Microscope device

Abstract

The present invention relates to 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), in which light generated by the light sources interacts with a sample (3), thereby generating a sample light beam (6), the sample light beam (6) being split by a first dichroic beam splitter (50) into a light beam (K) and a light beam (L), the light beam (K) and the light beam (L) having different spectral ranges of light, The light beam (K) is split by a second dichroic beam splitter (51) into a first light beam (A) having a first spectral range of light and a second light beam (B) having a second spectral range of light, the first light beam (A) is directed to a detector of a first camera (109) via a reflecting element (54), the second light beam (B) is directed to a detector of the first camera (109) via reflecting elements (52) and (53), - The present invention relates to a microscope apparatus, characterized in that the light beam (L) is split by a third dichroic beam splitter (56) into a third light beam (C) having a third spectral range of light and a fourth light beam (D) having a fourth spectral range of light, the third light beam (C) being guided to a detector of a second camera (117) via reflecting elements (58) and (59), and the fourth light beam (D) being guided to the detector of the second camera (117) via a reflecting element (57). Use of the microscope to obtain sequencing information.

Description

Technical Field

[0001] The present invention relates to a microscope using a camera that provides a fluorescence or transmitted light image with maximum contrast by utilizing the ability to distinguish four colors.

Background Art

[0002] Typically, camera detectors used in microscopy are monochrome. A multicolor microscope is disclosed in WO2020 / 038752, which relates to a microscope apparatus having a dual emission detection ability. It uses two cameras and disposes a dichroic beam splitter in the limited optical space between the microscope and the two cameras, and the cameras record two desired spectral regions. In order not to distort the transmitted spectral image, the dichroic is made as thin as possible and the reflection angle is made as small as possible and maintained. However, since a thin substrate tends to impair flatness and thus tends to impair the quality of the reflected image, the optimal thickness always reflects a compromise between the quality of the transmitted image and the quality of the reflected image.

[0003] A similar technique is disclosed in US Patent Application Publication No. 2018 / 0067327, where image rays are split by a dichroic beam splitter into two desired spectral regions, which are then led to one camera.

Summary of the Invention

Problems to be Solved by the Invention

[0004] An object of the present invention is to provide a microscope apparatus capable of separating four spectral regions with only two cameras.

Means for Solving the Problems

[0005] The object of the present invention is therefore a microscope apparatus comprising a microscope objective lens (1), one or more light sources, at least three dichroic beam splitters (50, 51, 56), and at least two cameras (109, 117), wherein the light generated by the light source interacts with a sample (3), thereby generating a sample ray (6), - The sample ray (6) is split into a ray (K) and a ray (L) by the first dichroic beam splitter (50), and the ray (K) and the ray (L) have different spectral ranges of light. - The light ray (K) is split by the second dichroic beam splitter (51) into a first light ray (A) having the spectral range of the first light and a second light ray (B) having the spectral range of the second light, the first light ray (A) is guided to the detector of the first camera (109) via the reflecting element (54), and the second light ray (B) is guided to the detector of the first camera (109) via the reflecting elements (52) and (53), - A microscope apparatus characterized in that a light ray (L) is split by a third dichroic beam splitter (56) into a third light ray (C) having a third spectral range of light and a fourth light ray (D) having a fourth spectral range of light, the third light ray (C) is guided to the detector of a second camera (117) via reflective elements (58) and (59), and the fourth light ray (D) is guided to the detector of the second camera (117) via reflective element (57).

[0006] Such microscopes are particularly useful for detecting multiple spectral ranges emitted by a sample, which is often the case in DNA / RNA molecule sequencing. To avoid damaging the sample, the interaction between light and the sample should be kept as short as possible. Since the apparatus of the present invention can simultaneously detect at least four different spectral ranges, the use of the microscope apparatus disclosed herein in sequencing-by-synthesis is a further object of the present invention. [Brief explanation of the drawing]

[0007] [Figure 1-2] This figure shows two embodiments of the light path of the microscope according to the present invention. [Figure 3] This figure shows a part of the apparatus upstream of the sample ray (6). [Modes for carrying out the invention]

[0008] In the apparatus of the present invention shown in Figure 1 or Figure 2, light emitted from a sample (either transmitted or emitted) is separated into four spectral regions by three dichroic beam splitters, and the resulting image rays A, B, C, and D are guided to two cameras (109, 117), so that each of the two cameras records two spectral regions side by side on its own sensor chip. This is facilitated by the availability of high-end camera chips for image fields much larger than those of current microscopes (e.g., 36 × 24 mm), covering a square field of view of up to 17 × 17 mm. Alternatively, TDI cameras are used.

[0009] The microscope according to the present invention may include at least one camera (109, 117) which is a charge-coupled device (CCD) type, an electron-multiplier charge-coupled device (EM-CCD) type, a complementary metal-oxide-semiconductor (CMOS) type, a chemically complementary metal-oxide-semiconductor (CMOS) type, a time-delay integral (TDI) type, or a combination thereof.

[0010] Preferably, the microscope is equipped to detect four spectral ranges, for example, 405, 488, 561, and 638 nm, or 375 nm, 473 nm, 532 nm, and 660 nm. For this purpose, preferably, the cut-on wavelengths are selected as 488, 561, and 638 nm to separate the emission into four spectral regions between the excitation wavelengths. Preferably, the spectral ranges of the first, second, third, and fourth rays (interchangeable as A, B, C, and D) are 473 nm to 532 nm; 532 nm to 594 nm, 594 nm to 660 nm; and 633 nm to 660 nm. It should be noted that these spectral ranges of light are given as examples and depend on the function of the beam splitter, i.e., are not necessarily limited to this sequence.

[0011] In a preferred embodiment, at least one of the dichroic beam splitters (50, 51, 56) is positioned (tilted) in the path of light 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) may be the same or different.

[0012] Therefore, the inclination angles of the first, second, and third dichroic beam splitters (50, 51, 56) with respect to each afterimage may 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 inclination angles of the first and third dichroic beam splitters (50, 56) with respect to their respective afterglow rays may be in opposite directions.

[0013] The light source preferably provides light having a spectral range of wavelengths of 300 to 1750 nm, preferably 300 to 800 nm, such as white light, laser light, or LED light. The sample may be exposed to the light "as is," or a fluorescent or phosphorescent agent may be applied to identify the region of interest. To avoid damaging the sample, a preferred light source that generates light of longer wavelengths, such as 525 nm or 635 nm, is used.

[0014] Therefore, the sample ray may be or include fluorescence or phosphorescence emitted from the sample (3), or radiation transmitted or reflected by the sample (3).

[0015] Furthermore, the microscope apparatus may include at least one focusing element (2) in the light path of the sample beam upstream of the first beam splitter (as shown in Figure 1). In an alternative example, as shown in Figure 2, two or more focusing elements (2) may be provided in the sample beam downstream of the first beam splitter.

[0016] The focusing element may consist of or include at least one lens, one objective lens, or a combination thereof.

[0017] In another embodiment, the microscope apparatus according to the present invention may include one or more optical elements (21, 22, 23, 24) in the light paths of the first, second, third, and / or fourth image rays A, B, C, D. Such optical elements may be focal plane or image plane shiftable and may optionally be inserted into and removed from the light path using appropriate apparatus. Suitable optical elements may have a refractive index higher than the surrounding medium and may consist of coated or uncoated glass or polymer. Furthermore, these optical elements may include filters for color correction of optical distortion caused by dichroic beam splitters.

[0018] Figures 1 and 2 show the apparatus of the present invention, which uses two identical optical configurations (48) and (49) for each of the two camera chips (109) and (117). Tube lens (2) and camera chip ( 109 ) and ( 117 A beam splitter (50), placed in a finite optical space between each of them, splits the image ray (6) into two rays (K=A+B) and (L=C+D). In optical configuration (48), the beam splitter (51) splits K into rays A and B, while in optical configuration (49), the beam splitter (56) splits L into rays C and D. The reflected ray (B) requires two more reflections at the mirrors (52) and (53), while the transmitted ray (A) Camera chip (109) detector It requires only one reflection at the surface (54) before reaching the target. The light ray (L) transmitted by the dichroic beam splitter (50) is divided into (C) by reflection at the dichroic beam splitter (56) and (D) transmitted by the dichroic beam splitter (56). Similar to (48), the transmitted portion (D) requires one reflection at the reflective element (57), while the reflected portion (C) requires two more reflections at the reflective surfaces (58) and (59).

[0019] As described in relation to previous modifications of the present invention, all rays reflected by the dichroic beam splitter may carry ghost image information. In Figures 1 and 2, this applies to spectral regions (A), (B), and (C), which require a suitable bandpass filter in front of the detector or suitable filter characteristics with respect to the reflective surfaces (54), (52), and (53), as well as (58) and (59), in order to overcome spatial constraints.

[0020] To minimize aberrations (primarily spherical, astigmatic, or coma), the inclination angles of the two dichroic elements (50) and (51) are kept at approximately 25° and its opposite angle to compensate for chromatic aberration.

[0021] The thickness of the dichroic beam splitter is kept as small as possible (usually 1 - 3 mm) to minimize the aberration related to the thickness in transmission, but is thick enough to maintain the flatness of the reflecting surface, which is extremely important for the image quality of the reflected light rays (Figure 1). As an alternative, in order to minimize (mainly spherical, astigmatic, or coma) aberration, one dichroic element is arranged between the objective lens (1) and the focusing element (2), enabling a larger tilt angle (preferably 45°) at this dichroic position and allowing for a larger thickness (usually 3 mm, in some cases 1 mm - 5 mm).

[0022] The three spectral regions (A), (B), and (C) that have undergone reflection by the dichroic element may all carry ghost images resulting from reflection on the rear (exit) side of their respective dichroic beam splitters (50, 51, and 56). These ghost images usually contain less than 1% of the transmitted image information, but when the reflected signal is weak and the sum of the transmitted signals is large, this can still lead to significant image degradation. The solution to this is to introduce appropriate band - pass filters or optical elements (21, 22, 23, and 24) into the light path.

[0023] Furthermore, the reflecting element (52) and / or (53) may be provided with a filter layer having the same optical characteristics as the first dichroic beam splitter (50) and / or the second dichroic beam splitter (51). The reflecting element (54) may be provided with a filter layer having the same optical characteristics as the first dichroic beam splitter (50). The reflecting elements (58) and / or (59) may be provided with a filter layer having the same optical characteristics as the third dichroic beam splitter (56).

[0024] Furthermore, the reflecting element (52) and / or the reflecting element (53) may be provided with a filter layer having the same optical characteristics as the second dichroic beam splitter (51).

[0025] Furthermore, the microscope apparatus of the present invention may include at least one focusing element (2) in the light path of the sample ray to produce an image (6). In an alternative example (shown in Figure 2), at least one focusing element (2) may be provided in the light paths of image L and / or K. The focusing element (2) may consist of or include at least one lens, or at least one objective lens, or a combination thereof.

[0026] If each dichroic beam splitter is a long-pass filter, a short-pass filter can substitute for a band-pass filter; conversely, if a dichroic beam splitter is a short-pass filter, a long-pass filter is required.

[0027] The microscope apparatus according to the present invention enables differentiation of images of color-labeled objects with respect to up to four spectral regions in both fluorescence emission and transmitted light absorption. Preferably, the optical path length is the same for all spectral ranges (color channels), and all images are located within the plane of their respective detector chips (cameras).

[0028] This holds true for an optimally corrected optical system. In the real world, this optical layout can be used to correct longitudinal color imperfections by appropriately adjusting the optical path length.

[0029] However, for uncolored samples, or for transmitted or reflected light images, intentional detuning of the path length can be used to observe two or more depths of focus simultaneously, and a contrast-enhanced image can be reconstructed from images taken at different focal positions. For example, a dichroic ensemble designed to separate emission excited by a 405 nm laser and a 488 nm laser splits the light from a white light-emitting diode into two spectral regions: below and above 488 nm.

[0030] By inserting the optical element (22) into the ray path (11) and providing means for removing the optical element (22) from the ray, ghost images in long-wavelength channels >488 nm are suppressed, so that the thickness of the optical element (22) determines the path length difference between rays A and B. The same configuration may include optical elements (21), (23) and / or (24).

[0031] By using a 40× objective lens and removing a 2mm thick filter substrate, a focal displacement of 416nm between the two images recorded by the camera chip (109) is obtained. Obviously, other means for providing a suitable path length difference between two or more color channels are also possible according to the present invention.

[0032] Use of the device The microscope apparatus of the present invention is particularly useful for methods of detecting multiple spectral ranges emitted during sequencing of DNA / RNA molecules, and especially for sequencing-by-synthesis methods for obtaining DNA or RNA sequence information of biological samples.

[0033] Preferably, the sequencing-by-synthesis method is carried out by hybridization of nucleotides given different dyes with DNA or RNA of a biological sample, the dyes emitting light when excited by one or more light sources in spectral ranges A, B, C, and D or a combination thereof. Such sequencing-by-synthesis methods and the required dyes are known to those skilled in the art.

Claims

1. Microscope objective lens (1), One or more light sources configured to generate light, A microscope apparatus comprising at least first (50), second (51), and third dichroic beam splitters (56), a first camera (109) equipped with a detector, and a second camera (117) equipped with a detector, The system is configured such that light generated by one or more light sources interacts with the sample (3), thereby generating a sample ray (6). The aforementioned microscope device is - The sample ray (6) is split into a first sample ray (K) and a second sample ray (L) by the first dichroic beam splitter (50), where the first sample ray (K) and the second sample ray (L) have different spectral ranges of light. - The first sample ray (K) is split by the second dichroic beam splitter (51) into a first ray (A) having a first spectral range of light and a second ray (B) having a second spectral range of light. - The second sample ray (L) is split by a third dichroic beam splitter (56) into a third ray (C) having a third spectral range of light and a fourth ray (D) having a fourth spectral range of light. It is configured in such a way, The microscope device further comprises first (54), second (52), third (53), fourth (58), fifth (59), and sixth reflective elements (57), and is configured to guide the first ray (A) to the detector of the first camera (109) via the first reflective element (54), and the second ray (B) to the detector of the first camera (109) via the second (52) and third reflective elements (53). The microscope apparatus is characterized in that it directs the third ray (C) to the detector of the second camera (117) via the fourth (58) and fifth reflective elements (59), and directs the fourth ray (D) 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 inclination angles of the first, second, and third dichroic beam splitters (50, 51, 56) with respect to each light ray are independently between +45° and -45°.

3. The microscope apparatus according to claim 1 or 2, characterized in that the first reflective element (54) comprises 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) comprises 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) comprises 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) comprises 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 one or more light sources are configured to provide light having a spectral range of 300 to 1750 nm.

8. The microscope apparatus according to any one of claims 1 to 7, further comprising at least one focusing element (2) within the light path of the sample ray (6).

9. The microscope apparatus according to claim 8, characterized in that the at least one focusing element (2) consists of 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, further comprising one or more optical elements (21, 22, 23, 24) in the ray paths of the first (A), second (B), third (C), and / or fourth ray (D).

11. The microscope apparatus according to claim 10, characterized in that one or more optical elements (21, 22, 23, 24) can be inserted into and removed from the light ray path.

12. The microscope apparatus according to any one of claims 1 to 11, characterized in that the sample light (6) includes fluorescence or phosphorescence emission or transmitted or reflected light emitted from the sample (3).

13. The microscope apparatus according to any one of claims 1 to 12, characterized in that the first ray (A) and the second ray (B), and / or the third ray (C) and the fourth ray (D) are emitted from different positions on the sample (3), and the rays at the different positions can be aligned by moving the sample (3) relative to the first camera (109) and the second camera (117).

14. Use of a microscope apparatus according to any one of claims 1 to 13 in the sequencing-by-synthesis method.

15. Use of the microscope apparatus according to claim 14 in a sequencing-by-synthesis method for obtaining DNA or RNA sequence information of a biological sample.

16. The use according to claim 15, characterized in that the sequencing-by-synthesis method is performed by hybridization of nucleotides, each given a different dye, with the DNA or RNA of the biological sample, and the dye emits light when excited by one or more light sources in the spectral ranges of the first, second, third, and fourth spectral ranges or combinations thereof.

Images