Scanning microscope for examining microscopic preparations and lighting device for same

p0001The invention relates to a scanning microscope arrangement for examining microscopic preparations. In particular, the invention relates to a scanning microscope for examining microscopic preparations arrangement comprising a laser and an optical means that images the light generated by the laser onto a sample to be examined. The scanning microscope can be configured as a confocal microscope.

p0002Furthermore, the invention relates to a lighting device for a scanning microscope.

p0003In scanning microscopy, a sample is scanned with a light beam. To this end, lasers are often used as a light source. From the<patcit id="pcit0001" dnum="EP0495930A"><text>EP 0495930</text></patcit>"A confocal microscope system for multicolor fluorescence, for example, an assembly with a single, more laser lines emitting lasers known Currently this are usually mixed gas laser, in particular ArKr lasers used..

p0004In use are also diode lasers and solid state lasers.

p0005From the patent specification <patcit id="pcit0002" dnum="US5161053A"><text>US-A-5,161,053</text></patcit> entitled "Confocal Microscope" is a confocal microscope is known, is transported in the light of an external light source using an optical fiber to the optical path of the microscope and the end of the fiber serves as a point light source, so that a mechanical shutter is unnecessary.

p0006The emission spectrum of lasers is limited to a narrow range of wavelengths, so that the simultaneous multi-line excitation light from several lasers must be united to form an illumination beam.

p0007The gas laser as Mehrlinieniaser most used are very complex and expensive. Moreover, they are very need of maintenance, which makes continuous use at many microscopic applications.

p0008The invention has for its object to provide a scanning microscope equipped with a plurality of spectral lines allows sample analysis, without having to rely on the use of multi-line laser.

p0009The objective problem is solved by an arrangement having the features of the characterizing part of claim 1 and 7, the example is characterized in that between the laser and the optical means an optical component is provided which is generated by the laser light at a single passage spectrally broadened.

p0010Another object of the invention is to provide a lighting device for a scanning microscope, which makes more spectral ranges available that were previously not addressed.

p0011The objective problem is solved by a lighting device which is characterized in that at the light exit opening to an optical component is mounted, which consists of photonic band gap material.

p0012The optical component in the form of a "photonic band gap" material has the advantage that a short laser pulse is broadened by the optically non-linear structure of the fiber and thus a spectrally broad, continuous spectrum of light produced. In "photonic band-gap material" is microstructured transparent material. Most by combining different dielectrics can be the resulting crystal impart a band structure for photons, which is reminiscent of the electronic band structure of semiconductors.

p0013The technique has recently been realized, the fibers are produced by drawing structured arranged glass tubes even when optical fibers. The fiber is a particular structure based on: In the grain small cannulas are left free, have a distance of about 2-3 microns and a diameter of about 1 micron and are usually filled with air. In the center of the fiber is no cannula. This type of fiber is called "photon crystal fibers", "holey fibers" or "micro structured fibers".

p0014"Photon crystal fibers" can be used in all areas for the production of a continuous spectral distribution over the entire visible wavelength. For this purpose, the light of a short pulse laser is coupled into the fiber. By the optically non-linear structure of the fiber, the frequency spectrum of the laser widened. The result is a spectrally broad, continuous spectrum of light.

p0015The optical component is constructed in a preferred embodiment of the scanning microscope of a plurality of microoptical structure elements which have at least two different optical densities. Very particularly preferred is an embodiment in which the optical element contains a first region and a second region, said first region having a homogeneous structure and in the second region a microscopic structure comprising micro-optical structure elements being formed. It is advantageous, furthermore, if the first area surrounding the second area. The micro-optical structural elements are preferably cannulas, webs, honeycombs, tubes or cavities.

p0016The optical component is, in another embodiment of adjacent glass or plastic material and cavities. especially preferable is the embodiment in which the optical component made of photonic band gap material and configured as an optical fiber, wherein preferably there is provided an optical diode, the back-reflection of the light beam suppresses the laser at the ends of the optical fiber

p0017A particularly preferred and simple to realize embodiment includes as an optical component, a conventional optical fiber with a fiber core, along a portion of said at least one taper. Optical fibers of this type are known as so-called. "Tapered fibers". Preferably, the optical fiber as a whole is 1 m long and has a taper over a length of 30 mm to 90 mm. The diameter of the fiber is in a preferred embodiment, 150 micron outside the region of the taper and the fiber core in this area about 8 microns. In the region of the taper of the diameter of the fiber is reduced to about 2 microns. The fiber core diameter is accordingly in the nanometer range.

p0018For use in microscopy, it is important to implement means for wavelength selection and light power stabilization. Therefore, can be advantageously such a fiber laser with acousto-optical or electro, adjustable filters (AOTF), with acousto- or electro-optical deflectors (AOD), or acousto-electro-optical beam splitters (AOBS) combine. These can be used for a for wavelength selection and for the suppression of the detection light (our German application<patcit id="pcit0003" dnum="DE19906757A1"><text>DE 199 06 757 A1</text></patcit>"An optical assembly").

p0019Especially in confocal microscopy can be the fiber exit end use as a point light source, making the use of an excitation shutter is superfluous. In such an embodiment, it would be particularly advantageous to coat the fiber end itself partly reflective, so that this part of the reflector forms a resonator end mirror.

p0020In further embodiments, devices are provided to compensate for light output variations. For example, a control loop can be installed for light output stabilization, the parasitic light output in the beam path of the microscope measures and maintains for example by varying the pumps layer performance or using a acousto or electro-optical element, the sample illumination light output constant. For this purpose, LCD attenuator could be used.

p0021Another advantage of the invention is when the Beleuchtungeeinrichtung is already designed accordingly, that it provides a plurality of spectral ranges for illumination. The laser, which is the lighting device for a scanning microscope, has attached to the light exit opening to an optical component. The optical component consists of photonic band-gap material. Further, the photonic band-gap material may be configured as an optical fiber.

p0022In the drawing, the subject invention is schematically shown and is described below with reference to FIGS. They show:<dl id="dl0001"><dt>Fig. 1</dt><dd>an arrangement with a confocal microscope,</dd><dt>FIG. 2</dt><dd>an arrangement in which has been dispensed an illumination pinhole,</dd><dt>Fig. 3</dt><dd>an arrangement with light output stabilization,</dd><dt>Fig. 4</dt><dd>an embodiment of the optical component</dd><dt>Fig. 5</dt><dd>another embodiment of the optical component.</dd></dl>

p0023<figref idrefs="f0001">Fig. 1</figref> shows a confocal microscope that uses an optical component 3 to the expansion of a laser pulse generated by a pulse laser. 1 The pulse laser 1 defines a pulsed laser beam 2, which is passed through the optical component. 3 The optical component 3 is a "photonic band-gap material. From the optical component 3 occurs spectrally broadband illumination light 4 from being imaged by a first optical system 5 to an illumination pinhole 6 and then strikes a beam splitter 7. From the beam splitter 7 reaches the spectrally broadband illumination light 4 to a second optical system 8 which generates a parallel light beam 4a is incident on a scanning mirror 9. the scanning mirror 9, a plurality of optics 10 and 11 downstream of the 4a forms the light beam. the light beam 4a arrives at a lens 12 from which it is mapped to a sample 13. the light reflected or emitted by the sample defines an observation beam path 4b. the light of the observation beam path 4b occurs again by the second optical system 8 and is imaged onto a detection pinhole 14, the detector in front of a 15 sets. through the optical component 3, it is possible to generate for the examination of the sample 13 necessary laser light according to the desired spectrum.

p0024This in <figref idrefs="f0002">FIG. 2</figref> Illustrated embodiment shows a confocal microscope, was to dispense with the illumination pinhole. 6 All the elements of the elements<figref idrefs="f0001">Fig. 1</figref> match are designated by the same reference numerals. In this embodiment, an AOTF 16 (acousto optical tunable filter) is used in place of the first optical system 5, which is connected to a corresponding AOTF drive 17th Since the optical component 3 can generate a broadband illumination light 4, it is necessary to provide means for wavelength selection and light power stabilization. Advantageously, one can acousto- or electro-optically tunable filter (AOTF) with acousto- or electro-optical deflectors (AOD) and acousto- or electro-optical beam splitters (AOBS) combine. These can be used for a for wavelength selection and for the suppression of the detection light. The AOTF 16, a beam sump 18 is also associated with the captures the spectral components not used the illumination light in order to avoid unnecessary disruption of the scanning microscope.

p0025According to the invention, as shown in <figref idrefs="f0003">Fig. 3</figref> shown, an optical fiber 20 is used as an optical component 3, which consists of the photonic band gap material. Plus from the laser 1, the pulsed laser beam 2 is coupled via a lens 19 in an entry end 20a of the optical fiber 20th Since the optical fiber 20 is constructed from the photonic band gap material, exits from an exit end 20b of a spectrally broadened laser pulse that is coupled out via an optical 21st Before the spectrally broadened laser pulse incident on the illumination pinhole 6, spectral filtering is performed. For this purpose, a plurality of color filters 24 are arranged on a turret 23rd Upper an engine 22 is the turret 23 rotatable so that the appropriate color filter 24 can be introduced into the beam path. Likewise, a linear array of the color filters 24 is conceivable, thereby the color filter 24 are moved by means of a linear motion in an illumination beam path 50th The illumination beam 50 after the illumination pinhole 6 is connected to the beam path of<figref idrefs="f0001">Fig. 1</figref> comparable. As in<figref idrefs="f0001">Fig. 1</figref> mentioned, directs the beam splitter 7, the light on the scanning mirror 9. A part of the light passes through the beam splitter 7 therethrough and defines a loss of beam path 50a. This portion of the light is lost for observation or measurement. For this reason, a detector 25 is provided in the optical path loss 50a which determines the loss of light and determines an electronic variable which is passed via a line 30 to a control electronics 26th The control electronics 26 is connected via a further line 32 to the pulse laser 1st Via the line 32 controls the control electronics 26, the intensity of the pulse laser 1 in such a way that at the specimen 13 is always impinges a constant light output. For example, a control loop for light output stabilization be provided such that they parasitically the light power in the optical path of the microscope measures and maintains for example by varying the pump light power or using an acousto or electro-optical element, the sample illumination light output constant. For this purpose, LCD attenuator could be used.

p0026<figref idrefs="f0004">Fig. 4</figref> schematically shows an embodiment of the optical device 3. In this embodiment, the optical component 3 consists of a conventional optical fiber 51 with an outer diameter of 925 microns and a fiber core 52 having a diameter of 6 microns. micron region of a 300 mm long taper 53 51 reduced to 1.8 microns of the external diameter of the optical fiber. In this area, the diameter of the fiber core is 52 only fractions of microns.

p0027<figref idrefs="f0005">Fig. 5</figref> shows an embodiment of the optical component 3. This consists of photonic band gap material having a special honeycomb-shaped microstructure 54 The honeycomb structure shown is particularly suitable for the generation of broadband light. The diameter of the inner cannula 55 is approximately 1.9 microns. The inner cannula 55 is surrounded by glass Steegen 56th The glass webs 56 form honeycombed cavities 57. These micro-optical structure elements together form a second region 58, which is executed by a first portion 59 of the glass envelope, is surrounded.

<u>LIST OF REFERENCE NUMBERS</u>

p0028<dl id="dl0002" compact="compact"><dt>1</dt><dd>pulse laser</dd><dt>2</dt><dd>Pulsed laser</dd><dt>3</dt><dd>optical component</dd><dt>4</dt><dd>Spectral broadband illumination light</dd><dt>4a</dt><dd>beam of light</dd><dt>4b</dt><dd>Observation beam path</dd><dt>5</dt><dd>optics</dd><dt>6</dt><dd>Illumination pinhole</dd><dt>7</dt><dd>beamsplitter</dd><dt>8th</dt><dd>optics</dd><dt>9</dt><dd>scanning mirror</dd><dt>10</dt><dd>optics</dd><dt>11</dt><dd>optics</dd><dt>12</dt><dd>lens</dd><dt>13</dt><dd>sample</dd><dt>14</dt><dd>Detection pinhole</dd><dt>15</dt><dd>detector</dd><dt>16</dt><dd>AOTF (acousto optical tunable filter)</dd><dt>17</dt><dd>AOTF control</dd><dt>18</dt><dd>ray marsh</dd><dt>19</dt><dd>optics</dd><dt>20</dt><dd>photonic band-gap optical fiber</dd><dt>20a</dt><dd>entry end</dd><dt>20b</dt><dd>exit end</dd><dt>21</dt><dd>optics</dd><dt>22</dt><dd>motor</dd><dt>23</dt><dd>revolver</dd><dt>24</dt><dd>color filter</dd><dt>25</dt><dd>detector</dd><dt>26</dt><dd>control electronics</dd><dt>30</dt><dd>management</dd><dt>32</dt><dd>management </dd><dt>50</dt><dd>Illumination beam path</dd><dt>50a</dt><dd>Loss optical path</dd><dt>51</dt><dd>optical fiber</dd><dt>52</dt><dd>fiber core</dd><dt>53</dt><dd>rejuvenation</dd><dt>54</dt><dd>microstructure</dd><dt>55</dt><dd>cannula</dd><dt>56</dt><dd>glass webs</dd><dt>57</dt><dd>cavities</dd><dt>58</dt><dd>second region</dd><dt>59</dt><dd>first region</dd></dl>