Microscope device

Abstract

The invention relates to a microscope device comprising a microscope objective (1); one or more light sources; at least 3 dichroic beam splitters (50, 51, 56) and at least 2 cameras (109, 117), characterized in that light generated by the light source interacts with a sample (3) thereby generating a sample light beam (6), wherein - the sample light beam (6) is split into a light beam (K) and a light beam (L) with a first dichroic beam splitter (50), wherein the light beam (K) and the light beam (L) have light of different spectral ranges, and wherein - the light beam (K) is split into a first light beam (A) with light of a first spectral range and a second light beam (B) with light of a second spectral range with a second dichroic beam splitter (51), and wherein the first light beam (A) is guided via a reflective element (54) onto a detector of the first camera (109), and wherein the second light beam (B) is guided via reflective elements (52) and (53) onto a detector of the first camera (109), and wherein - the light beam (L) is split into a third light beam (C) with light of a third spectral range and a fourth light beam (D) with light of a fourth spectral range with a third dichroic beam splitter (56), and wherein the third light beam (C) is guided via reflective elements (58) and (59) onto a detector of the second camera (117), and wherein the fourth light beam (D) is guided via a reflective element (57) onto a detector of the second camera (117). Use of a microscope for obtaining sequencing information.

Description

Background Technology

[0001] This invention relates to a camera-based microscope that employs four-color discrimination capability to provide highly contrasting images of fluorescence transmitted light separately.

[0002] Typically, the camera detectors used in microscopes are monochromatic. WO 2020 / 038752 discloses a multicolor microscope, which relates to a microscope apparatus with dual emission detection capability. It employs two cameras and places a dichroic beam splitter within a limited optical space between the microscope and the two cameras, which records two desired spectral regions. To avoid distortion of the transmitted spectral image, the dichroic beam splitter is kept as thin as possible and its reflection angle as small as possible. However, since a thin substrate tends to compromise flatness and thus the quality of the reflected image, the optimal thickness always reflects a trade-off between the quality of the transmitted and reflected images.

[0003] A similar technique is disclosed in US 2018 / 0067327, in which an image beam is split into two desired spectral regions by a dichroic beam splitter, and then the spectral regions are directed to a camera. Summary of the Invention

[0004] One object of the present invention is to provide a microscope device capable of separating four spectral regions using only two cameras.

[0005] Therefore, the object of the present invention is a microscope device, including...

[0006] A microscope objective (1); one or more light sources; at least three dichroic beam splitters (50, 51, 56) and at least two cameras (109, 117), characterized in that light generated by the light sources interacts with a sample (3) to produce a sample beam (6), wherein...

[0007] - The sample beam (6) is split into beam (K) and beam (L) using a first dichroic beam splitter (50), wherein beam (K) and beam (L) have different spectral ranges, and wherein

[0008] - The beam (K) is split into a first beam (A) having a first spectral range and a second beam (B) having a second spectral range using a second dichroic beam splitter (51), wherein the first beam (A) is guided to the detector of the first camera (109) via a reflective element (54), and wherein the second beam (B) is guided to the detector of the first camera (109) via reflective elements (52) and (53), and wherein

[0009] - The beam (L) is split into a third beam (C) having a third spectral range and a fourth beam (D) having a fourth spectral range by a third dichroic beam splitter (56), wherein the third beam (C) is directed to the detector of the second camera (117) via reflective elements (58) and (59), and wherein the fourth beam (D) is directed to the detector of the second camera (117) via reflective element (57).

[0010] Such microscopy equipment is particularly useful for detecting multiple spectral ranges emitted by a sample, a common practice in DNA / RNA molecular sequencing. To avoid damaging the sample, the interaction between light and the sample should be kept as short as possible. Since the device of this invention can simultaneously detect at least four different spectral ranges, the use of the microscopy equipment disclosed herein in the synthesis sequencing process is another objective of this invention. Attached Figure Description

[0011] Figure 1 and 2 Two embodiments of the microscope optical path of the present invention are shown.

[0012] Figure 3 A portion of the device upstream of the sample beam (6) is shown. Detailed Implementation

[0013] In such Figure 1 In the apparatus of the present invention shown in Figure 2, light (transmitted or emitted) originating from the sample is split into four spectral regions by three dichroic beam splitters, and the resulting image beams A, B, C, and D are directed to two cameras (109, 117), whereby each of the two cameras records two spectral regions side-by-side on its sensor chip. This is facilitated by the fact that high-end camera chips can provide a much larger image field of view (e.g., 36 x 24 mm) than current microscopes, which at most cover a square field of view of 17 x 17 mm. Alternatively, a TDI camera is used.

[0014] The microscope according to the invention may include at least one camera (109, 117), which is a charge-coupled device (CCD) type, an electron multiplication charge-coupled device (EM-CCD) type, a complementary metal-oxide-semiconductor (CMOS) type, a scientific complementary metal-oxide-semiconductor (CMOS) type, a time delay integration (TDI) type, or a combination thereof.

[0015] Preferably, the microscope is equipped to detect four spectral ranges, such as 405, 488, 561, and 638 nm or 375 nm, 473 nm, 532 nm, and 660 nm. For this purpose, the cutoff wavelengths are preferably selected as 488, 561, and 638 nm to separate the emission into four spectral regions between the excitation wavelengths. Preferably, the corresponding spectral ranges of the first, second, third, and fourth beams (which may be interchanged with the terms A, B, C, and D) are between 473 nm and 532 nm; between 532 nm and 594 nm; between 594 nm and 660 nm; and between 633 nm and 660 nm. It should be noted that these spectral ranges are given by way of example and depend on the function of the beam splitter, i.e., not necessarily limited to this sequence.

[0016] In a preferred embodiment, at least one of the dichroic beam splitters (50, 51, 56) is arranged (tiled) in the optical path to minimize or avoid color errors in the light detected by the camera. Since color errors depend on the spectral range / wavelength of the light, the angular positions of the dichroic beam splitters (50, 51, 56) can be the same or different.

[0017] Therefore, the tilt angles of the first, second, and third dichroic beam splitters (50, 51, 56) with the corresponding residual images can be independently between +45° and -45°, preferably 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 third dichroic beam splitters (50, 56) with the corresponding residual beams can be in opposite directions.

[0018] The light source preferably provides light with a wavelength spectral range of 300 to 1750 nm, preferably 300-800 nm, such as white light, laser light, or LED light. The sample can be exposed to light "as is," or it can be provided with fluorescent or phosphorescent agents to mark the region of interest. To avoid damaging the sample, it is preferable to use a light source that produces light with longer wavelengths, such as 525 and 635 nm.

[0019] Therefore, the sample beam may be or include fluorescent or phosphorescent radiation originating from the sample (3), or radiation transmitted or reflected by the sample (3).

[0020] Furthermore, the microscope equipment may provide at least one focusing element (2) in the beam path of the sample beam upstream of the first beam splitter. Figure 1 (As shown in the image). As an alternative, such as Figure 2 As shown, two or more focusing elements (2) can be provided in the sample beam downstream of the first beam splitter.

[0021] The focusing element may include or contain a lens or at least one objective lens or a combination thereof.

[0022] In another embodiment, the microscope apparatus according to the invention may provide one or more optical elements (21, 22, 23, 24) in the beam paths of the first, second, third, and / or fourth image beams A, B, C, D. Such optical elements are capable of focal plane or image plane shifting and can be optionally inserted into and removed from the beam path using suitable means. Suitable optical elements have a higher refractive index than the surrounding medium and may be composed of coated or uncoated glass or polymer. Furthermore, the optical elements may be provided with filters for color correction of any optical distortion caused by the dichroic beam splitter.

[0023] Figure 1 and Figure 2 The device of the present invention is shown, which uses two identical optical arrangements (48) and (49) for each of the two camera chips (109) and (117). A beam splitter (50) – placed in a limited optical space between the tube lens (2) and the camera chip (9) (18) respectively – splits the image beam (6) into two beams (K = A + B) and (L = C + D). In optical arrangement (48), beam splitter (51) splits K into beams A and B, while in optical arrangement (49), beam splitter (56) splits L into beams C and D. The reflected beam (B) needs to be reflected twice more at mirrors (52) and (53), while the transmitted beam (A) only needs to be reflected once at surface (54) before it reaches detector (109). (L) – the beam transmitted by the dichroic (50) – is split into: (C), which is reflected at the dichroic (56), and (D), which is transmitted at the dichroic (56). In (48), the transmissive portion (D) requires a single reflection at the reflective element (57), while the reflective portion (C) requires two additional reflections at the reflective surfaces (58) and (59).

[0024] As described with respect to the foregoing variant of the invention, all beams reflected by the dichroic beam splitter can carry ghosting information. Figure 1 and 2 This applies to spectral regions (A), (B), and (C), which require suitable bandpass filters in front of the detector, or suitable filter characteristics of the corresponding reflective surfaces (54), (52), (53), and (58)(59) to overcome spatial limitations.

[0025] To minimize aberrations (primarily spherical, astigmatic, or coma), the tilt angles of the two dichroic elements (50) and (51) are kept at approximately 25° and at opposite angles to compensate for chromatic aberration.

[0026] The thickness of the dichroic beam splitter is kept as small as possible (typically 1-3 mm) to minimize thickness-related aberrations in transmission, but thick enough to maintain the flatness of the reflecting surface, which is crucial for image quality in the beam reflection section. Figure 1 Alternatively, in order to minimize aberrations (primarily spherical, astigmatic, or coma), a dichroic element is placed between the objective (1) and the focusing element (2), thereby allowing for a larger tilt angle (preferably 45°) at the dichroic position and allowing for a larger thickness (typically 3 mm, possibly 1 mm to 5 mm).

[0027] All three spectral regions (A), (B), and (C) that have already undergone reflection at the dichroic element may carry ghosting, which is caused by reflections on the rear (outgoing) side of the corresponding dichroic beamsplitters (50, 51, and 56). Although these ghostings typically contain less than 1% of the transmitted image information, they can still lead to significant image degradation when the reflected signal is weak and the sum of the transmitted signals is large. A countermeasure to this is to introduce appropriate bandpass filters or optical elements (21, 22, 23, and 24) into the beam path.

[0028] Furthermore, the reflective element (52) and / or (53) may be provided with a filter layer having the same optical properties as the first dichroic beam splitter (50) and / or the second dichroic beam splitter (51). The reflective element (54) may be provided with a filter layer having the same optical properties as the first dichroic beam splitter (50). The reflective element (58) and / or (59) may be provided with a filter layer having the same optical properties as the third dichroic beam splitter (56).

[0029] In addition, the reflective element (52) and / or the reflective element (53) may be provided with a filter layer having the same optical properties as the second dichroic beam splitter (51).

[0030] Furthermore, the microscope apparatus of the present invention may provide at least one focusing element (2) in the beam path of the sample beam in order to create an image (6). Alternatively (e.g.) Figure 2 As shown in the diagram, at least one focusing element (2) can be provided in the beam path to the image L and / or K. The focusing element (2) may include or contain a lens or at least one objective lens or a combination thereof.

[0031] When the corresponding dichroic beam splitter is a long-pass filter, a short-pass filter can replace the bandpass filter; if the dichroic beam splitter is short-pass, then a long-pass filter is required.

[0032] The microscope apparatus according to the invention allows for the differentiation of images of color-marked objects relative to up to four spectral regions in terms of both fluorescence emission and transmitted light absorption. Preferably, the optical path length is the same for all spectral ranges (color channels), and all images lie within the plane of the corresponding detector chip (camera).

[0033] This applies to optimally calibrated optical systems. In the real world, optical layouts can be used to correct longitudinal color defects by adjusting the optical path length accordingly.

[0034] However, in unstained samples or for transmitted or reflected light images, arbitrary detuning of the optical path length can be used to simultaneously observe two or more depths of focus and reconstruct contrast-enhanced images from images taken at different focal positions. For example, a dichroic ensemble designed to separate emission excited by 405 and 488 nm lasers splits the light from a white LED into two spectral regions below and above 488 nm.

[0035] Ghosting in longer wavelength channels greater than 488 nm is suppressed by inserting an optical element (22) into the beam path (11), and the thickness of the optical element (22) determines the optical path length difference between beams A and B by providing means for removing the optical element (22) from the beam. The same arrangement can be provided with optical elements (21), (23) and / or (24).

[0036] With a 40x objective lens, removing the 2mm thick filter substrate provides a focus shift for the two 416nm images recorded by the camera chip (109). Clearly, according to the invention, other means for providing a suitable optical path length difference between two or more color channels are also possible.

[0037] Uses of equipment

[0038] The microscope apparatus of the present invention is particularly useful for detecting multiple spectral ranges emitted during DNA / RNA molecular sequencing, especially in methods for synthesizing sequencing processes to obtain DNA or RNA sequence information of biological samples.

[0039] Preferably, the synthetic sequencing process is performed by hybridizing nucleotides provided with different dyes with the DNA or RNA of a biological sample, wherein the dyes emit light in spectral ranges A, B, C, and D, or combinations thereof, when excited by one or more light sources. Such synthetic sequencing processes and the required dyes are known to those skilled in the art.

Claims

1. A microscope apparatus, comprising: A microscope objective (1); one or more light sources; at least three dichroic beam splitters (50, 51, 56) and at least two cameras (109, 117), characterized in that the microscope apparatus further comprises a first (54), a second (52), a third (53), a fourth (58), a fifth (59) and a sixth reflective element (57), and wherein the light generated by the light source interacts with the sample (3) to produce a sample beam (6), wherein - The sample beam (6) is split into beam (K) and beam (L) using a first dichroic beam splitter (50), wherein beam (K) and beam (L) have different spectral ranges, and wherein - The beam (K) is split into a first beam (A) having a first spectral range and a second beam (B) having a second spectral range using a second dichroic beam splitter (51), wherein the first beam (A) is guided to the detector of the first camera (109) via a first reflective element (54), and wherein the second beam (B) is guided to the detector of the first camera (109) via second and third reflective elements (52) and (53), and wherein - The beam (L) is split into a third beam (C) having a third spectral range and a fourth beam (D) having a fourth spectral range by a third dichroic beam splitter (56), wherein the third beam (C) is directed to the detector of the second camera (117) via the fourth and fifth reflective elements (58) and (59), and wherein the fourth beam (D) is directed to the detector of the second camera (117) via the sixth reflective element (57).

2. The microscope apparatus according to claim 1, characterized in that, The first, second, and third dichroic beam splitters (50, 51, 56) are independently tilted between +45° and -45° with respect to their respective beams.

3. The microscope apparatus according to claim 1 or 2, characterized in that, The first reflective element (54) is provided with a filter layer having the same optical properties as the first dichroic beam splitter (50).

4. The microscope apparatus according to any one of claims 1 to 3, characterized in that, The fourth reflective element (58) and / or the fifth reflective element (59) are provided with a filter layer having the same optical properties as the third dichroic beam splitter (56).

5. The microscope apparatus according to any one of claims 1 to 4, characterized in that, The second reflective element (52) and / or the third reflective element (53) are provided with a filter layer having the same optical properties as the first dichroic beam splitter (50).

6. The microscope apparatus according to any one of claims 1 to 5, characterized in that, The second reflective element (52) and / or the third reflective element (53) are provided with a filter layer having the same optical properties as the second dichroic beam splitter (51).

7. The microscope apparatus according to any one of claims 1 to 6, characterized in that, The light source provides light with a spectral range of 300 to 1750 nm.

8. The microscope apparatus according to any one of claims 1 to 7, characterized in that, At least one focusing element (2) is provided in the beam path of the sample beam (6).

9. The microscope apparatus according to claim 8, characterized in that, The focusing element (2) includes at least one lens or at least one objective lens or a combination thereof.

10. The microscope apparatus according to any one of claims 1 to 9, characterized in that, One or more optical elements (21, 22, 23, 24) are provided in the beam paths of the first, second, third and / or fourth beams A, B, C, D.

11. The microscope apparatus according to claim 10, characterized in that, The optical element can be inserted into and removed from the beam path.

12. The microscope apparatus according to any one of claims 1 to 11, characterized in that, The sample beam (6) includes fluorescent or phosphorescent radiation, transmitted light, or reflected light originating from the sample (3).

13. The microscope apparatus according to any one of claims 1 to 12, characterized in that, The first and second beams and / or the third and fourth beams originate from different positions on the sample (3); wherein the beams at different positions are aligned by moving the sample (3) relative to the camera.

14. Use of the microscope apparatus according to any one of claims 1 to 13 in the synthesis sequencing process.

15. The use of the microscope apparatus according to claim 14 in the synthesis sequencing process to obtain DNA or RNA sequence information of a biological sample.

16. The use according to claim 14 or 15, characterized in that, The synthesis sequencing process is performed by hybridizing nucleotides with different dyes with the DNA or RNA of a biological sample, wherein the dyes emit light in spectral ranges A, B, C, and D or combinations thereof when excited by one or more light sources.

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