BACKGROUND

[0001] The invention relates to a camera-based microscope which employs four-color distinction capabilities to provide a maximally contrast-rich fluorescence respectively transmitted light image.

[0002] Typically, camera detectors used in microscopy are monochrome. A multicolor microscope is disclosed in WO 2020/038752, which relates to a microscope device having dual emission detection capabilities. It employs two cameras and places a dichroic beam splitter into the finite optical space between the microscope and the two cameras, which record the two desired spectral regions. In order not to distort the transmitted spectral image, the dichroic is kept as thin and the reflection angle as small as possible. However, since a thin substrate tends to compromise flatness and hence the quality of the reflected image, an optimal thickness always reflects a compromise between the quality of the transmitted and the reflected image.

[0003] A similar technology is disclosed in US 2018/0067327 wherein an image beam is divided by a dichroitic beam splitter into two desired spectral regions which are then guided onto one camera.

SUMMARY

[0004] It is an object of the invention to provide for a microscope device capable of separating four spectral regions with two cameras only.

[0005] According to the invention, this object is achieved by a a microscope device comprising: a microscope objective (1); one or more light sources; at least 3 dichroic beam splitters (5, 10, 16) and at least 2 cameras (4, 17) characterized in that the light generated by the light source interacts with the sample (3) thereby producing a sample beam wherein - a first image having a spectral range of light (A) is separated from the sample beam with a first dichroic beam splitter (5) and guided via reflection element (8) to the light detector (4) of the first camera (9), thereby resulting in residual beam X - a second image having a spectral range of light (B) is separated from residual beam X with a second dichroic beam splitter (10) and guided via reflection element (13) to light detector (4) of the first camera (9) thereby resulting in residual beam Y

- a third image having a spectral range of light (C) is separated from residual beam Y with a third dichroic beam splitter (16) and guided via reflection element (19, 20) to the light detector (4) of the second camera (17) thereby resulting in residual beam D

- guiding residual beam D as forth image having a forth spectral range of light via reflection element (18) to the light detector (4) of the second camera (17).

[0006] Such microscope devices are especially useful for detecting multiple spectral ranges emitted by a sample which is often the case in sequencing DNA/RNA molecules. To avoid damaging of the sample, the interaction between the light and the sample should be kept as short as possible. Since the device of the invention can detect at least 4 different spectral ranges simultaneously, the use of the microscope device as disclosed herein in a sequenc- ing-by-synthesis process it is a further object of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Fig. 1 shows the general path of light of the microscope of the invention

DETAILED DESCRIPTION

[0008] In the device of the invention as shown in Fig. 1, light originating from the sample (either transmitted or emitted) is separated into four spectral regions by three dichroic beam splitters, and the resulting image beams A, B, C and D are directed to two cameras (4, 17), whereby each of the two cameras records two spectral regions side-by-side on its sensorchip. This is facilitated by the fact that high-end camera chips are available for image fields much bigger (for example 36 x 24 mm) than the image field of current microscopes, which maximally covers a square field of 17 x 17 mm. Preferable, TDI cameras are used.

[0009] Preferable, the microscope is equipped to detect four spectral ranges, for example 405, 488, 561 and 638 nm or 375 nm, 473 nm, 532 nm, und 660 nm.). To this end, preferably the cut-on wavelengths are chosen to 488, 561 and 638 nm so as to separate the emission into the four spectral regions between the excitation wavelengths. Preferable, the respective spectral ranges of light of the images are between 473 nm and 532 nm; 532 nm and 594 nm, 594 nm and 660 nm; 633 nm and 660 nm.

[0010] In a preferred embodiment, at least one of the third dichroic beam splitters (5, 10, 16) is arranged (tiled) in the path of light as to minimize or avoid the chromatic error of the light detected by the camaras. Since the chromatic error depends on the spectral range / the wavelength of the light, the angular position of the dichroic beam splitters (5, 10, 16) may be same or different.

[0011] Accordingly, the tilt angles of first, second and third dichroic beam splitter (5, 10, 16) with the respective residual images may be independently between + 45° and - 45°, preferable independently between + 30° and - 30° or independently between + 25° and - 25°. or independently between + 15° and - 15°. In any case, the tilt angles of the first and second dichroic beam splitters (5, 10) and/or second and third dichroic beam splitters (10, 16) with the respective residual beams may be in opposing directions.

[0012] The light sources preferable provide light having a spectral range of wavelengths of 300 to 800 nm like white light, laser light, or LED light. The sample may be subjected to the light “as is” or may be provided with fluorescence or phosphorenscence agent to mark regions or interest. To avoid damaging the sample, preferable light sources producing light with longer wavelengths are used, such as 525 and 635 nm.

[0013] Accordingly, the sample beam may be or comprise fluorescence or phosphorenscence radiation originating from the sample (3) or radiation transmitted or reflected by the sample (3).

[0014] Further, the microscope device may be provided with at least one focusing element (2) into the beam-path of the sample beam upstream of the first beam splitter.

[0015] Preferable, at least one focusing element is placed into the beam-path of the sample beam. The focusing elements may consist or comprise at one lens or at least one objective or a combination thereof. In Fig. 1 this is shown with focusing element (2) providing image (6).

[0016] In another embodiment, the microscope device according to the invention may be provided with one or more optical element (21, 22, 23, 24) into the beam-path of first, second, third and/or forth images A, B, C, D. Such optical elements are capable of focal plane or image plane shift and can optionally be inserted and removed from the beam-paths with an appropriate device. Suitable optical elements have a higher refractive index than the surrounding medium and may consist of coated or uncoated glass or polymer. Further, the optical elements can be provided with a filter for chromatic correction of any optical distortion which caused by the dichromatic beam splitters

[0017] Fig 1 shows a microscope in its most basic form, comprising an objective (1) and a tube-lens (2), which generates an image of an object (3) in the focal plane (4) of the tube-lens (2). A first dichroic beam-splitter (5) splits the image-beam (6) by separating a first spectral region (A) from the residual image beam (X = B + C + D), and directs it, by means of a plane reflecting element (8), to one (in Fig. 1 the left) half of the camera detector-chip (9). By means of appropriate optical adjustments, the chip (9) is aligned such that its position lies flat in the image plane (4) of the optical system. A second dichroic beam splitter (10) splits the transmitted fraction (X) into a second spectral region (B) and the remaining image beam (Y = C + D). By means of another plane reflecting element (13), B is directed to the other (in Fig. 1 the right) half of the camera detector-chip (9). In order to minimize (mostly spherical, astigmatic or comatic) aberrations, the tilt-angle of the two dichroic elements (5) and (10) is kept to at about 25° and at opposing angles so as to compensate for chromatic aberrations. The thickness of the dichroic beam splitters is kept as small as possible (usually 1 to 3 mm) so as to minimize thickness-related aberrations in transmission, but thick enough to maintain flatness of the reflecting surfaces, which is of paramount importance for image quality of the reflected fraction of the beam.

[0018] The residual image beam (Y), after being transmitted by the two dichroic beam splitters (5) and (10), is split into two spectral regions (C) and (D) by means of another dichroic beam splitter (16). The transmitted beam (D) is reflected onto the second camera-chip (17) with the help of a single reflecting element (18), whereas the reflected beam (C) requires 2 more reflections by means of the two reflecting elements (19) and (20).

[0019] All three images / spectral regions (A), (B) and (C), which have undergone reflections at dichroic elements, carry ghost-images, resulting from reflections on the rear (exit) side of the respective dichroic beam splitters (5, 10 and 16). While these ghost-images usually contain less than 1% of the transmitted image information, this may still lead to significant image degradation in cases where the reflected signal is weak and the sum of the transmitted signals is large. The cure for this is to bring appropriate bandpass-filters or optical elements (21, 22 and 23) into the beam-path, and/or equip the reflecting elements with the same filter- layer as dichroic the beam splitter. Preferable, reflection element (8) is provided with a filter layer having the same optical properties as first dichroic beam splitter (5) and /or reflection element (13) is provided with a filter layer having the same optical properties as second dichroic beam splitter (10).

[0020] In case the respective dichroic beam splitters are long-pass filters, a short-pass filter can replace the bandpass, if the dichroic beam splitter is a short-pass, one needs long- pass filter.

[0021] The microscope device according to the present invention allows to differentiate the image of a color-labelled object with respect to up to four spectral regions, both in fluorescence-emission and in transmitted light absorption. Preferable, the optical path-lengths are identical for all spectral ranges (color-channels) and all images lie in the plane of the respective detector chip (camera).

[0022] This holds for an optimally corrected optical system. In the real world, the optical layout may be used to correct for longitudinal color-imperfections by adjusting the optical path-lengths accordingly.

[0023] In an unstained sample or for a transmitted or reflected light image, however, a volitional detuning of the path-lengths may be used to look at two or more focal depths simultaneously and to reconstruct contrast-enriched images from images taken at different focal positions. For example, a dichroic ensemble, designed for separating the emission excited by a 405 and a 488 nm laser, divides the light of a white light emitting diode into two spectral regions below and above 488 nm. By suppressing ghost-images in the longer wavelength-channel > 488 nm by inserting a optical element (22) into the beam-path (11), and by providing means to remove the optical element (22) from the beam, the thickness of the filter-element (22) determines the path-length difference of beams A and B. The same arrangement can be provided with optical element (21) and/or (23).

[0024] With a 40x objective, removing a optical-substrate of thickness 2 mm, provides a focal displacement the two images recorded by camera chip (9) of 416 nm. Obviously other means for providing a suitable path-length difference between two or more color channels are also possible according to the invention.

USE OF DEVICE [0025] The microscope devices of the invention are especially useful in methods for detecting multiple spectral ranges which are emitted during sequencing of DNA/RNA molecules, in particular in sequencing -by- synthesis processes to obtain DNA or RNA sequence information of a biological sample. [0026] Preferable, the sequencing-by-synthesis process is performed by hybridization of nucleotides provided with different dyes to the DNA or RNA of the biological sample and wherein the dyes emit light upon excitation by the one or more light sources in the spectral ranges A, B, C and D. Such sequencing-by-synthesis process and the required dyes are known to the person skilled in the art