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EP-4235255-B1 - AN OPTICAL ARRANGEMENT AND METHOD FOR IMAGING A SAMPLE

EP4235255B1EP 4235255 B1EP4235255 B1EP 4235255B1EP-4235255-B1

Inventors

  • QUINTAS GLASNER DE MEDEIROS, Gustavo
  • NORLIN, NILS
  • HUFNAGEL, LARS

Dates

Publication Date
20260506
Application Date
20140408

Claims (11)

  1. An optical arrangement (10) for imaging a sample (20) comprising: - at least one first objective lens (30) and at least one second objective lens (40); - at least one illumination source (50, 50') for producing a first illumination beam (60) and a second illumination beam (60'); - a detector (70) for imaging radiation (80, 80') from the sample (20); and - a plurality of movable mirrors (90a, 90b, 90c) being movable between at least a first position and a second position along a direction (92); wherein a first movable mirror (90a) of the plurality of movable mirrors (90a, 90b, 90c) is configured, in the first position, to reflect the radiation (80) towards a movable mirror arrangement (90c) to further reflect the radiation (80) onto the detector (70), and a second movable mirror (90b) of the plurality of movable mirrors (90a, 90b, 90c) is configured, in the first position, not to interrupt a passage of the first illumination beam (60) to the first objective lens (30) to illuminate the sample (20), and the first moveable mirror (90a) of the plurality of movable mirrors (90a, 90b, 90c) is configured, in the second position, not to interrupt a passage of the second illumination beam (60') to the second objective lens (40) to illuminate the sample (20); and the second movable mirror (90b) of the plurality of movable mirrors (90a, 90b, 90c) is configured, in the second position, to reflect the radiation (80) towards the movable mirror arrangement (90c) to further reflect the radiation (80) onto the detector (70).
  2. The optical arrangement (10) of claim 1, wherein the plurality of movable mirrors (90a, 90b, 90c) is translatable or rotatable.
  3. The optical arrangement (10) of claim 1 or 2, wherein at least one of the plurality of movable mirrors (90a, 90b, 90c) is double-sided.
  4. The optical arrangement (10) of any one of claims 1 to 3, comprising an image processor (100) connected to the detector (70).
  5. The optical arrangement (10) of any one of claims 1 to 4, suitable for imaging a fluorescing sample.
  6. The optical arrangement (10) of any one of claims 1 to 5, comprising at least one optical filter (91a, 91b, 91c).
  7. The optical arrangement (10) of any one of claims 1 to 6, comprising at least a third objective lens (31) for illuminating the sample (20) or collecting radiation (80") from the sample (20).
  8. A method for imaging a sample (20) comprising the steps of: - positioning a plurality of movable mirrors (90a, 90b, 90c) to a first position; - illuminating (200; 230), by means of a first objective lens (30), the sample (20) using a first illumination beam (60); - imaging first radiation (80) through a second objective lens (40) into a detector (70), the imaging comprising reflecting the first radiation (80) with a first movable mirror (90a) of the plurality of movable mirrors (90a, 90b, 90c) and further with a mirror arrangement (90c) of the plurality of movable mirrors (90a, 90b, 90c); - detecting (210; 240) at the detector (70) the first radiation (80) from the sample (20); - processing (220) the first radiation (80) to obtain a first data set (120); - repositioning the plurality of movable mirrors (90a, 90b, 90c) along a direction (92) to a second position; - illuminating, by means of the second objective lens (40), the sample (20) using a second illumination beam (60'); - imaging second radiation (80') through the first objective lens (30) into the detector (70), the imaging comprising reflecting the second radiation (80') with a second movable mirror (90b) and further with the mirror arrangement (90c) of the plurality of movable mirrors (90a, 90b, 90c); - detecting, at the detector (70) the second radiation (80') from the sample (20); - processing the second radiation (80') to obtain a second data set (120), and - combining the first data set (130) and the second data set (120) to produce an image of the sample (20).
  9. The method of claim 8, further comprising the steps of illuminating the sample (20) with a third illumination beam (60"), and detecting, at the detector (70), third radiation (80") from the sample (20).
  10. The method of claim 8 or 9, wherein the first illumination beam (60) and the second illumination beam (60') are produced from a single illumination source (50).
  11. The method of any one of claims 8 to 10, further comprising the step of optically filtering of at least one of the first illumination beam (60) or the second illumination beam (60').

Description

Field of the invention The invention relates to an optical arrangement and a method for imaging a sample using at least two illumination beams. Background to the invention A microscope is a scientific instrument that is used for the visualization of objects, which can be either small cells or have details that are too small to be resolved by the naked eye. There are many types of microscopes available on the market. The most common of these and the first to be invented is the so-called optical microscope, which uses light in a system of lenses to magnify images of the samples. The image from the optical microscope can be either viewed through an eyepiece or, more commonly nowadays, captured by a light-sensitive camera sensor to generate a so-called micrograph. There are a wide range of sensors available to catch the images. Non-limiting examples are charge-coupled devices (CCD) and scientific complementary metal-oxide semiconductor (sCMOS) based technologies, which are widely used. These sensors allow the capture and storage of digital images to the computer. Typically there is a subsequent processing of these images in the computer to obtain the desired information. The illumination sources as used in optical microscopes have been developed over the years and wide varieties of illumination sources are currently available, which can emit light or other type of radiation at different wavelengths. Optical filters can be placed between the illumination source and the sample to be imaged in order to restrict the wavelength of the radiation illuminating the sample. Modern biological microscopy uses fluorescent probes for imaging specific structures within a cell as the sample. In contrast to normal trans-illuminated light microscopy, the sample in fluorescent microscopy is illuminated through one or more objective lenses with a narrow set of light wavelengths. These narrow set of light wavelengths interact with fluorophores in the sample, which then emit light of a different wavelength. This emitted fluorescent light is detected in a detector and is used to construct the image of the sample. The use of multiple images enables a 3-dimensional reconstruction of the sample to be made. This 3-D reconstruction can be done by generating images at different positions on the sample, as the sample moves relatively to one or more objective lens. Depending on the number of detection units necessary, several detectors may be required. These detectors are quite expensive and a microscope designer will wish to reduce the number of detectors. The use of a single detector, which is moved during the imaging process, can be disadvantageous in that the movement of the detector itself can slightly effect the position of the sample, due to vibrations. Alternately the sample itself may move for other reasons whilst the detector is being placed into another position. This movement of the detector requires a precise and fast movement of a part of hardware, which is comparatively massive and in turn leads to further increase in development costs and/or in extra parts of equipment. A number of papers and patents have been published on various aspects of microscopy. For example, European patent EP 1 019 769 (Carl Zeiss, Jena) teaches a compact confocal feature microscope, which can be used as a microscope with a single objective lens or with multiple objective lenses. The microscope has separate directions of illumination and detection. The direction of detection in the objective lens is aligned inclined at a set angle in relation to the direction of illumination. Another example of a microscope is taught in the paper by Krzic al. "Multi View Light-Sheet Microscope for Rapid in toto Imaging", Nature Methods, July 2012, vol. 9 No. 7, pages 730 - 733. This paper teaches a multi-view selective-plane illumination microscope comprising two detection and illumination objective lenses. The microscope allows in toto fluorescence imaging of the samples with subcellular solution. The fixed geometrical arrangement of the imaging branches enables multi-view data fusion in real time. The document DE 195 09 885 A1 discloses an endoscope. The endoscope comprises several objective systems. The endoscope comprises at its end illumination windows and observation windows. Some of the disclosed embodiments employ mirror arrangements for separating two images transmitted by one transmission system. Another embodiment employs two mirrors, which are each pivotable around their own axes for enabling bending of an end portion of the endoscope. A further embodiment employs a pivotable mirror for switching between two beam paths. The document discloses objectives that are used for observation only. The mirrors are not connected to the usage of the objectives, i.e., whether for illumination or for observation. The document US4,440,475A discloses an electron probe microanalyzer including a system for observing the sample. The observation system comprises a high-magni