Abstract:
The present invention discloses a light source for illumination in scanning microscopy, and a scanning microscope. The light source and the scanning microscope contain an electromagnetic energy source that emits light of one wavelength, and a means  5  for spatially dividing the light into at least two partial light beams. An intermediate element for wavelength modification is provided in at least one partial light beam.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS  
         [0001]    This invention claims priority of the German patent application 100 56 382.1 which is incorporated by reference herein.  
         FIELD OF THE INVENTION  
         [0002]    The invention concerns a light source for illumination in scanning microscopy.  
           [0003]    The invention further concerns a scanning microscope. The scanning microscope can also be configured as a confocal microscope.  
         BACKGROUND OF THE INVENTION  
         [0004]    In scanning microscopy, a sample is illuminated with a light beam in order to observe the reflected or fluorescent light emitted from the sample. The focus of the illuminating light beam is moved in a specimen plane with the aid of a controllable beam deflection device, generally by tilting two mirrors; the deflection axes are usually at right angles to one another, so that one mirror deflects in the X and the other in the Y direction. The tilting of the mirrors is brought about, for example, using galvanometer positioning elements. The power level of the light coming from the specimen is measured as a function of the position of the scanning beam. The positioning elements are usually equipped with sensors for ascertaining the present mirror position.  
           [0005]    In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam.  
           [0006]    A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto a pinhole (called the excitation stop), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection stop, and the detectors for detecting the detected or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen arrives via the beam deflection device back at the beam splitter, passes through it, and is then focused onto the detection stop behind which the detectors are located. Detected light that does not derive directly from the focus region takes a different light path and does not pass through the detection stop, so that a point datum is obtained which, by sequential scanning of the specimen, results in a three-dimensional image. Usually a three-dimensional image is obtained by image acquisition in layers.  
           [0007]    The power level of the light coming from the specimen is measured at fixed time intervals during the scanning operation, and thus scanned one grid point at a time. The measured value must be unequivocally associated with the pertinent scan position so that an image can be generated from the measured data. Preferably, for this purpose the status data of the adjusting elements of the beam deflection device are also continuously measured, or (although this is less accurate) the setpoint control data of the beam deflection device are used.  
           [0008]    In a transmitted-light arrangement it is also possible, for example, to detect the fluorescent light, or the transmission of the exciting light, on the condenser side. The detected light beam does not then pass via the scanning mirror to the detector (non-descan configuration). For detection of the fluorescent light in the transmitted-light arrangement, a condenser-side detection stop would be necessary in order to achieve three-dimensional resolution as in the case of the descan configuration described. In the case of two-photon excitation, however, a condenser-side detection stop can be omitted, since the excitation probability depends on the square of the photon density (proportional intensity 2 ), which of course is much greater at the focus than in neighboring regions. The fluorescent light to be detected therefore derives, with high probability, almost exclusively from the focus region; this makes superfluous any further differentiation between fluorescent photons from the focus region and fluorescent photons from the neighboring regions using a stop arrangement.  
           [0009]    The resolution capability of a confocal scanning microscope is determined, among other factors, by the intensity distribution and spatial extension of the focus of the illuminating light beam. An arrangement for increasing the resolution capability for fluorescence applications is known from PCT/DE/95/00124. In this, the lateral edge regions of the illumination focus volume are illuminated with light of a different wavelength that is emitted by a second laser, so that the specimen regions excited there by the light of the first laser are brought back to the ground state in stimulated fashion. Only the light spontaneously emitted from the regions not illuminated by the second laser is then detected, the overall result being an improvement in resolution. This method has become known as STED (stimulated emission depletion).  
           [0010]    Two lasers are usually used in STED microscopy, i.e. one to excite a specimen region and another to generate the stimulated emission. In particular for generating the stimulated emission, high light outputs and at the same time a maximally flexible wavelength selection are needed. Optical parametric oscillators (OPOs) are often used for this purpose. OPOs are very expensive, and moreover require high-powered pumping lasers. These are usually mode-coupled pulsed lasers, which are also very expensive. Costs for the exciting light source must also be added. All the lasers must furthermore be exactly aligned so as to arrive exactly at the individual specimen regions. In the case of pulsed excitation, it is important for the light pulses generating the stimulated emission to arrive within a specific time frame—which depends on the lifetime of the excited states of the specimen material—after the exciting light pulses. Synchronizing the pulsed lasers with one another is complex, and the result is often unsatisfactory and unstable.  
         SUMMARY OF THE INVENTION  
         [0011]    It is the object of the invention to create a light source for illumination in scanning microscopy which is easy to handle, reliable, flexible and allows for STED microscopy in a less expensive way.  
           [0012]    This object is achieved by a light source for illumination in scanning microscopy comprising:  
           [0013]    an electromagnetic energy source that emits light of one wavelength,  
           [0014]    a means for spatially dividing the light into at least two partial light beams, which is placed after the electromagnetic energy source; and  
           [0015]    an intermediate element for wavelength modification in at least one partial light beam.  
           [0016]    A further object of the invention is to create an a scanning microscope which provides a flexible, reliable and easy to handle illumination and which allows for STED microscopy in a less expensive way.  
           [0017]    The further object is achieved by a scanning microscope comprising:  
           [0018]    an electromagnetic energy source that emits light of one wavelength,  
           [0019]    a means for spatially dividing the light into at least two partial light beams, which is placed after the electromagnetic energy source,  
           [0020]    an intermediate element for wavelength modification in at least one partial light beam,  
           [0021]    a beam deflection device for guiding the two partial light beams over a specimen and  
           [0022]    a microscope optical system for focusing the partial light beams.  
           [0023]    The use of the light source according to the present invention makes the illumination system for microscopy, and in particular STED microscopy, much simpler and much less expensive, since only one electromagnetic energy source is required.  
           [0024]    In a particular embodiment, one partial light beam serves for optical excitation of a first region of a specimen. A further partial light beam, whose wavelength is modified with the aid of an intermediate element, is used to generate the stimulated emission in a further region of the specimen. The first region and the further region overlap at least partially. The wavelength of the second partial light beam is modified with an intermediate element. This intermediate element is preferably an optical parametric oscillator (OPO).  
           [0025]    The invention has the further advantage that in the case of pulsed excitation, for example for purposes of multi-photon excitation, it is possible to dispense with synchronization among the pulsed light sources if the electromagnetic energy source that causes both the excitation and the stimulated emission is a pulsed laser. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0026]    The subject matter of the invention is depicted schematically in the drawings and will be described below with reference to the Figures, in which:  
         [0027]    [0027]FIG. 1 shows a light source according to the present invention;  
         [0028]    [0028]FIG. 2 shows a scanning microscope according to the present invention with elevated resolution using STED, in the descan configuration;  
         [0029]    [0029]FIG. 3 shows a scanning microscope according to the present invention with elevated resolution using STED, in the non-descan configuration and with multi-photon excitation;  
         [0030]    [0030]FIG. 4 a  schematically depicts the overlapping partial light beams of a specimen; and  
         [0031]    [0031]FIG. 4 b  also schematically depicts the overlapping partial light beams of a specimen. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0032]    [0032]FIG. 1 shows a light source  1  according to the present invention. A pulsed laser that is embodied as a titanium: sapphire laser is provided as electromagnetic energy source  3 . Light  17  of the pulsed laser is split into a first and second partial light beam  19  and  21  with the means for spatial division of the light, which is embodied as beam splitter  5 . Partial light beam  21  passes via mirror  7  to intermediate element  9 , which is embodied as an optical parametric oscillator. Partial light beam  23  emerging from optical parametric oscillator  9  is guided via mirror  11  to dichroic beam combiner  13  and combined there with first partial light beam  19  to form illuminating light  15  that emerges from light source  1 . Mirrors  7  and  11  are mounted tiltably so that the relative positions of the components of the illuminating light can adjusted to one another.  
         [0033]    [0033]FIG. 2 shows a scanning microscope according to the present invention that is embodied as a confocal scanning microscope. In the embodiment shown here, light source  1  contains, in the beam path of partial light beam  23 , not only an optical parametric oscillator  9  but also a means for influencing the focus shape, which is embodied as a λ/2 plate and through which only the central portion of the cross section of partial light beam  23  passes. Partial light beam  19  also arrives at an optical parametric oscillator  25 . The partial light beam emerging therefrom has a different wavelength and is labeled  27 . Partial light beam  23  that has passed through the λ/2 plate passes to dichroic beam combiner  13  and is combined there with partial light beam  27  to form illuminating light  29  that emerges from light source  1 .  
         [0034]    Illuminating light  29  is reflected from a beam combiner  31  to beam deflection device  33 , which contains a gimbal-mounted scanning mirror  32  that guides illuminating light  29 , through scanning optical system  35 , optical system  37 , and microscope optical system  39 , over or through specimen  41 . In the case of non-transparent specimens  41 , illuminating light  29  is guided over the specimen surface. In the case of biological specimens  41  (preparations) or transparent specimens, illuminating light  29  can also be guided through specimen  41 . This means that different focal planes of specimen  41  are scanned successively by illuminating light  29 . Illuminating light  29  is depicted as a solid line. Detected light  43  emerging from the specimen arrives through microscope optical system  39  and via the beam deflection device  33  at the beam splitter  31 , passes through the latter, and strikes detector  47 , which is embodied as a photomultiplier. Detected light  43  emerging from specimen  41  is depicted as a dashed line. In detector  47 , electrical detection signals proportional to the power level of detected light  43  emerging from the specimen are generated, and are forwarded to a processing unit (not depicted). Arranged in front of the detector is a bandpass filter  48  that blocks light of the wavelengths of partial light beams  23  and  27 .  
         [0035]    Illumination pinhole  46  and detection pinhole  45 , which are usually provided in a confocal scanning microscope, are depicted schematically for the sake of completeness. Certain optical elements for guiding and shaping the light beams are omitted, however, for better clarity; these are sufficiently known to those skilled in this art.  
         [0036]    [0036]FIG. 3 shows a scanning microscope according to the present invention in non-descan configuration with multi-photon excitation. Illumination is provided substantially by light source  1  shown in FIG. 1, which additionally contains a means for influencing the focus shape which is embodied as λ/2 plate  61  and through which only the central portion of the cross section of partial light beam  53  passes. Partial light beam  53  that has passed through λ/2 plate  61  is reflected via mirror  55  to dichroic beam combiner  31 , and combined there with partial light beam  19  to form illuminating light  51  that emerges from light source  1 . Sample  41  is illuminated in a manner analogous to that described in FIG. 2. Excitation of a region of specimen  41  is effected with the component of illuminating light  51  that exhibits the wavelength of partial light beam  19 . The stimulated emission is generated with the component of illuminating light beam  51  that has the wavelength of partial light beam  23 . λ/2 plate  61  causes this component of illuminating light beam  51  to have an internally hollow focus, the result being clipping of the emission volume in all spatial directions and thus an increase in resolution in the axial and lateral directions.  
         [0037]    In this embodiment, detection takes place on the condenser side. Detected light  57  emerging from specimen  41  is focused by condenser  59  and directed to detector  49 , which is embodied as a photomultiplier. Arranged in front of the detector is a bandpass filter  48  that blocks light of the wavelength of partial light beam  23 .  
         [0038]    [0038]FIG. 4 a  illustrates the physical locations of first and second partial light beams  19  and  23  within or on the surface of specimen  41  being examined. Second partial light beam  23  possesses a larger beam diameter than first partial light beam  19 , so that in the focus region, first partial light beam  19  is completely surrounded by second partial light beam  23 . Second partial light beam  23  has an internally hollow focus. The overlap between first and second partial light beams  19  and  23  defines in the focus region a three-dimensional overlap region  63  that is depicted in FIG. 4 a  as a crosshatched surface. The region that lies in the focus region of first partial light beam  19  and within the hollow portion of second partial light beam  23  defines emission volume  65 .  
         [0039]    [0039]FIG. 4 b  also illustrates the physical locations of first and second partial light beams  19  and  23  within or on the surface of specimen  41  being examined. Second partial light beam  23  and first partial light beam  19  intersect in their respective edge regions. The overlap in the edge regions of first and second light beams  19  and  23  defines in the focus region a three-dimensional overlap region  63  that is depicted in FIG. 4 b  as a crosshatched surface.  
         [0040]    The invention was described with reference to a particular embodiment. It is nevertheless self-evident that changes and modifications can be made without thereby leaving the range of protection of the claims recited hereinafter.