Patent Description:
Typically, camera detectors used in microscopy are monochrome.

<CIT> relates to a microscope device having dual emission capability, wherein detrimental effects of image aberrations and image distortions are reduced by reflecting the first beam of the first spectral range in a manner so as to invert its handedness and reflecting the second beam of the second spectral range in a manner so as to preserve its handedness, thereby obtaining a fully symmetrical configuration, so that corresponding image points in both spectral channels all experience the same field dependent operations. This allows to image the two spectrally different images adjacent to each other onto the same chip of a given detector camera.

<CIT> discloses a microscope according to the preamble of claim <NUM>.

It is an object of the invention to provide for a microscope device capable of producing sample images with relatively high contrast.

According to the invention, this object is achieved by a microscope device as defined in claim <NUM>.

The sample is illuminated with transmitted light of two different spectral ranges, which are directed to separate area-detectors. Congruent images on the two detectors are achieved by using a common tube lens for both spectral ranges, with the light of the two spectral ranges being separated by a dichroic beam splitter in the image beam paths between the tube lens and the detectors. The congruent images can be combined to generate a total sample image with enhanced contrast. By using a common tube lens for both beam paths, i.e. by separating the image beam path into two spectral arms in the finite optical space after the last lens system, the optical effects compromising congruence of the two image beam paths are greatly diminished compared to a configuration with separate tube lenses for each spectral arm. Such a configuration with separate tube lenses cannot result in congruent images, since lenses in practice are never exactly identical. Their focal lengths - and hence magnification - will always differ, and optical imperfections lead to different distortions of two images.

Using a joint tube lens for both spectral arms reduces optical asymmetry, but cannot fully avoid it. A planar optical element (the dichroic beam splitter), passed at an angle greater <NUM>° (as done by one of the two spectral arms), causes image distortions to that beam which increase with increasing angle and with substrate thickness. In order to separate incoming and reflected beam, the angle α between the incoming beam and the reflected beam should assume a finite value of at least <NUM>° (the angle at which the beam splitter is passed by the incoming beam is half of the angle α between the incoming beam and the reflected beam, i.e. it is α/<NUM>). Reducing the substrate thickness, on the other hand, also works only to a certain degree, since the reflective coating tends to spoil the planarity of a given substrate, and the effect becomes more pronounced with decreasing substrate thickness. Thus, while reducing the substrate thickness would reduce the detrimental effect on the transmitted beam, it may result in an increased bending the substrate through the reflecting surface layer and hence compromise the reflected beam. A substrate-thickness of <NUM> - <NUM> at an angle α/<NUM> of the dichroic beam splitter with regard to the incident (common) image beam of <NUM> to <NUM>° is a good compromise, as illustrated in <FIG>.

Hereinafter, examples of the invention will be illustrated by reference to the attached drawings, wherein:.

In <FIG> an example of a microscope device according to the invention is shown schematically. The arrangement <NUM> shown in <FIG> comprises a microscope objective <NUM>, a sample holder (or stage) <NUM> for holding and moving a sample <NUM>, a first light source <NUM> for oblique transmitted light illumination of the sample <NUM> with light within a first spectral range at a first angle with regard to the sample surface and a second light source <NUM> for oblique transmitted light illumination of the sample <NUM> with light within a second spectral range different from the first spectral range at a second angle. The light collected by the microscope objective <NUM> from the sample <NUM> is converted into an image by a tube lens <NUM>. A dichroic beam splitter <NUM> is provided in the convergent image beam path <NUM> so as to reflect light within one of the two spectral ranges (e.g., within the first spectral range), thereby creating a reflected image beam <NUM>, while transmitting light in the other one of the two spectral ranges (e.g., within the second spectral range), thereby creating a transmitted image beam <NUM>. The reflected image beam <NUM> reaches a first detector camera <NUM> for recording a first image of the sample based on light within the first spectral range (assuming that the beam splitter <NUM> reflects light within the first spectral range), while the transmitted image beam paths <NUM> reaches a second camera detector <NUM> for recording a second image of the sample <NUM> based on light within the second spectral range. In the example of <FIG> the beam paths of the light within the first spectral range are indicated by dotted lines, while beam paths of the light in the second spectral range are indicated by solid lines.

The beam splitter <NUM> may be implemented as a long-pass filter (in this case the first spectral range would be at shorter wavelengths than the second spectral range), or it may be implemented as a short-pass (in this case the first spectral range would be at longer wavelengths than the second spectral range).

A blocking filter <NUM> (shown in <FIG> only) may be placed into the reflected image beam <NUM> between the beam splitter <NUM> and the first detector <NUM>, so as to block light within the second spectral range (which may be reflected by the beam splitter <NUM> to some extent although the beam splitter <NUM> is designed to transmit light within the second spectral range), thereby preventing ghost images on the first detector <NUM> caused by reflected light within the second spectral range.

In order to minimize the remaining optical asymmetry between the transmitted image beam path <NUM> and the reflected image beam path <NUM>, the beam splitter <NUM> should have a relatively low thickness of less than <NUM>, and preferably at least <NUM>, and should be inclined with respect to the common image beam path <NUM> at as flat an angle as separation of incoming and reflected beam permits. The latter requirement is fulfilled by selecting the angle of the beam splitter <NUM> with regard to the incident beam <NUM> such that the angle between the common incident beam <NUM> and the reflected image beam <NUM> lies between <NUM> and <NUM> degrees.

The blocking filter <NUM> should be inclined at about the same angle, for example, within ±<NUM> % with regard to the reflected image beam <NUM> as is the beam splitter <NUM> with regard to the common incident beam <NUM>; further, the blocking filter <NUM> should have the same thickness as the beam splitter <NUM>. Thereby the optical asymmetry between the reflected image beam <NUM> and the transmitted image beam <NUM> is further minimized, since in this case the reflected image beam <NUM> passes through a transmitting optical element (namely the blocking filter <NUM>) which is very similar to the beam splitter <NUM> through which the transmitted image beam <NUM> is transmitted, so that the reflected image beam <NUM> is affected by such transmission in a manner very similar to the manner the transmitted image beam <NUM> is affected by the transmission through the beam splitter <NUM>. Thereby, the resulting optical distortion is very similar for both the transmitted image beam <NUM> and the reflected image beam <NUM>, so that the resulting images on the first detector <NUM> and the second detector <NUM>, respectively, have a very similar point spread function ("PSF"), so that the resulting images will be congruent. In other words, the optical distortion resulting from transmission of the convergent beam through the beam splitter <NUM> on the one hand and the blocking filter <NUM> on the other hand will affect both spectral channels in the same manner.

In the example of <FIG>, the first and second light source <NUM>, <NUM> are arranged for realizing an oblique transmitted light illumination of the sample <NUM> from opposite lateral directions, with the first light source <NUM> providing for oblique illumination from the left side, and the second light source <NUM> providing for oblique illumination from the right side, with the same inclination angle β with regard to the sample surface. The angle β can be optimized for a given chosen objective by allowing as many diffraction orders as possible to pass the objective. More precisely, the illumination light from the first light source <NUM> and the illumination light from the second light source <NUM> are directed onto the sample <NUM> in such a manner that the light bundles are mirror-symmetric to each other with regard to a plane, which is normal to the sample and which includes the optical axis <NUM> of the microscope objective <NUM>. Contrast increases with increasing inclination angle β, i.e. the more diffracted and refracted light is collected by the objective.

The first image obtained by the first camera <NUM>, resulting from illumination of the sample <NUM> by light from the first light source <NUM>, and the second image recorded by the second detector <NUM>, obtained by illumination of the sample <NUM> with light from the second light source <NUM>, are combined by an analyzer unit <NUM> in a manner so as to increase the contrast compared to the case of a single oblique transmission illumination (i.e., illumination of the sample <NUM> only either with the first light source <NUM> or the second light source <NUM>). One way to combine the first and second image would be to simply add the first and second image. However, a better contrast enhancement can be obtained by dividing the difference between the first image and the second image by the sum of the first image and the second image.

The dual color system illustrated above can also be used in an epi-illumination fluorescence microscope, which includes an epi-illumination light source <NUM> for illuminating the sample <NUM> via the microscope objective <NUM> so as to achieve fluorescence excitation (for example, a beam splitter <NUM> may be used for directing the epi-illumination light onto the microscope objective <NUM>) wherein the fluorescence emission light from the sample <NUM> is collected by the microscope objective <NUM> and is focused by the tube lens <NUM> for being directed to at least one of the first detector <NUM> and the second detector <NUM> by using the dichroic beam splitter <NUM>.

It is to be understood that the light sources <NUM>, <NUM> and <NUM> may be integrated within a single light source or may by implements by different spectral ranges obtained from a multiband light source.

Thus, microscope devices like that shown in <FIG> do not only allow to obtain high contrast images by oblique transmitted light illumination, but also allow to obtain fluorescence images in at least two different spectral ranges without the need for insertion or removal of optical elements in the beam paths, thereby allowing convenient switching between different optical modes. In particular, the enhanced contrast images may be obtained by using high quality fluorescence microscope objectives without negatively affecting the fluorescence beam path. Consequently, the proposed oblique transmitted light illumination with two separate detectors is superior compared to other methods of obtaining contrast transmission images. For example, phase contrast microscopy or differential interference contrast (DIC) microscopy are not well-suited for being used with high quality fluorescence microscopy, since, for example, phase contrast microscopy requires specific objectives, which are not well-suited for fluorescence measurements, and DIC microscopy requires polarizers to be inserted not only in the transmission illumination beam path but also in the image beam path, which polarizers then would have to be removed prior to fluorescence measurement.

Claim 1:
A microscope device comprising:
a microscope objective (<NUM>),
a first light source (<NUM>) for transmitted light illumination of the sample (<NUM>) with light within a first spectral range and a second light source (<NUM>) for transmitted light illumination of the sample with light within a second spectral range different from the first spectral range,
a tube lens (<NUM>) for forming a sample-image from the light collected by the microscope objective,
a first camera detector (<NUM>),
and
an analyzer unit (<NUM>),
wherein the analyzer unit is configured to combine a first image of the sample and a second image of the sample so as to generate a total sample image with enhanced contrast, characterised in that:
the microscope comprises a second camera detector (<NUM>), and
a dichroic beam splitter (<NUM>) in the imaging beam path (<NUM>) between the tube lens (<NUM>) and the camera detectors (<NUM>, <NUM>),
wherein the dichroic beam splitter (<NUM>) reflects light within the first spectral range onto the first camera detector (<NUM>) and transmits light within the second spectral range onto the second camera detector (<NUM>), so that the first camera detector (<NUM>) detects the first image of the sample from light within the first spectral range and the second camera detector (<NUM>) detects the second image of the sample from light within the second spectral range,
wherein the angle (a/<NUM>) of the surface normal of the dichroic beam splitter (<NUM>) with regard to the incident beam (<NUM>) lies between <NUM>° and <NUM>°.