US20090250632A1 - Method and Arrangement for Collimated Microscopic Imaging - Google Patents

Method and Arrangement for Collimated Microscopic Imaging Download PDF

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US20090250632A1
US20090250632A1 US12/444,290 US44429007A US2009250632A1 US 20090250632 A1 US20090250632 A1 US 20090250632A1 US 44429007 A US44429007 A US 44429007A US 2009250632 A1 US2009250632 A1 US 2009250632A1
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illumination
sample
fluorescence
arrangement according
partial regions
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Michael Kempe
Gerhard Krampert
Matthias Wald
Ralf Wolleschensky
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Carl Zeiss Microscopy GmbH
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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • 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
    • G02B21/0032—Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers
    • 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
    • G02B21/0052—Optical details of the image generation
    • G02B21/0072—Optical details of the image generation details concerning resolution or correction, including general design of CSOM objectives
    • 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
    • G02B21/0052—Optical details of the image generation
    • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence

Definitions

  • the present invention relates to methods and arrangements for microscopic imaging with structured illumination. Increased spatial resolution is achieved by means of nonlinear interactions between samples, which is known from the prior art. By means of the method and the corresponding arrangements of this invention, this sample interaction is made use of for imaging in such a way that confocal imaging is possible with a resolution that goes beyond the diffraction boundary in all spatial directions with parallel data acquisition.
  • the intention is to achieve a fluorescing region which is smaller than that which can be achieved by diffraction-limited excitation.
  • FIG. 1 The basic principle is illustrated in FIG. 1 with the steps and schematically depicted spatial distributions which are used or which result.
  • the arrangements known from the prior art are based on point scanning methods in which light distributions with zero settings in the center (doughnut modes, as they are called) which are preferred for depopulation, switching and de-excitation are applied.
  • the laser beam is scanned over the sample and the steps shown in FIG. 1 are carried out sequentially in every spatial point.
  • Previous arrangements according to the first method e.g., Klar et al, PNAS 97 (2000), 8206-8210) and third method (Hoffmann et al., PNAS 102 (2005), 17565-17569), all of which function according to the scheme mentioned above, have been described.
  • a decisive disadvantage of this arrangement is the sequential data acquisition. Owing to the fact that the increased spatial resolution results in reduced excitation volumes, the fluorescence emission is reduced so that the pixel integration time must generally be longer (with a fivefold increase in resolution only laterally, the expected fluorescence emission is about twenty-five times lower, e.g., with a homogeneous dye distribution over the extent of diffraction-limited excitation). Further, some known switchable dyes such as Dronpa can be switched only with limited light power so as to afford many switching cycles (Habuchi et al., PNAS 102 (2005) 9511-9516). In this case, the third method mentioned above requires a considerably longer exposure time per pixel for switching off than that required for fluorescence excitation alone.
  • a method and an arrangement in which a high-resolution image is achieved directly (without image processing) with parallel data acquisition is described in the following.
  • the extrafocal background can be eliminated by variable confocal detection.
  • This method and arrangement prevent the above-mentioned disadvantages of the multi-spot arrangement according to the prior art.
  • an optimal parallelization maximum density of the simultaneously excited regions of the sample
  • a relatively simple technical implementation are made possible by the invention.
  • FIG. 1 is a diagram schematically illustrating sequential steps of known point scanning methods
  • FIG. 2 is a diagram illustrating the switching beam distribution
  • FIG. 3 illustrates the camera pixels and light distribution, object point allocation in the scanning steps, the resulting line image, and the resulting two-dimensional image after a Y-scanning step
  • FIG. 4 shows the lateral (X/X) and axial (optical axis Z) light distribution in the object plane
  • FIG. 5 schematically shows the axial light distribution which would occur without axially structured switching light, together with the axial structuring of the switching light;
  • FIG. 7 schematically shows a modified line scanning arrangement
  • FIG. 8 illustrates a phase mask for lateral structuring
  • FIG. 9( a ) illustrates an achromatic color splitter which results from the pupil light distributions in FIG. 8 ;
  • FIG. 9( b ) illustrates the achromatic color splitter located in the beam in a conjugate pupil plane
  • FIG. 10( a ) shows the distribution without a mask (corresponds to excitation distribution);
  • FIG. 10( b ) shows the distribution with a mask according to FIG. 8( b );
  • FIG. 10( c ) shows the distribution with a mask according to FIG. 8( a );
  • FIG. 10(d) shows incoherent overlapping results in the distribution shown
  • FIG. 11( a ) is a graph illustrating intensity versus time showing switching on and off
  • FIG. 11( b ) is a graph illustrating intensity versus time showing switching off
  • FIG. 12 is a schematic view showing a preferred illumination unit for a laser scanning microscope for use with Dronpa;
  • FIG. 13 shows the integration of an illumination unit according to the invention in a line-scanning microscope
  • FIGS. 14( a )-( c ) illustrate adjustment gratings for masks
  • FIG. 15 shows the intensity distribution of the illumination in the object plane using the phase mask from FIG. 8( a ).
  • FIG. 16 shows a molecule population in the excited state with saturated switching off
  • FIG. 17 illustrates an embodiment example of the illumination
  • FIG. 18( a ) illustrates an intensity structure
  • FIG. 18( b ) illustrates an axial structuring of the illumination light in the object plane.
  • An arrangement which meets the demands mentioned above is based on a line-shaped excitation and confocal detection by means of a line camera behind a slit diaphragm.
  • a de-excitation/depopulation/switching (always referred to hereinafter, by way of example, as switching) is realized in such a way that only a series of spots along a line can emit fluorescent light.
  • the distance between these spots is at least as great as (but advantageously no greater than) the diffraction-limited resolution of the optical system.
  • a possibility for generating an illumination of this kind (which is simultaneously also axially structured) consists in the diffraction of coherent light at a periodic structure and interference of the diffraction orders in the object plane, as is described in the application DE102004034962A1 which is hereby incorporated in the present disclosure. This results in diffraction-limited light distributions on the camera line which are separated from one another, impinge on corresponding pixels and are detected separately at the latter (see FIG. 2 ).
  • FIG. 2 shows, from top to bottom, the switching beam distribution in the object plane, the excitation distribution in the object plane, the superposition of excitation and switching light in the object plane, the camera pixels, and the light distribution in the camera plane.
  • X represents the zeroth diffraction order referring to the diagram and description in FIG. 9 a ) and FIG. 11 .
  • XX is the +/ ⁇ first diffraction order according to the diagram and description in FIG. 9 a ) and FIG. 11 .
  • a highly resolved image is now obtained by scanning the object along line (x) and perpendicular to line (y).
  • This scanning can be carried out by moving the illumination distributions, e.g., by means of a galvanometer scanner and/or by moving the object.
  • the illumination beam and the detection beam must pass over the same beam deflection elements in order to obtain a stationary light distribution on the detector (descanned detection).
  • the object is simply moved (object scanning), the light distributions are stationary in every case.
  • FIG. 3 shows, one below the other, the camera pixels and light distribution, object point allocation in scanning steps 1 to 10 , the resulting line image, and the resulting two-dimensional image after a Y-scanning step.
  • the PSF can correspond to the diffraction-limited PSF (structured switching light only in lateral direction).
  • FIG. 4 shows the lateral (X/X) and axial (optical axis Z) light distribution in the object plane.
  • an axial resolution enhancement can also be achieved ( FIG. 5 ) by means of additional structuring of the switching light in axial direction.
  • FIG. 5 shows schematically the axial light distribution which would occur without axially structured switching light, together with the axial structuring of the switching light.
  • the switching light distribution results, e.g., from the use of an optical element from FIG. 8 b ) or FIG. 8 c ) in an arrangement according to FIG. 12 (including the associated descriptions).
  • the light distribution actually obtained in this case is shown at the bottom in FIG. 5 .
  • a distribution such as that in FIG. 4 , bottom, or FIG. 5 , bottom, can be useful. Therefore, it is advisable to be able to implement both scenarios in one device.
  • An option for the use of increased parallelism while simultaneously preserving a certain confocality of the imaging is to image a plurality of separate lines on the sample and to carry out detection by means of an area detector.
  • a confocal detection can be achieved by selectively reading out the pixels associated with the lines.
  • the confocality can be adjusted by also taking into account adjacent pixels. For example, by reading out the two pixel rows adjacent to the line and summing the corresponding associated pixel elements, the effective “slit diaphragm” can be increased by a factor of 3 with a corresponding decrease in confocality.
  • the same elements in the vicinity of the pupil as those shown in FIGS.
  • the light must be split into n partial beams impinging in the pupil at different angles by means of an element arranged in front of the main color splitter.
  • An element, mentioned above, placed in front of the main color splitter can be a suitably shaped diffractive element (or a combination of a plurality of elements) with corresponding imaging or an arrangement for the geometric splitting and beam deflection of the partial beams.
  • a preferred arrangement uses a modified line-scanning system as shown in FIG. 7 and, for example, in DE 10257237A1 and EP1617271 A2 which are hereby incorporated in the present disclosure.
  • the AchroGate achromatic color splitter is replaced by one permitting a structuring of the switching light in the object plane—this can be a suitable achromatic design or a dichroic beamsplitter.
  • the illumination unit is replaced by one which enables the required nonlinear sample interactions and the structuring of the switching light.
  • the illumination and the data acquisition are adapted in such a way that the exposure of the sample and the detection with the suitable sequence of switching/excitation/detection/switching on (see FIG. 1 ) is carried out within a suitable period of time.
  • the data evaluation is adapted so that a correlation of pixels to object points is carried out as shown schematically in FIG. 3 .
  • a preferred way to generate a lateral structuring is made possible by means of a phase mask according to FIG. 8 a ) which should be imaged in the vicinity of the pupil of the objective.
  • the mask is divided in the middle, the right half and the left half producing different phase shifts in the light passing through.
  • a sine-shaped modulation of the line in the intermediate space of the double line (to produce a modulation of the excitation in the intermediate space of the double line, see FIG. 2 , top) is generated by the interference of the two line distributions at the edge of the pupil.
  • the lines must have a spacing equal to 2 ⁇ 3 of the diameter of the pupil radius. It is important that the components of the light which generate the double line and those which generate the modulated center line overlap incoherently in the sample. This can be ensured by delaying the arrival of the corresponding light components in the object by a time greater than the coherence length.
  • a suitable phase grating can be used in the vicinity of a conjugate image plane in front of the mask in order to obtain the distribution of the light on the phase mask shown in FIG. 8 a ).
  • the center line then corresponds to the zeroth diffraction order, and the two lines at the edge of the pupil correspond to the first diffraction order of the corresponding grating.
  • the optimal position of the light distributions in the object plane relative to one another is ensured at the same time through the use of diffraction orders of a grating.
  • FIG. 8 b another partial beam of the switching light is modified with a phase mask according to FIG. 8 b ) located in the vicinity of a plane conjugate to the objective pupil.
  • Masks 8 b ) and 8 c ) have a central region symmetric to the optical axis and to an axis perpendicular to the optical axis which produces a different phase delay of the passing light compared to the outer regions to the right and left sides.
  • a phase delay of ⁇ of an outer portion of the line-shaped pupil illumination relative to an inner portion of the line-shaped pupil illumination is carried out.
  • the optimal radius of the phase jump is 1/ ⁇ square root over (2) ⁇ of the pupil radius of the objective. In practice, a somewhat smaller radius is often optimal.
  • a phase plate such as that shown in FIG. 8 c ) is suitable for optimizing the radius. Adaptation of the radius can be carried out by translation.
  • FIG. 9 a shows an achromatic color splitter which results from the pupil light distributions in FIG. 8 and which is also compatible with the distributions for excitation and switching on (lines).
  • This achromatic color splitter is located in the beam in a conjugate pupil plane as is shown in FIG. 9 b ).
  • the fluorescent light emitted by the sample fills the pupil and therefore passes the splitter (except at the reflecting strips).
  • the outer mirror surfaces (X) are used for transmitting the +/ ⁇ first diffraction order of S 1 in FIG. 11
  • the inner mirror surface (XX) is used to transmit the zeroth diffraction order of S 1 in FIG. 12 and the beam paths S 2 , S 3 .
  • the mirror surfaces do not all lie in the same focal plane.
  • the defocusing due to the magnification of the outer mirror surfaces (which are not in the focus) compared to the inner mirror surface must be taken into account.
  • FIG. 9 shows experimental results (sections through the light distributions in the sample) with the corresponding masks with an oil-immersion objective having a numerical aperture (NA) of 1.4 at 488 nm.
  • FIG. 10 a shows the distribution without a mask (corresponds to excitation distribution)
  • FIG. 10 b shows the distribution with a mask according to FIG. 8 b
  • FIG. 10 c shows the distribution with a mask according to FIG. 8 a ) (only the center portion).
  • an incoherent overlapping results in the distribution shown in FIG. 10 d ) which shows a minimum which is surrounded in the y- and z-directions. This minimum extends to about 170 nm (y) and 400 nm (z), which corresponds roughly to the inverse of the diffraction-limited limiting frequencies (lateral: ⁇ /(2 ⁇ NA) ⁇ 170 nm, axial:
  • the point pattern of intensity minima occurring in the focus in the sample caused by a corresponding nonlinear sample interaction is a point pattern of excitable regions (GSD/switching) or of regions in which there is still significant excitation (STED). These regions are appreciably smaller after suitable exposure optimized for the dye (wavelength, intensity, exposure time) than a diffraction-limited light distribution and therefore allow a scanning of the sample with increased resolution.
  • the adjustment of the illumination is carried out based on knowledge of the dye and can be optimized at the device based on the resolution of test structures (e.g., beads).
  • variable optical system which makes it possible to adapt the periods of the pattern to the periods of the detector elements is advantageously provided in the illumination beam path or detection beam path.
  • the position of the imaged spots i.e., their center of gravity
  • the excitation and switching on are carried out with diffraction-limited light distributions which are generated without a mask.
  • these are lines which have the most homogeneous possible intensity curve along the line and extend in a diffraction-limited manner perpendicular to the line.
  • the excitation is carried out either before the switching/de-excitation (STED) or after the dye has been switched off (GSD/switching) by the switching light in a structured manner. Only in the case of switching is it generally necessary to subsequently reverse the switching off by a switch-on laser, because the other processes spontaneously revert to the initial state of the dye (see also FIG. 1 with respect to the timing).
  • the time sequence can be achieved by retarding the corresponding partial beams.
  • the time sequence must be achieved by means of fast switches (preferably AOTFs).
  • the optimal parameters for excitation and switching on are determined beforehand from knowledge of the dye characteristics.
  • the aim is to reverse the switching off as completely as possible; optimized excitation and simultaneous detection of the emitted fluorescence signal is carried out by maximizing the signal-to-noise ratio.
  • Different boundary conditions must be taken into account for the different interaction mechanisms:
  • a suitable candidate for switching is the Dronpa protein (Habuchi et al., PNAS 102 (2005) 9511-9516).
  • the switching off and the excitation are carried out with a wavelength in the range of about 450 nm to 520 nm.
  • Switching on can be carried out in the range of 350 nm to 420 nm.
  • the mutual switching on and off is shown in FIG. 11 a ).
  • a detailed view of switching off is represented in FIG. 11 b ). This illustrates the long exposure times of about 5 ms for achieving a sufficient switching off. While the exposure time can be shortened by increasing the intensity, this would reduce the number of possible switching cycles compared to FIG. 11 a ) in which about 100 switching cycles are shown.
  • FIG. 12 shows a schematic view of a preferred illumination unit for a laser scanning microscope for use with Dronpa.
  • S 1 represents the beam path for generating the switching light distribution (see FIGS. 2 , 7 and 5 ).
  • the dye is switched on in this case either before switching off or after excitation.
  • S 2 is the excitation beam path
  • S 3 is another beam path for switching on a switched off dye, if necessary. Accordingly, S 1 (switching off) is carried out first, followed by S 2 (excitation) and S 3 (switching on) either at the very start or at the very end, wherein detection is carried out at S 2 .
  • the wavelength 477 nm at S 2 can also be replaced with another excitation wavelength (e.g., 488 nm).
  • STED and GSD can be realized by means of S 1 and S 2 , STED can be realized by S 2 followed by S 1 , and detection can be realized in S 1 , GSD by S 1 followed by S 2 , and detection in S 2 .
  • S 1 after the beam shaping (e.g., by means of a Powel lens) and after passing through a splitter DC in the zeroth diffraction order of a grating followed by the mask 7 a ) (phase jump), the two laterally limiting outer lines of the switching beam are generated in the sample and the structuring of the center line is generated by means of the +/ ⁇ first diffraction order.
  • the mask 7 b ) or c ) (after reflection at the splitter DC and separate beam path until after the mask 7 c )) generates the limiting lines of the switching beam in axial direction by means of the phase jump described above.
  • Slightly different wavelengths in this case, 488 nm, 477 nm can be used for the beam paths through 7 a ) and 7 c ), but they must both lie within the range of the response behavior of the respective dye.
  • FIG. 13 The integration of an illumination unit according to the invention in a line-scanning microscope is shown in FIG. 13 . It contains the above-mentioned adjusting plate in the detection beam path for positioning the spot on the row of detectors.
  • An additional zoom in the system serves to adjust the imaging of the illuminated line in the object on the row detectors. Accordingly, a selected portion of the line can be imaged on the row detectors.
  • the definitive correlation of the illumination spot and camera pixels must be ensured in every case by the interaction of the two zooms.
  • additional lasers are connected and the design of the dichroic splitter DC is provided for a plurality of colors (or as a neutral splitter). Further, it may be necessary (depending upon the wavelength difference) to change masks (although simultaneous operation would not then be possible).
  • a simple adaptation consists in changing the grating constant so that the diffraction orders occur at the same location in the pupil. This can be achieved by a translation of a grating as is shown in FIG. 14 a ). With moderate wavelength changes (on the order of 5%), the masks can also be used simultaneously with a plurality of wavelengths.
  • FIG. 14 b shows a preferred form of adapting by changing the effective gap ratio at the grating while maintaining the same grating constant. Adapting the radius of the phase jump of the mask in FIG. 8 b ) is particularly critical for optimal structuring in axial direction.
  • a precision optimization of this radius e.g., based on the recording of PSF, with the objective to be used is possible with a mask design such as that shown in FIG. 12 c ).
  • the effective radius of the phase edge can be adapted by translation of the mask.
  • FIG. 15 shows the intensity distribution of the illumination in the object plane using the phase mask from FIG. 8 a ).
  • the quantity n of excited molecules decreases exponentially with the illumination intensity and the exposure duration.
  • FIG. 16 shows the resulting molecule population in the excited stated with saturated switching off, which provides a measurement for the PSF.
  • FIG. 17 An embodiment example of the illumination is shown in FIG. 17 . Side views of the beam path are shown at left and at the top.
  • a light bundle which is adapted to a Powell lens and has a Gaussian intensity profile is provided by fiber coupling.
  • the light bundle has two wavelengths, 477 nm and 488 nm.
  • the Powell lens (divergence 30°) generates a line-shaped illumination with a homogeneous intensity distribution along the line in the focal plane of the following achromatic objective with a focal distance of 10 mm. This plane is conjugate to the object plane of the microscope.
  • a first color splitter splits the two homogenized laser lines.
  • a light path, advantageously that with 488 nm, has a line grating with 55 l/mm which generates the zeroth and ⁇ first diffraction orders in a determined intensity ratio of 8:1.
  • phase element which is located in a plane conjugate to the microscope pupil.
  • the phase element is described in FIG. 8 a ).
  • this phase element causes an intensity structure according to FIG. 18 a ).
  • the grating is advantageously formed as a phase grating.
  • the grating has a variable groove depth as is shown in FIG. 14 b ). Since the illumination of the grating is line-shaped, the efficiency of the diffraction orders can be adjusted by lateral displacement of the grating.
  • the second light path only has the same 2f imaging as the first, but without a grating.
  • the phase procedure in the pupil of the second light path is implemented by a phase mask according to FIG. 8 c ).
  • This enables an axial structuring of the illumination light in the object plane ( FIG. 18 b )).
  • the lateral extension of the masks is about 12 ⁇ 12 mm 2 .
  • the diffraction orders in the first light path are at ⁇ 4 mm, 0 mm and 4 mm.
  • the two light paths are recombined after these procedures.
  • the illumination pupil is adapted to the pupil of the microscope by means of relay optics.
  • the interface is the main color splitter.
  • the imaging scale of the relay optics is 4.35.
  • the variability of the relay optics serves for precision adjustment of the imaging scale.
  • the beam combiner is followed by a neutral splitter which serves to couple in the light for fluorescence excitation (477 nm) and also the light for erasing the structure impressed in the object (405 nm).

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US10051240B2 (en) 2010-06-14 2018-08-14 Howard Hughes Medical Institute Structured plane illumination microscopy
US10393661B2 (en) 2014-04-25 2019-08-27 Nikon Corporation Structured illumination microscopic device and structured illumination observation method
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