is then focused on the detection diaphragm behind which the detectors are located.
  • Detection light that does not originate directly from the focus region takes a different light path and does not pass through the detection aperture, so that point information is obtained which leads to a three-dimensional image by sequential scanning of the object.
  • a three-dimensional image is usually achieved by recording image data in layers, the path of the scanning light beam ideally describing a meander on or in the object. (Scanning a line in the x direction with a constant y position, then stopping x scanning and swiveling to the next line to be scanned by y adjustment and then, with a constant y position, scanning this line in the negative x direction, etc.).
  • the sample table or the objective is moved after scanning a layer and thus the next layer to be scanned is brought into the focal plane of the objective.
  • FCS fluorescence correlation spectroscopy
  • FCS measurements with a confocal scanning microscope, in which diffusion speeds are determined, are not directionally sensitive, in addition, a problem in that the point spread function (point image function, PSF) - the intensity distribution of the illuminating light in the focus volume - is cylinder-symmetrical and has a similar profile in the z direction to that in the lateral direction.
  • PSF point image function
  • the invention is therefore based on the object of proposing a scanning microscope which allows the direction-dependent measurement of diffusion speeds or flow speeds of components of a sample, for example of molecules. Ideally, it provides a method to easily separate the influence of movement and reaction dynamics on the correlation curve.
  • the object is achieved by a scanning microscope, which is characterized in that the intensity of the illuminating light and / or the detection sensitivity is spatially modulated within the measurement volume.
  • the invention has the advantage that, with suitable spatial modulation, movements in different directions can be distinguished from one another and effects of other dynamics can be separated by a suitable combination of such measurements. You can not only movements along the spatial axes x, y and z but in any direction are analyzed and diffusion of z. B. directional transport can be distinguished.
  • the measurement volume is preferably defined by at least one raster point, the person skilled in the art understands by measurement volume a suitable standardized spatial distribution of the combined probability of causing an existing fluorescence molecule to fluoresce and to detect this fluorescence.
  • the term grid point does not refer to a point without extension in the mathematical sense.
  • the measurement volume can also comprise a plurality of raster points, the detection light emanating from the raster points being detectable simultaneously — in parallel — with preferably a plurality of detectors.
  • the intensity of the illuminating light and / or the detection sensitivity is spatially periodically modulated.
  • the modulation of the intensity of the illuminating light and / or of the detection sensitivity can preferably be switched on and off or its spatial shape can be changed.
  • This has the advantage that fluorescence correlation spectra of the same measurement volume recorded with different modulation can be evaluated by mutual comparison.
  • the fluorescence correlation spectra of the same measurement volume recorded with the modulation switched on and switched off, possibly after corrections (e.g. background), are divided by one another.
  • the modulation of the intensity of the illuminating light and / or the detection sensitivity can be changed over time.
  • fluorescence correlation spectra of the same measurement volume recorded with different modulations can be evaluated by mutual comparison.
  • "effective" spatial modulations of the measurement volume can be generated.
  • the intensity of the illuminating light is preferably modulated and / or the detection sensitivity can be generated by interference.
  • the light waves contributing to the interference have an adjustable phase relationship with one another, so that the spatial modulation can be changed by varying the phase relationship.
  • the modulation of the intensity of the illuminating light can be generated by interference between two illuminating light beams with opposite directions of propagation.
  • the modulation of the detection sensitivity can be generated by interference of two detection light beams with opposite directions of propagation.
  • the scanning microscope preferably has a 4Pi arrangement with two opposing objectives, between which the sample is located.
  • the illuminating light is preferably split with a beam splitter into two partial illuminating beams which each pass through an interferometer arm and pass through one of the objectives to the sample.
  • the detection light emanating from the sample passes through the objectives in initially two detection light beam bundles to a beam combiner and is then fed together to a detector.
  • the beam splitter for the detection light beams preferably acts as a beam combiner.
  • This embodiment is particularly suitable for measuring flow velocities in the axial direction by continuously changing the phase relationship of the light passing through the interferometer arms over time.
  • the speed of the change over time is e.g. B. varies until it is adapted to the flow rate.
  • an amplitude filter is provided which generates the modulation of the intensity of the illuminating light and / or the detection sensitivity.
  • a side maxima side lobes
  • phase filter or a combination of phase and amplitude filters can be provided in another variant, which the Modulation of the intensity of the illuminating light and / or the detection sensitivity generated.
  • a lens array for example a microlens array
  • the combination of a plurality of beam splitters is provided, which generates the modulation of the intensity of the illuminating light and / or of the detection sensitivity.
  • the scanning microscope is preferably designed as a confocal scanning microscope.
  • the incident laser beam is split into two beams by a beam splitter (not shown).
  • One of the partial beams is fed directly into the 4Pi resonator, where another beam splitter (BS) generates two beams, which are focused by the two objectives (OL) into the same point in the sample and create an axial pattern there by interference.
  • BS beam splitter
  • OL two objectives
  • the other partial beam can be used, which is divided into several adjacent beams by means of micro lens grids (LA).
  • LA micro lens grids
  • the position of the beams can be changed within the beam profile. This creates an additional, movable lateral pattern in the sample.
  • DP Dove prism
  • Fig. 2a shows the point spread function (PSF) of a 1-photon 4Pi confocal microscope, calculated for 488nm excitation light, a numerical aperture of 1.2, water immersion and a detection pinhole from the Size of an Airy disk.
  • PSF point spread function
  • 3a shows a laterally modulated PSF, resulting from the coherent superposition of several excitation foci, which are arranged at a distance of 200 nm on the x-axis.
  • the phases of neighboring foci are shifted from each other by ⁇ .
  • Their amplitude depends on the distance to the center of the Gaussian envelope and drops to 1 / e 2 times after 400nm.
  • the PSF is shown in the focal plane (xy) and in the xz direction. The small figures below show the change in the pattern when the foci are moved within the envelope.
  • FIG. 5a shows the resulting PSF from the combination of lateral and axial patterns.
  • the envelope created by time averaging is shown in (b).
  • FIG. 6a shows the effective by combination of the pattern shown in Figure 5 with the lock-in detection method at the difference frequency. 1 - 2 resulting PSF.
  • the diagonal structure can be used to detect movement along the bisector of the coordinate system, for example to completely determine the diffusion tensor.

Landscapes

  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Microscoopes, Condenser (AREA)

Abstract

L'invention concerne un microscope à balayage pourvu d'un dispositif de spectroscopie à corrélation de fluorescence. Ce microscope à balayage se caractérise en ce que l'intensité de la lumière d'éclairage et/ou la sensibilité de détection sont modulées spatialement à l'intérieur du volume de mesure.
PCT/EP2004/050984 2003-06-04 2004-06-02 Microscope a balayage pourvu d'un dispositif de spectroscopie a correlation de fluorescence WO2004109360A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2006508311A JP2006526798A (ja) 2003-06-04 2004-06-02 蛍光相関分光法のための装置を有する走査顕微鏡

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DE10325506.0 2003-06-04
DE10325506 2003-06-04
DE10329756A DE10329756B4 (de) 2003-06-04 2003-07-02 Rastermikroskop mit einer Vorrichtung zur Fluoreszenz-Korrelations-Spektroskopie
DE10329756.1 2003-07-02

Publications (2)

Publication Number Publication Date
WO2004109360A2 true WO2004109360A2 (fr) 2004-12-16
WO2004109360A3 WO2004109360A3 (fr) 2005-02-10

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2004/050984 WO2004109360A2 (fr) 2003-06-04 2004-06-02 Microscope a balayage pourvu d'un dispositif de spectroscopie a correlation de fluorescence

Country Status (2)

Country Link
JP (1) JP2006526798A (fr)
WO (1) WO2004109360A2 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0491289A1 (fr) * 1990-12-18 1992-06-24 Stefan Dr. Hell Microscope double-confocal à balayage
US6262423B1 (en) * 1996-12-22 2001-07-17 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E. V. Scanning microscope in which a sample is simultaneously and optically excited at various points

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0491289A1 (fr) * 1990-12-18 1992-06-24 Stefan Dr. Hell Microscope double-confocal à balayage
US6262423B1 (en) * 1996-12-22 2001-07-17 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E. V. Scanning microscope in which a sample is simultaneously and optically excited at various points

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DIASPRO A ET AL: "TWO-PHOTON MICROSCOPY AND SPECTROSCOPY BASED ON A COMPACT CONFOCAL SCANNING HEAD" JOURNAL OF BIOMEDICAL OPTICS, Bd. 6, Nr. 3, Juli 2001 (2001-07), Seiten 300-310, XP002306388 *
SCHRADER M ET AL: "OPTICAL TRANSFER FUNCTIONS OF 4PI CONFOCAL MICROSCOPES: THEORY AND EXPERIMENT" OPTICS LETTERS, Bd. 22, Nr. 7, 1. April 1997 (1997-04-01), Seiten 436-438, XP002306389 USA *
SOINI J T ET AL: "IMAGE FORMATION AND DATA ACQUISITION IN A STAGE SCANNING API CONFOCAL FLUORESCENCE MICROSCOPE" APPLIED OPTICS, OPTICAL SOCIETY OF AMERICA,WASHINGTON, US, Bd. 36, Nr. 34, 1. Dezember 1997 (1997-12-01), Seiten 8929-8934, XP000728467 ISSN: 0003-6935 *
WINFRIED WIEGR[BE: "FLUORESCENCE CORRELATION SPECTROSCOPY: PROBING MOLECULAR INTERACTIONS INSIDE LIVING CELLS" AMERICAN LABORATORY, September 2000 (2000-09), Seiten 44-47, XP002306387 *

Also Published As

Publication number Publication date
JP2006526798A (ja) 2006-11-24
WO2004109360A3 (fr) 2005-02-10

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