Resumen
The invention describes an extensive sample flat laser beam microscope (1) comprising a cuvette (2), a laser emitter, and an optical assembly (5) that receives the fluorescence emission from the sample (M). The optical assembly comprises: a lens matrix (51) located adjacent one face of the cuvette (2); a tube lens (52) encompassing several lenses (51a) of the lens matrix (51): a set (53) of galvanometric mirrors that transmits to a focusing unit (54) a sub-image of the sample (M) received from the tube lens (52); and the focusing unit (54) that focuses the sub-image on a camera sensor (55). Thus, by successively selecting several lenses (51a), and scanning the sample (M) for each of said lenses (51a), a complete 3D image of the sample (M) is constructed. (Machine-translation by Google Translate, not legally binding)
Reivindicaciones
1. A light sheet fluorescence microscope (1) for large samples comprising a tray (2) configured to house a sample (M), a laser emitter configured to emit a light sheet beam (4) along a direction of illumination (DI), and an optical assembly (5) arranged along a direction of detection (DD) perpendicular to the light sheet beam (4) configured to receive fluorescence emission from the sample (M) caused by said light sheet beam (4), characterised in that the optical assemble comprises: a lens array (51), wherein each lens (51a) has a field of view substantially smaller than the size of the tray (2) and an axis parallel to the direction of detection (DD), and wherein the lens array (51) is located adjacent to a face of the tray (2); a tube lens (52) with an axis parallel to the direction of detection (DD) and whose field of view (FOV) covers several lenses (51a) of the lens array (51); a set (53) of galvanometric mirrors configured to receive, from the tube lens (52), a sub-image of the sample (M) corresponding to a lens (51a) selected from the lens array (51) and to transmit said sub-image of the sample (M) to a focusing unit (54); and the focusing unit (54) configured to focus the sub-image of the sample (M) on a camera sensor (55), such that successively selecting several lenses (51a) from the lens array (51) using the set (53) of galvanometric mirrors and, by scanning the sample (M) using the light sheet beam (4) for each of these lenses (51a), a complete 3D image of the sample (M) housed in the tray (2) is constructed. 2. The light sheet fluorescence microscope (1) according to claim 1, wherein the focusing unit (54) comprises a first transmission lens (54a) that receives, from the set (53) of galvanometric mirrors, the sub-image of the sample (M) and transmits it to a tuneable lens (54b), the tuneable lens (54b) that focuses said sub-image, and a second transmission lens (54c) that receives from the tuneable lens (54b) said focused sub-image and transmits it to the camera sensor (55). 3. The light sheet fluorescence microscope (1) according to any one of the preceding claims further comprising, between the focusing unit (54) and the camera sensor (55), a spatial light modulation unit (56) configured to correct spherical aberrations of the sub-image of the sample (M). 4. The light sheet fluorescence microscope (1) according to claim 3, wherein the spatial light modulation unit (56) comprises a third transmission lens (56a) that receives from the second transmission lens (54c) the sub-image of the sample (M) and transmits it to a spatial light modulator (56b), the spatial light modulator (56b) that corrects spherical aberrations of the sub-image, and a fourth transmission lens (56c) that receives from the spatial light modulator (56b) the sub-image with the spherical aberrations corrected and transmits it to the camera sensor (55). 5. The light sheet fluorescence microscope (1) according to any one of the preceding claims further comprising a dichroic mirror (58) arranged behind the tuneable lens (54b) and configured to introduce an external laser beam which meets the sample (M) following an optical path inverse to that followed by the sub-image of the sample (M). 6. The light sheet fluorescence microscope (1) according to claim 5 further comprising an external laser beam source oriented to a second set of galvanometric mirrors (59) that direct said pulsed laser beam to the dichroic mirror (58). 7. A method for imaging large samples using the light sheet fluorescence microscope (1) of any one of claims 1-2, characterised in that it comprises performing the following steps for each lens of the lens array (51): - configuring the set (53) of galvanometric mirrors to transmit to the focusing unit (54) a sub-image of the sample (M) collected by a selected lens (51a); - scanning the sample (M) using the light sheet beam (4) to obtain a partial 3D sub-image of a portion of the sample (M) located within the field of view (FOV) of the selected lens (51a); and - constructing a complete 3D image of the sample (M) by combining the plurality of partial 3D sub-images obtained by each lens (51a), wherein the scanning of the sample (M) using the light sheet beam comprises, in turn, the following steps: - emitting a light sheet beam (4) at a selected position along the direction of illumination (DI); - transmitting to the focusing unit (54), using the set (53) of galvanometric mirrors, the sub-image of the sample (M) collected by the selected lens; - focusing the sub-image using the focusing unit (54) and transmitting it to a camera sensor (55); - repeating these operations for a plurality of positions of the light sheet beam (4) so that they cover the entire sample (M). 8. The method for imaging large samples according to claim 7 further comprising the step of correcting aberrations of each focused sub-image of the sample (M) by means of a spatial light modulation unit (56). 9. The method for imaging large samples according to any one of claims 7-8 further comprising the step of introducing an external laser beam which meets the sample (M) following an optical path inverse to that followed by the sub-image of the sample (M).
