CN113655026B - Elliptic hemispherical surface large-view high-flux two-photon microscope - Google Patents
Elliptic hemispherical surface large-view high-flux two-photon microscope Download PDFInfo
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Abstract
The invention discloses an elliptic hemispherical surface large-field high-flux two-photon microscope, which comprises: the system comprises a near infrared femtosecond pulse laser coupling group, a beam splitting delay module coupling group, a scanning unit coupling group, a beam combining and splicing module, an ellipsoidal curved surface large-view-field reflective objective lens, a dichroic sheet and a photomultiplier detection array. The invention realizes the large view field of the elliptic curved surface through the design of the reflective objective lens, and can convert the laser scanning field of the plane rectangle into the laser scanning field of the elliptic hemispherical surface; the high-flux scanning can be realized by adopting a multi-path laser parallel scanning and multi-path fluorescence parallel detection mode, the non-adjacent areas are divided into a group by utilizing the four-color principle, and the energy efficiency of multi-polarization beam combination is maximized by utilizing the area gaps and the combined half-wave plates; delay is introduced between the laser groups, and the source regions of fluorescent signals can be effectively distinguished in time and space by the scheme that regions at the same time are not adjacent and adjacent regions are not identical, so that signal crosstalk caused by fluorescent scattering is avoided to a great extent.
Description
Technical Field
The invention relates to the field of microscopic imaging instruments, in particular to an elliptic hemispherical curved surface large-field high-flux two-photon microscope.
Background
Because of the deep biological tissue imaging depth and high spatial resolution, the two-photon microscope is well applied to the imaging of the functional structure of the nerve tissue of the animal cerebral cortex at present, and the development of neuroscience (brain science) is promoted.
Scientists wish to be able to simultaneously view and record functional signals from more neurons over a larger area, and new demands are being made on two-photon imaging technology. Firstly, the resolution of neurons is required to be achieved, the optical resolution is required to be 1-2 microns, and the numerical aperture is corresponding to 0.3-0.5; secondly, the image refreshing speed needs at least 5 frames per second to effectively capture the calcium function signals of the neurons; on the premise of meeting the spatial and temporal resolution, a larger visual field range is imaged, so that the number of neurons recorded at a time is larger.
At present, the maximum field diameter of the international two-photon imaging is about 5mm, but the imaging speed of the full field is less than 1 frame per second, and the requirements of functional signal detection cannot be met. Under real-time imaging and single-cell resolution conditions, the field of view area of two-photon imaging is only 1mm×1mm, and also covers only a single brain functional region for mice. Whereas the full cerebral cortex area of mice is about 200mm2 and the brain surface is uneven, approaching an elliptical hemisphere, which is a great challenge for optical microscopy imaging.
The animal in-vivo cerebral cortex single neuron resolution dynamic large-field imaging needs to solve the two problems, namely, the field is bent while realizing large imaging field area, and the field is matched with cerebral cortex curvature, so that the field has great difference with the conventional field objective field design concept; secondly, the large-field high-resolution image requires a very large number of pixels, and time resolution is guaranteed, so that the required imaging data flux is huge, and therefore, the laser scanning and fluorescence detection flux is required to be improved by orders of magnitude.
The parallel scanning and detection are common means for improving laser scanning and fluorescence detection flux, a large-area visual field is divided into a plurality of areas, a plurality of laser focuses are respectively and independently scanned, and a multichannel detector or an area array detector is combined for detecting fluorescence signals of all the areas, so that imaging flux can be remarkably improved. However, the current parallel scanning scheme has large laser energy loss when the view is spliced by multiple beam combination, and the laser energy is reduced by half when the laser passes through the depolarization beam combination prism once; the signal crosstalk caused by fluorescence scattering exists in the parallel detection of multiple channels, specifically, the fluorescence scattering area is large, and fluorescence falls on adjacent areas simultaneously, so that the signal crosstalk among the channels is caused.
Therefore, a more reliable solution is now needed.
Disclosure of Invention
The technical problem to be solved by the invention is to provide an elliptic hemispherical surface large-field high-flux two-photon microscope aiming at the defects in the prior art.
In order to solve the technical problems, the invention adopts the following technical scheme: an elliptical hemispherical surface large field of view high flux two-photon microscope comprising: the system comprises a near infrared femtosecond pulse laser coupling group, a beam splitting delay module coupling group, a scanning unit coupling group, a beam combining and splicing module, an ellipsoidal curved surface large-view-field reflective objective lens, a dichroic sheet and a photomultiplier detection array;
the laser emitted by the near infrared femtosecond pulse laser coupling group sequentially passes through the beam splitting delay module coupling group, the scanning unit coupling group and the beam combining and splicing module and then transmits the dichroic sheet, then the fluorescence generated by excitation of the sample is collected by the ellipsoidal curved surface large-field reflective objective and then reflected to the photomultiplier detection array by the dichroic plate;
the ellipsoidal curved surface large-field reflective objective lens comprises a main lens, a secondary lens and a triple lens which are sequentially arranged along a light path, wherein the optical surface of the main lens is a hyperboloid, the optical surface of the secondary lens is a flat elliptic surface, the optical surface of the triple lens is a flat elliptic surface, and laser entering the ellipsoidal curved surface large-field reflective objective lens is sequentially reflected by the main lens, the secondary lens and the triple lens and then irradiates on a sample;
The near infrared femtosecond pulse laser group comprises 4 near infrared femtosecond pulse lasers;
the beam splitting delay module group comprises 4 beam splitting delay modules, and the beam splitting delay modules comprise delay light paths and beam splitting light paths;
the scanning unit group comprises 4 scanning units, each scanning unit comprises 4 independent scanning modules, and each scanning module independently realizes two-dimensional scanning of 1 rectangular sub-scanning area;
each near infrared femtosecond pulse laser corresponds to 1 beam splitting delay module, and each beam splitting delay module corresponds to 1 scanning unit;
4 paths of delay lasers with time intervals of T/4 are formed after the lasers emitted by the near infrared femtosecond pulse lasers pass through the delay light path, each path of delay lasers passes through the beam splitting light path and then is equally divided into 4 paths of sub lasers, 4 paths of sub lasers from the same path of delay lasers enter the same scanning unit, and each path of sub lasers corresponds to 1 scanning module and is used for realizing the scanning of 1 sub scanning area, so that the scanning of 16 sub scanning areas is realized through the one-to-one correspondence of 16 sub lasers and 16 scanning modules; wherein, T is the pulse period of the laser light emitted by the near infrared femtosecond pulse laser, in a further preferred embodiment, t=12.5 ns, and the time delay between two groups adjacent in time is 3.125ns; the sub-scanning areas obtained by scanning by each scanning module in the same scanning unit are positioned at the same time point, and the sub-scanning areas obtained by scanning by the scanning modules in different scanning units are positioned at different time points;
The beam combination splicing module is used for realizing beam combination splicing of 16 sub lasers emitted by the 16 scanning modules, and forms a rectangular scanning field distributed in a 4 x 4 array by combining and splicing the 16 sub scanning areas, and the boundaries of the 4 sub scanning areas positioned at the same time point are not adjacent, and any 4 areas adjacent to the boundaries are positioned at different time points in time.
Preferably, the optical surface of the primary mirror is an eighth-order hyperboloid, the optical surface of the secondary mirror is a secondary flat elliptic surface, and the optical surface of the three mirrors is a sixth-order flat elliptic surface.
Further preferably, the numerical aperture of the ellipsoidal curved surface large-field reflection type objective lens is in the range of 0.3-0.5. The imaging view field of the reflective objective lens is an ellipsoidal curved surface, the radius of curvature of a long axis is 9-12 mm, the radius of curvature of a short axis is 6-9 mm, and the plane projection size of the view field is 6mm multiplied by 6mm.
Preferably, the delay light path includes a first reflecting mirror and a second reflecting mirror, laser emitted by the near infrared femtosecond pulse laser sequentially passes through the first reflecting mirror and the second reflecting mirror to form delay laser output, and the distance between the first reflecting mirror and the second reflecting mirror is adjusted to enable the output delay laser to generate different delay amounts;
The beam splitting light path comprises a first beam splitting element, a second beam splitting element and a third beam splitting element, delay laser output by the second reflector enters the first beam splitting element and is equally divided into two paths, one path of delay laser is transmitted to the first beam splitting element and then reaches the second beam splitting element and is equally divided into two paths by the second beam splitting element, and the other path of delay laser is reflected by the first beam splitting element and then reaches the third beam splitting element and is equally divided into two paths by the third beam splitting element, so that the delay laser is equally divided into 4 paths.
Preferably, the first light-splitting element, the second light-splitting element and the third light-splitting element are all depolarizing light-splitting prisms or 50/50 light-splitting sheets.
Preferably, the scanning module comprises a fast axis resonant scanning mirror and a slow axis galvanometer mirror.
Preferably, the beam combining and splicing module comprises 4 primary beam combining and splicing modules and 1 secondary beam combining and splicing module, wherein each primary beam combining and splicing module corresponds to 1 scanning unit respectively so as to combine and splice 4 sub-lasers emitted by 4 scanning modules in the 1 scanning units;
the secondary beam combining and splicing module is used for combining and splicing 4 groups of lasers emitted by the 4 primary beam combining and splicing modules again and then inputting the 4 groups of lasers into the dichroic sheet.
Preferably, the primary beam combining and splicing module comprises 2 primary beam combining Shu Pinjie sub-optical paths, a primary polarization beam combining prism, a first primary lens, a primary combining half-wave plate and a second primary lens, wherein laser emitted from the 2 primary beam combining Shu Pinjie sub-optical paths passes through the primary polarization beam combining prism to be combined and then sequentially passes through the first primary lens, the primary combining half-wave plate and the second primary lens to be output, and the primary combining half-wave plate comprises 4 different half-wave plates;
the 2 primary combining Shu Pinjie sub-optical paths have the same structure and comprise a first primary sub-half wave plate, a second primary sub-half wave plate, a primary sub-polarization beam combining prism, a first primary sub-lens, a primary sub-combining half wave plate and a second primary sub-lens, wherein the primary sub-combining half wave plate comprises two different half wave plates, and 2 primary combining Shu Pinjie sub-optical paths are respectively marked as a first primary combining Shu Pinjie sub-optical path and a second primary combining Shu Pinjie sub-optical path;
the 4 sub lasers emitted by the 4 scanning modules in the same scanning unit are respectively recorded as: a first path of sub-laser, a second path of sub-laser, a third path of sub-laser and a fourth path of sub-laser,
the first path of sub-laser and the second path of sub-laser are transmitted to the first-stage polarization beam combining prism after being combined by a first-stage combining Shu Pinjie sub-optical path, and the method specifically comprises the following steps:
The first path of sub-laser passes through the first primary sub-half wave plate and then transmits the primary sub-polarization beam combining prism, the second path of sub-laser passes through the second primary sub-half wave plate and then is reflected by the primary sub-polarization beam combining prism, and then passes through the first primary sub-lens together with the first path of sub-laser transmitted by the primary sub-polarization beam combining prism, 2 sub-scanning areas are formed on two different half wave plates of the primary sub-combining half wave plate on the back focal plane of the first primary sub-lens, and the distance of one sub-scanning area is formed between the 2 sub-scanning areas; the two sub-lasers after beam combination pass through the second primary sub-lens and then are changed into collimated lasers, and then the primary polarization beam combining prism is transmitted, wherein the polarization states of the 2 paths of lasers after passing through the primary sub-combining half-wave plate are the same;
the third sub-laser and the fourth sub-laser are reflected by the primary polarization beam combining prism after being combined by the secondary primary combining Shu Pinjie sub-optical path, and then are combined with the combined laser transmitted by the primary polarization beam combining prism; and then passing through the first primary lens together, forming 4 sub-scanning areas on four different half wave plates of a primary combined half wave plate on the back focal plane of the first primary lens, separating the 4 sub-scanning areas by a distance of one sub-scanning area, and finally passing through the second primary lens and then converting the laser back into collimated laser and outputting the collimated laser to the secondary combined beam splicing module, wherein the polarization states of the 4 paths of laser after passing through the primary combined half wave plate are the same.
Preferably, the secondary beam combining and splicing module comprises 2 paths of secondary beam combining Shu Pinjie sub-optical paths, a secondary polarization beam combining prism and a secondary lens, and laser emitted from the 2 secondary beam combining Shu Pinjie sub-optical paths passes through the secondary polarization beam combining prism to be combined, passes through the secondary lens and then is incident on the dichroic sheet;
the 2 secondary combined Shu Pinjie sub-optical paths have the same structure and comprise a secondary sub-polarization beam combining prism, a first secondary sub-lens and a second secondary sub-lens, and the 2 secondary combined Shu Pinjie sub-optical paths are respectively marked as a first secondary combined Shu Pinjie sub-optical path and a second secondary combined Shu Pinjie sub-optical path;
the 4 sub-laser beam combination lasers emitted by the 4 primary beam combination splicing modules are respectively recorded as follows: the first path of sub-beam combination laser, the second path of sub-beam combination laser, the third path of sub-beam combination laser and the fourth path of sub-beam combination laser;
the first sub-beam combining laser and the second sub-beam combining laser are transmitted through the second-level combining Shu Pinjie sub-optical path beam combining prism after being combined, and the method specifically comprises the following steps:
after the first sub-beam-combining laser transmits the secondary sub-polarization beam-combining prism, the first sub-beam-combining laser and the second sub-beam-combining laser reflected by the secondary sub-polarization beam-combining prism are combined and then pass through the first secondary sub-lens together, 8 sub-scanning areas are formed on the back focal plane of the first secondary sub-lens, the 8 sub-scanning areas are equally divided two line scanning areas formed by splicing the 4 sub-scanning areas of the first sub-beam-combining laser and the second sub-beam-combining laser, the 4 sub-scanning areas in each line of scanning areas are adjacent in sequence, and the distance of one line of scanning areas is reserved between the two line of scanning areas;
The third path of sub-beam combination laser and the fourth path of sub-beam combination laser are reflected by the secondary polarization beam combination prism after being combined by the secondary combining Shu Pinjie sub-beam path, then are combined with the combined beam laser transmitted by the secondary polarization beam combination prism, finally pass through the secondary lens together, and a complete rectangular scanning field distributed in a 4 multiplied by 4 array formed by 16 sub-scanning areas is formed on the back focal plane of the secondary lens.
Preferably, the photomultiplier tube detection array is an array of 4×4 photomultiplier tubes, and each photomultiplier tube corresponds to fluorescence detection of an imaging field of view of 1 scanning module.
The invention realizes the large view of the elliptic curved surface through the design of the reflective objective lens, and can convert the plane rectangular laser scanning field into the elliptic hemispherical laser scanning field, thereby adapting to the bending shape of the animal brain cortex.
The invention adopts a multi-path laser parallel scanning and multi-path fluorescent channel parallel detection mode to realize high flux scanning, and in the process of splicing and jointing beams of a plurality of sub-visual field areas, the sub-visual field areas of the 16 paths of 4X4 arrays are divided into 4 groups by utilizing a four-color principle, and the boundaries of the 4 sub-areas of the same group are not adjacent. The beam combination and splicing of the same group of 4 sub-areas are firstly carried out, and by utilizing the characteristic that the boundaries of the sub-areas are not adjacent, the scanning visual field can fall on half wave plates with different crystal directions, the polarization state after polarization beam combination is adjusted to be consistent, and the polarization direction is the optimal polarization direction of the next polarization beam combination. The method realizes the maximization of the energy transmittance of multi-polarization beam combination, the efficiency of each beam combination is better than 90 percent, and the laser energy is reduced by half when the beam combination passes through the depolarization beam combination prism every time in the traditional method. The beam combination and splicing among different groups adopts a traditional beam combination mode.
In each beam combination process, the relative position between the scanning sub-fields is controlled by adjusting the angle of incidence to the polarization beam splitter prism or the depolarization beam splitter prism, so that the designed post-splice position distribution effect is achieved.
The laser beams corresponding to the 4 sub-areas of the same group are at the same time point in time through the time delay module, the laser beams of different groups are different in time, the time delay between two groups adjacent in time is 3.125ns, which is equivalent to equally dividing a pulse period of 12.5ns into 4 parts, and each group of laser is positioned at one time point.
The photomultiplier detection array is an array consisting of 4×4 photomultipliers, and each array element photomultiplier corresponds to fluorescence detection of an imaging field of view of 1 scanning unit. Because of the scattering of fluorescence, when the laser scans the visual field boundary, the generated fluorescence enters the detection array element and also falls into the adjacent detection array element with a certain probability. Because the scanning lasers of adjacent areas have time delay differences, the generated fluorescence also has the same time difference, so that the source areas of the fluorescence can be distinguished in time, and signal crosstalk caused by fluorescence scattering is reduced.
In general, the invention can effectively distinguish the source areas of fluorescent signals in time and space by the thought that the areas at the same time point of the scanning areas are not adjacent and the adjacent areas are not the same, thereby avoiding signal crosstalk between the areas to a great extent.
The beneficial effects of the invention are as follows:
the elliptic curve surface large-view high-flux two-photon microscope provided by the invention realizes the elliptic curve surface large-view through the design of the reflective objective lens, and can convert a planar rectangular laser scanning field into an elliptic hemispherical laser scanning field, so that the elliptic curve surface large-view high-flux two-photon microscope can adapt to the bending shape of animal brain cortex;
the invention adopts a multi-path laser parallel scanning and multi-path fluorescence parallel detection mode to realize high flux scanning, in the process of splicing the beams in a plurality of sub-visual field areas, the non-adjacent areas are divided into a group by utilizing a four-color principle, and the energy efficiency of multi-polarization beam combination is maximized by utilizing the area gap and the combined half-wave plate; and delay is introduced between the laser groups, and the source regions of fluorescent signals can be effectively distinguished in time and space through the scheme that regions at the same time are not adjacent and adjacent regions are not identical, so that signal crosstalk between the regions caused by fluorescent scattering can be avoided to a great extent.
Drawings
Fig. 1 is a schematic diagram of the general principle structure of an elliptic hemispherical curved surface large-field high-flux two-photon microscope according to an embodiment of the present invention.
Fig. 2 is an optical path diagram of the beam splitting delay module.
Fig. 3 is a schematic diagram of time distribution of each laser on a time axis after passing through a delay light path.
Fig. 4 is a schematic diagram of the distribution of the scanned field of view after scanning and beam combination and splicing of each laser.
Fig. 5 is an optical path diagram of a primary beam combining and splicing module.
Fig. 6 is an optical path diagram of the two-stage beam combining and splicing module.
Fig. 7 is a schematic diagram of an ellipsoidal curved surface large-field-of-view reflective objective lens.
FIG. 8 is a schematic diagram of the distribution of laser-excited fluorescence of each path at a photomultiplier tube detection array.
Reference numerals illustrate:
1-near infrared femtosecond pulse laser coupling; 1.1 to 1.4-near infrared femtosecond pulse laser;
2-beam splitting delay module group; 2.1-2.4-beam splitting delay module; 2.11—a first mirror; 2.12—a second mirror; 2.13—a first spectroscopic element; 2.14—a second light splitting element; 2.15-a third spectroscopic element;
3-a scanning unit group; 3.11-3.14-scanning module;
4-a beam combining and splicing module; 4.1-4.4-first-level beam combination splicing module; 4.5-a secondary beam combining and splicing module; 4.1.01-first combining Shu Pinjie sub-optical paths; 4.1.02-a second combining Shu Pinjie sub-optical path; 4.1.1 and 4.1.7-a first level sub-half wave plate; 4.1.2, 4.1.8-second level sub-half wave plate; 4.1.3 and 4.1.9-primary sub-polarization beam combining prisms; 4.1.4, 4.1.10-first order sub-lenses; 4.1.5, 4.1.11-first order sub-combining half wave plate; 4.1.6, 4.1.12 —a second order sub-lens; 4.1.13-primary polarization beam combining prism; 4.1.14-first primary lens; 4.1.15-first order combined half wave plate; 4.1.16-second order lens;
4.5.01-first second-order combining Shu Pinjie sub-optical paths; 4.5.02-second-order Shu Pinjie sub-optical path; 4.5.1, 4.5.4-two-stage sub-polarization beam combining prism; 4.5.2, 4.5.5-first secondary lenses; 4.5.3, 4.5.6 —a second order sub-lens; 4.5.7-a secondary polarization beam combining prism; 4.5.8-baffle; 4.5.9-secondary lenses;
5-ellipsoidal curved surface large-field-of-view reflective objective lens; 5.1-a primary mirror; 5.2-secondary mirror; 5.3-three mirrors;
6-dichroic plate;
7-photomultiplier detection array;
A. b, C, D —laser; a1-first path laser; a2-a second sub-laser; a3-a third sub-laser; a4-fourth path sub-laser.
Detailed Description
The present invention is described in further detail below with reference to examples to enable those skilled in the art to practice the same by referring to the description.
It will be understood that terms, such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
Example 1
Referring to fig. 1, an elliptic hemispherical surface large field high flux two-photon microscope of the present embodiment includes: the device comprises a near infrared femtosecond pulse laser group 1, a beam splitting delay module group 2, a scanning unit group 3, a beam combining and splicing module 4, an ellipsoidal curved surface large-view-field reflective objective lens 5, a dichroic sheet 6 and a photomultiplier detection array 7;
The laser emitted by the near-infrared femtosecond pulse laser unit group 1 sequentially passes through the beam splitting delay module group 2, the scanning unit group 3 and the beam combining and splicing module 4 and then is transmitted to the dichroic sheet 6, then the laser irradiates the sample through the ellipsoidal curved surface large-view reflecting type objective 5, and fluorescence generated by excitation of the sample is collected through the ellipsoidal curved surface large-view reflecting type objective 5 and then is reflected to the photomultiplier detection array 7 by the dichroic sheet 6.
In this embodiment, the near-infrared femtosecond pulse laser set 1 includes 4 near-infrared femtosecond pulse lasers;
the beam splitting delay module group 2 comprises 4 beam splitting delay modules, and the beam splitting delay modules comprise delay light paths and beam splitting light paths;
the scanning unit group 3 comprises 4 scanning units, each scanning unit comprises 4 independent scanning modules, and each scanning module independently realizes two-dimensional scanning of 1 rectangular sub-scanning area;
each near infrared femtosecond pulse laser corresponds to 1 beam splitting delay module, and each beam splitting delay module corresponds to 1 scanning unit;
the laser emitted by the 4 near-infrared femtosecond pulse lasers passes through a delay light path to form 4 paths of delay lasers with time intervals of T/4 in sequence, each path of delay laser is equally divided into 4 paths of laser after passing through a beam splitting light path, 4 paths of laser from the same path of delay laser enter the same scanning unit, and each path of laser corresponds to 1 scanning module for realizing the scanning of 1 sub-scanning area, so that the scanning of 16 sub-scanning areas is realized through the one-to-one correspondence of 16 paths of laser and 16 scanning modules; wherein, T is the pulse period of the laser emitted by the near infrared femtosecond pulse laser; the sub-scanning areas obtained by scanning by each scanning module in the same scanning unit are positioned at the same time point, and the sub-scanning areas obtained by scanning by the scanning modules in different scanning units are positioned at different time points;
The beam combination splicing module 4 is used for realizing beam combination splicing of 16 sub lasers emitted by the 16 scanning modules, and forms a rectangular scanning field distributed in a 4×4 array by combining and splicing the 16 sub scanning areas, and enables boundaries of the 4 sub scanning areas positioned at the same time point to be non-adjacent, and any 4 areas adjacent to the boundaries to be positioned at different time points in time.
The foregoing is a general inventive concept, and is further described below, with reference to more particular embodiments and drawings.
Referring to fig. 2-8, in this embodiment, the near-infrared femtosecond pulse laser set 11 includes four near-infrared femtosecond pulse lasers 1.1, 1.2, 1.3 and 1.4, which are identical in model number, and respectively emit femtosecond laser A, B, C, D into respective beam splitting delay modules 2.1-2.4.
In this embodiment, the beam splitting delay module group 2 includes 4 beam splitting delay modules 2.1-2.4, and the beam splitting delay modules include delay light paths and beam splitting light paths, as shown in fig. 2 (only 2.1, 2.2-2.4 are the same); the delay light path comprises a first reflecting mirror 2.11 and a second reflecting mirror 2.12, laser A emitted by the near infrared femtosecond pulse laser sequentially passes through the first reflecting mirror 2.11 and the second reflecting mirror 2.12 and forms delay laser output, and the output delay laser generates different delay amounts L/c by adjusting the distance L between the first reflecting mirror 2.11 and the second reflecting mirror 2.12, wherein c is the light speed;
The beam splitting optical path comprises a first beam splitting element 2.13, a second beam splitting element 2.14 and a third beam splitting element 2.15, delay laser output by the second reflecting mirror 2.12 enters the first beam splitting element 2.13 and is equally divided into two paths, one path of the delay laser is transmitted to the first beam splitting element 2.13 and then reaches the second beam splitting element 2.14, the other path of the delay laser is equally divided into two paths by the second beam splitting element 2.14, the other path of the delay laser is reflected by the first beam splitting element 2.13 and then reaches the third beam splitting element 2.15, and the delay laser is equally divided into two paths by the third beam splitting element 2.15, and thus the delay laser is equally divided into 4 paths. In a preferred embodiment, the first 2.13, second 2.14 and third 2.15 light splitting elements are all depolarizing prisms or 50/50 light splitting sheets.
In a preferred embodiment, the repetition rate of the femtosecond laser pulses is typically 80MHz, i.e., 12.5ns period. By adjusting the respective delay amounts of the beam splitting delay modules 2.1-2.4, four groups of laser pulses A-D are separated in time, and the delay between two paths adjacent in time is 3.125ns, as shown in FIG. 3, which is equivalent to dividing the pulse period of 12.5ns into 4 parts, and each group of laser is positioned at one time point.
The laser light of each laser is equally divided into 4 paths, and thus 16 paths of laser light are divided in total. Each path of laser light further enters the parallel scanning unit group 33 and is incident on the corresponding scanning unit. The laser of A1-A4 corresponds to the scanning units 3.11-3.14, the laser of B1-B4 corresponds to the scanning units 3.21-3.24, the laser of C1-C4 corresponds to the scanning units 3.31-3.34, and the laser of D1-D4 corresponds to the scanning units 3.41-3.44.
Each scanning module 3.11-3.14 comprises a fast axis 8kHz resonance scanning mirror and a slow axis galvanometer mirror, so that two-dimensional rapid scanning of each individual subarea is realized, and the scanning view field surface of each subarea is rectangular.
The 16 sub-area scan field of view surfaces need to be combined and spliced by the beam combining and splicing module 4, and a complete scan field is formed before entering the objective lens 5, as shown in fig. 4, and is a4×4 scan field of view array. The arrangement characteristics of the subareas formed by each path of laser scanning are as follows: the boundaries of the subareas of the same group of lasers are not adjacent, such as A1 to A4 are not adjacent; the sub-regions with adjacent boundaries are derived from different groups of lasers, such as A1, B1, C1 and D1, which are adjacent and come from four groups of lasers; the following is a detailed description.
The beam combination and splicing module 4 comprises 4 primary beam combination and splicing modules 4.1-4.4 and 1 secondary beam combination and splicing module 4.5, wherein each primary beam combination and splicing module corresponds to 1 scanning unit respectively so as to combine and splice 4 sub-lasers emitted by 4 scanning modules in the 1 scanning units;
the second-stage beam combining and splicing module 4.5 is used for combining and splicing the 4 groups of lasers emitted by the 4 first-stage beam combining and splicing modules again, and then inputting the 4 groups of lasers into the dichroic sheet 6.
The 16 paths of scanning branches are subjected to beam combination and view splicing through the beam combination splicing module 4, and finally a scanning field shown in fig. 4 is formed. Four scanning branches of the same group of lasers are subjected to beam combination and splicing through a first-level beam combination and splicing module, for example, A1-A4 are subjected to beam combination and splicing through a first-level beam combination and splicing module 4.1, and B, C, D groups are subjected to beam combination and splicing through 4.2, 4.3 and 4.4 respectively.
The first-order beam-combining and splicing module 4.1 is taken as an example to describe how to perform beam-combining and splicing, and 4.2 to 4.4 are the same as the first-order beam-combining and splicing module.
Referring to fig. 5, the primary beam combining and splicing module includes 2 primary beam combining Shu Pinjie sub-optical paths 4.1.01 and 4.1.02, a primary polarization beam combining prism 4.1.13, a first primary lens 4.1.14, a primary combining half-wave plate 4.1.15 and a second primary lens 4.1.16,2 primary beam combining Shu Pinjie sub-optical path 4.10, after being combined by the primary polarization beam combining prism 4.1.13, laser beams sequentially pass through the first primary lens 4.1.14, the primary combining half-wave plate 4.1.15 and the second primary lens 4.1.16 and then are output, and the primary combining half-wave plate 4.1.15 includes 4 different half-wave plates;
the 2 first-stage combined Shu Pinjie sub-optical paths 4.1.01 and 4.1.02 have the same structure and comprise a first-stage sub-half-wave plate 4.1.1, a second-stage sub-half-wave plate 4.1.2, a first-stage sub-polarization beam combining prism 4.1.3, a first-stage sub-lens 4.1.4, a first-stage sub-combined half-wave plate 4.1.5 and a second-stage sub-lens 4.1.6, wherein the first-stage sub-combined half-wave plate 4.1.5 comprises two different half-wave plates, and the 2 first-stage combined Shu Pinjie sub-optical paths are respectively marked as a first-stage combined Shu Pinjie sub-optical path 4.1.01 and a second-stage combined Shu Pinjie sub-optical path 4.1.02;
The 4 sub lasers emitted by the 4 scanning modules 3.11-3.14 in the same scanning unit are respectively recorded as: a first path of sub-laser A1, a second path of sub-laser A2, a third path of sub-laser A3 and a fourth path of sub-laser A4,
the first path of sub-laser A1 and the second path of sub-laser A2 transmit the first-stage polarization beam combining prism 4.1.13 after being combined by the first-stage combining Shu Pinjie sub-optical path 4.1.01, specifically:
the first path of sub-laser A1 passes through a first primary sub-half-wave plate 4.1.1 and then transmits a primary sub-polarization beam combining prism 4.1.3, the second path of sub-laser A2 passes through a second primary sub-half-wave plate 4.1.2 and then is reflected by the primary sub-polarization beam combining prism 4.1.3, then is combined with the first path of sub-laser A1 which transmits the primary sub-polarization beam combining prism 4.1.3, and then passes through a first primary sub-lens 4.1.4 together, 2 sub-scanning areas are formed on two different half-wave plates of the primary sub-combining half-wave plate 4.1.5 on the back focal plane of the first primary sub-lens 4.1.4, and the distance of one sub-scanning area is formed between the 2 sub-scanning areas; the first-stage sub-half-wave plate 4.1.1 and the second-stage sub-half-wave plate 4.1.2 are adjusted to maximize the beam combination efficiency of laser, the angle of incidence of the laser to the first-stage sub-polarization beam combination prism 4.1.3 is adjusted to enable two sub-scanning areas to be at proper positions, namely the distance of one sub-scanning area is spaced between 2 sub-scanning areas, and as the scanning fields formed by A1 and A2 are not adjacent to each other, gaps exist, the scanning fields can further fall into two different half-wave plates completely, and the two half-wave plates form the first-stage sub-combination half-wave plate 4.1.5 in a gluing mode;
The two sub-lasers after beam combination are changed into collimated lasers after passing through the second first-stage sub-lens 4.1.6, and then the collimated lasers are transmitted through the first-stage polarization beam combining prism 4.1.13, wherein the polarization states of the 2 paths of lasers after passing through the first-stage sub-combining half-wave plate 4.1.5 are the same;
the polarization states of the A1 and the A2 after polarization beam combination are different and are exactly perpendicular to each other, and the polarization beam combination is carried out later, so that the polarization beam combination is carried out again, and the beam combination effect is lost. The crystal directions of the two half-wave plates glued in the first-stage sub-combination half-wave plate 4.1.5 are selected to be in different directions, so that the polarization states of the laser in the A1 and A2 areas become consistent after passing through the first-stage sub-combination half-wave plate 4.1.5, and the laser polarization states are just adjusted to the optimal polarization state required by the next polarization beam combination.
Similarly, the third sub-laser A3 and the fourth sub-laser A4 are combined by the second primary combining Shu Pinjie sub-optical path 4.1.02, then reflected by the primary polarization beam combining prism 4.1.13, so that the A3 and the A4 are combined with the combined laser A1 and the A2 transmitted through the primary polarization beam combining prism 4.1.13, and then pass through the first primary lens 4.1.14 together, 4 sub-scanning areas are formed on four different half-wave plates of the primary combining half-wave plate 4.1.15 on the back focal plane of the first primary lens 4.1.14, the 4 sub-scanning areas are separated by a distance of one sub-scanning area, and finally pass through the second primary lens 4.1.16 and then are converted into collimated laser, and the collimated laser is output to the secondary beam combining and splicing module 4.5, wherein the polarization states of the 4 laser after passing through the primary combining half-wave plate 4.1.15 are the same.
The beam combining efficiency is maximized by the first-order sub-combining half-wave plates 4.1.5 and 4.1.11, and the mutual positions of the scanning fields of view of the four scanning sub-areas on the back focal plane of the first-order lens 4.1.14 are designed proper positions by adjusting the angles of incidence to 4.1.13. The four sub-fields of view remain non-contiguous in boundary and thus fall entirely onto four different half-wave plates. The four half-wave plates form a first-order combined half-wave plate 4.1.15 by gluing. By controlling and selecting the crystal orientation of each half wave plate, the polarization states of the four sub-fields after passing through 4.1.15 are the same, and the optimal polarization state is needed for the next polarization beam combination. The laser light is then converted back to collimated laser light through a second primary lens 4.1.16.
The beam combination and the view field splicing are completed by the first-stage beam combination splicing module A1 to A4, and the beam combination mode of B, C, D groups is the same and is not described.
Referring to fig. 6, four groups of A, B, C, D lasers are combined and spliced by the secondary beam combining and splicing module 4.5.
The secondary beam combination and splicing module 4.5 comprises 2 paths of secondary beam combination Shu Pinjie sub-light paths 4.5.01 and 4.5.02, a secondary polarization beam combination prism 4.5.7 and a secondary lens 4.5.9,2, wherein laser emitted from the secondary beam combination Shu Pinjie sub-light path 4.5.0 is combined by the secondary polarization beam combination prism 4.5.7, passes through the secondary lens 4.5.9 and then is incident on the dichroic sheet 6;
The 2 secondary combined Shu Pinjie sub-optical paths 4.5.01 and 4.5.02 have the same structure and comprise a secondary sub-polarization beam combining prism 4.5.1, a first secondary sub-lens 4.5.2 and a second secondary sub-lens 4.5.3,2, and the two secondary combined Shu Pinjie sub-optical paths 4.5.0 are respectively marked as a first secondary combined Shu Pinjie sub-optical path 4.5.01 and a second secondary combined Shu Pinjie sub-optical path 4.5.02;
the 4 sub-laser beam combination lasers emitted by the 4 first-level beam combination splicing modules 4.1-4.4 are respectively recorded as follows: the first path of sub-beam combination laser A, the second path of sub-beam combination laser B, the third path of sub-beam combination laser C and the fourth path of sub-beam combination laser D;
the first sub-beam combining laser a and the second sub-beam combining laser B transmit the second polarized beam combining prism 4.5.7 after being combined by the first secondary beam combining Shu Pinjie sub-optical path 4.5.01, specifically:
after the first sub-beam combination laser A transmits the second sub-polarization beam combination prism 4.5.1, the first sub-beam combination laser A and the second sub-beam combination laser B reflected by the second sub-polarization beam combination prism 4.5.1 are combined and then pass through the first sub-lens 4.5.2 together, 8 sub-scanning areas are formed on the back focal plane of the first sub-lens 4.5.2, the 8 sub-scanning areas are equally divided two line scanning areas formed by splicing the 4 sub-scanning areas of the first sub-beam combination laser A and the second sub-beam combination laser B, the 4 sub-scanning areas in each line scanning area are adjacent in sequence, and the distance of one line scanning area is formed between the two line scanning areas;
The third sub-beam combining laser C and the fourth sub-beam combining laser D are reflected by the second-stage polarization beam combining prism 4.5.7 after being combined by the second-stage combining Shu Pinjie sub-optical path 4.5.02, and then combined with the combined laser transmitted by the second-stage polarization beam combining prism 4.5.7, and finally pass through the second-stage lens 4.5.9 together, and a complete rectangular scan field distributed in a 4×4 array formed by 16 sub-scan areas is formed on the back focal plane of the second-stage lens 4.5.9, as shown in fig. 4.
The group A and the group B laser are combined through the secondary polarization beam combining prism 4.5.7, and the polarization states of the group A and the group B laser are adjusted to be optimal through the combined half wave plates in the respective primary beam combining and splicing modules, so that the beam combining efficiency is ensured. The angle of incidence to the secondary polarizing beam-combining prism 4.5.7 is adjusted so that each of the two sets of 8 sub-areas formed on the back focal plane of the first secondary sub-lens 4.5.2 is in the proper position for the design. And the same applies to the beam combination and splicing of the lasers in the group C and the group D.
With continued reference to fig. 6, in a preferred embodiment, the secondary beam combining splice module 4.5 further includes a baffle 4.5.8, with the baffle 4.5.8 blocking half of the laser light lost by the depolarized beam combining. In a further preferred embodiment, the baffle 4.5.8 can also be replaced by a camera for monitoring the current splicing situation of the fields of view in real time, and if the position of the field of view of a certain subarea deviates, maintenance personnel can find and adjust in time.
In a preferred embodiment, the secondary polarizing beam combining prism 4.5.7 can also be replaced with a 50/50 split flat plate.
The dichroic plate 6 is highly efficient in transmitting the laser band, while the visible fluorescence band will be reflective, with high reflectivity. The laser beam after the scanning view beam combination passes through the dichroic plate 6 and then reaches the reflective objective lens, an excitation scanning field is formed in the sample, and the excited fluorescence in the sample reaches the dichroic plate 6 through the reflective objective lens and is reflected to the photomultiplier tube detection array for photoelectric signal conversion.
Referring to fig. 7, a planar scan field (fig. 4) obtained by splicing a plurality of scan sub-areas is converted into a curved scan field of an elliptical hemispherical surface by an ellipsoidal curved surface large-field reflective objective lens 55.
In this embodiment, the ellipsoidal curved surface large-view field reflective objective lens 5 includes a primary lens 5.1, a secondary lens 5.2 and a tertiary lens 5.3 sequentially arranged along the optical path, the optical surface of the primary lens 5.1 is an eight-order hyperboloid, the optical surface of the secondary lens 5.2 is a secondary flat elliptical surface, the optical surface of the tertiary lens 5.3 is a six-order flat elliptical surface, and the laser entering the ellipsoidal curved surface large-view field reflective objective lens 5 is sequentially reflected by the primary lens 5.1, the secondary lens 5.2 and the tertiary lens 5.3 and then irradiates the sample. The numerical aperture range of the ellipsoidal curved surface large-field reflection type objective lens 5 is 0.3-0.5. The imaging view field of the reflective objective lens is an ellipsoidal curved surface, the radius of curvature of a long axis is 9-12 mm, the radius of curvature of a short axis is 6-9 mm, and the plane projection size of the view field is 6mm multiplied by 6mm.
The 16 scanning laser beams excite the sample to generate corresponding fluorescence. AA1 to AA4 are fluorescence excited by A1 to A4 lasers respectively, BB1 to BB4 are fluorescence excited by B1 to B4 lasers respectively, CC1 to CC4 are fluorescence excited by C1 to C4 lasers respectively, and DD1 to DD4 are fluorescence excited by D1 to D4 lasers respectively.
The photomultiplier detection array 7 is an array of 4×4 photomultipliers, and each array element photomultiplier corresponds to fluorescence detection of an imaging field of view of 1 scanning unit, as shown in fig. 8. The fluorescence from the curved field of view is converted by the scan field of the reflecting objective 5 and then returned to the planar matrix distribution at the detector array 7.
Because of the scattering of fluorescence, when the laser scans the visual field boundary, the generated fluorescence enters the detection array element and also falls into the adjacent detection array element with a certain probability. Because the scanning lasers of adjacent areas have time delay differences, the generated fluorescence also has the same time difference, so that the source areas of the fluorescence can be distinguished in time, and signal crosstalk caused by fluorescence scattering is reduced.
In general, the invention can effectively distinguish the source areas of fluorescent signals in time and space by the thought that the areas at the same time are not adjacent and the adjacent areas are not identical, thereby greatly avoiding signal crosstalk between the areas.
The embodiment of the invention describes a scanning and detecting method of a 16-path 4 x 4 array, and according to the four-color principle, for any area distributed on a plane, the areas are always divided into four types and the areas of the same type are not adjacent, so that the energy transmittance of the combined beam is improved by a unique beam combining and splicing mode provided by the invention, and the fluorescence signal crosstalk is reduced by a time-space division method provided by the invention, so that the embodiment of the invention can be popularized to any number of parallel scanning and detecting.
Although embodiments of the present invention have been disclosed above, it is not limited to the use of the description and embodiments, it is well suited to various fields of use for the invention, and further modifications may be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the particular details without departing from the general concepts defined in the claims and the equivalents thereof.
Claims (7)
1. An elliptical hemispherical surface large field high flux two-photon microscope, comprising: the system comprises a near infrared femtosecond pulse laser coupling group, a beam splitting delay module coupling group, a scanning unit coupling group, a beam combining and splicing module, an ellipsoidal curved surface large-view-field reflective objective lens, a dichroic sheet and a photomultiplier detection array;
The laser emitted by the near infrared femtosecond pulse laser coupling group sequentially passes through the beam splitting delay module coupling group, the scanning unit coupling group and the beam combining and splicing module and then transmits the dichroic sheet, then the fluorescence generated by excitation of the sample is collected by the ellipsoidal curved surface large-field reflective objective and then reflected to the photomultiplier detection array by the dichroic plate;
the ellipsoidal curved surface large-field reflective objective lens comprises a main lens, a secondary lens and a triple lens which are sequentially arranged along a light path, wherein the optical surface of the main lens is a hyperboloid, the optical surface of the secondary lens is a flat elliptic surface, the optical surface of the triple lens is a flat elliptic surface, and laser entering the ellipsoidal curved surface large-field reflective objective lens is sequentially reflected by the main lens, the secondary lens and the triple lens and then irradiates on a sample;
the near infrared femtosecond pulse laser group comprises 4 near infrared femtosecond pulse lasers;
the beam splitting delay module group comprises 4 beam splitting delay modules, and the beam splitting delay modules comprise delay light paths and beam splitting light paths;
the scanning unit group comprises 4 scanning units, each scanning unit comprises 4 independent scanning modules, and each scanning module independently realizes two-dimensional scanning of 1 rectangular sub-scanning area;
Each near infrared femtosecond pulse laser corresponds to 1 beam splitting delay module, and each beam splitting delay module corresponds to 1 scanning unit;
4 paths of delay lasers with time intervals of T/4 are formed after the lasers emitted by the near infrared femtosecond pulse lasers pass through the delay light path, each path of delay lasers passes through the beam splitting light path and then is equally divided into 4 paths of sub lasers, 4 paths of sub lasers from the same path of delay lasers enter the same scanning unit, and each path of sub lasers corresponds to 1 scanning module and is used for realizing the scanning of 1 sub scanning area, so that the scanning of 16 sub scanning areas is realized through the one-to-one correspondence of 16 sub lasers and 16 scanning modules; wherein, T is the pulse period of the laser emitted by the near infrared femtosecond pulse laser; the sub-scanning areas obtained by scanning by each scanning module in the same scanning unit are positioned at the same time point, and the sub-scanning areas obtained by scanning by the scanning modules in different scanning units are positioned at different time points;
the beam combination splicing module is used for realizing beam combination splicing of 16 sub lasers emitted by the 16 scanning modules, and forming a rectangular scanning field distributed in a 4 x 4 array by combining and splicing the 16 sub scanning areas, and enabling the boundaries of the 4 sub scanning areas positioned at the same time point to be non-adjacent, wherein any 4 areas adjacent to the boundaries are positioned at different time points in time;
The optical surface of the main mirror is an eighth-order hyperboloid, the optical surface of the secondary mirror is a secondary flat elliptic surface, and the optical surface of the three mirrors is a sixth-order flat elliptic surface;
the scanning module comprises a fast axis resonance scanning mirror and a slow axis galvanometer mirror.
2. The elliptic hemispherical surface large-field high-flux two-photon microscope according to claim 1, wherein the delay light path comprises a first reflecting mirror and a second reflecting mirror, laser emitted by the near infrared femtosecond pulse laser sequentially passes through the first reflecting mirror and the second reflecting mirror to form delay laser output, and the distance between the first reflecting mirror and the second reflecting mirror is adjusted to enable the output delay laser to generate different delay amounts;
the beam splitting light path comprises a first beam splitting element, a second beam splitting element and a third beam splitting element, delay laser output by the second reflector enters the first beam splitting element and is equally divided into two paths, one path of delay laser is transmitted to the first beam splitting element and then reaches the second beam splitting element and is equally divided into two paths by the second beam splitting element, and the other path of delay laser is reflected by the first beam splitting element and then reaches the third beam splitting element and is equally divided into two paths by the third beam splitting element, so that the delay laser is equally divided into 4 paths.
3. The elliptical curved surface large field of view high throughput two-photon microscope of claim 2, wherein the first, second and third light splitting elements are all depolarizing prisms or 50/50 light splitting sheets.
4. The elliptic hemispherical surface large-field high-flux two-photon microscope according to claim 3, wherein the beam combination and splicing module comprises 4 primary beam combination and splicing modules and 1 secondary beam combination and splicing module, each primary beam combination and splicing module corresponds to 1 scanning unit respectively, so as to combine and splice 4 sub lasers emitted by 4 scanning modules in 1 scanning unit;
the secondary beam combining and splicing module is used for combining and splicing 4 groups of lasers emitted by the 4 primary beam combining and splicing modules again and then inputting the 4 groups of lasers into the dichroic sheet.
5. The elliptic hemispherical surface large-field high-flux two-photon microscope according to claim 4, wherein the primary beam combining and splicing module comprises 2 primary combined Shu Pinjie sub-optical paths, a primary polarization beam combining prism, a first primary lens, a primary combined half-wave plate and a second primary lens, and laser emitted from the 2 primary combined Shu Pinjie sub-optical paths sequentially passes through the first primary lens, the primary combined half-wave plate and the second primary lens after being combined by the primary polarization beam combining prism and then is output, and the primary combined half-wave plate comprises 4 different half-wave plates;
The 2 primary combining Shu Pinjie sub-optical paths have the same structure and comprise a first primary sub-half wave plate, a second primary sub-half wave plate, a primary sub-polarization beam combining prism, a first primary sub-lens, a primary sub-combining half wave plate and a second primary sub-lens, wherein the primary sub-combining half wave plate comprises two different half wave plates, and 2 primary combining Shu Pinjie sub-optical paths are respectively marked as a first primary combining Shu Pinjie sub-optical path and a second primary combining Shu Pinjie sub-optical path;
the 4 sub lasers emitted by the 4 scanning modules in the same scanning unit are respectively recorded as: a first path of sub-laser, a second path of sub-laser, a third path of sub-laser and a fourth path of sub-laser,
the first path of sub-laser and the second path of sub-laser are transmitted to the first-stage polarization beam combining prism after being combined by a first-stage combining Shu Pinjie sub-optical path, and the method specifically comprises the following steps:
the first path of sub-laser passes through the first primary sub-half wave plate and then transmits the primary sub-polarization beam combining prism, the second path of sub-laser passes through the second primary sub-half wave plate and then is reflected by the primary sub-polarization beam combining prism, and then passes through the first primary sub-lens together with the first path of sub-laser transmitted by the primary sub-polarization beam combining prism, 2 sub-scanning areas are formed on two different half wave plates of the primary sub-combining half wave plate on the back focal plane of the first primary sub-lens, and the distance of one sub-scanning area is formed between the 2 sub-scanning areas; the two sub-lasers after beam combination pass through the second primary sub-lens and then are changed into collimated lasers, and then the primary polarization beam combining prism is transmitted, wherein the polarization states of the 2 paths of lasers after passing through the primary sub-combining half-wave plate are the same;
The third sub-laser and the fourth sub-laser are reflected by the primary polarization beam combining prism after being combined by the secondary primary combining Shu Pinjie sub-optical path, and then are combined with the combined laser transmitted by the primary polarization beam combining prism; and then passing through the first primary lens together, forming 4 sub-scanning areas on four different half wave plates of a primary combined half wave plate on the back focal plane of the first primary lens, separating the 4 sub-scanning areas by a distance of one sub-scanning area, and finally passing through the second primary lens and then converting the laser back into collimated laser and outputting the collimated laser to the secondary combined beam splicing module, wherein the polarization states of the 4 paths of laser after passing through the primary combined half wave plate are the same.
6. The elliptic hemispherical surface large-field high-flux two-photon microscope according to claim 5, wherein the secondary beam combination and splicing module comprises 2 secondary beam combination Shu Pinjie sub-optical paths, a secondary polarization beam combination prism and a secondary lens, and laser emitted from 2 secondary beam combination Shu Pinjie sub-optical paths passes through the secondary lens after being combined by the secondary polarization beam combination prism and then is incident on the dichroic plate;
the 2 secondary combined Shu Pinjie sub-optical paths have the same structure and comprise a secondary sub-polarization beam combining prism, a first secondary sub-lens and a second secondary sub-lens, and the 2 secondary combined Shu Pinjie sub-optical paths are respectively marked as a first secondary combined Shu Pinjie sub-optical path and a second secondary combined Shu Pinjie sub-optical path;
The 4 sub-laser beam combination lasers emitted by the 4 primary beam combination splicing modules are respectively recorded as follows: the first path of sub-beam combination laser, the second path of sub-beam combination laser, the third path of sub-beam combination laser and the fourth path of sub-beam combination laser;
the first sub-beam combining laser and the second sub-beam combining laser are transmitted through the second-level combining Shu Pinjie sub-optical path beam combining prism after being combined, and the method specifically comprises the following steps:
after the first sub-beam-combining laser transmits the secondary sub-polarization beam-combining prism, the first sub-beam-combining laser and the second sub-beam-combining laser reflected by the secondary sub-polarization beam-combining prism are combined and then pass through the first secondary sub-lens together, 8 sub-scanning areas are formed on the back focal plane of the first secondary sub-lens, the 8 sub-scanning areas are equally divided two line scanning areas formed by splicing the 4 sub-scanning areas of the first sub-beam-combining laser and the second sub-beam-combining laser, the 4 sub-scanning areas in each line of scanning areas are adjacent in sequence, and the distance of one line of scanning areas is reserved between the two line of scanning areas;
the third path of sub-beam combination laser and the fourth path of sub-beam combination laser are reflected by the secondary polarization beam combination prism after being combined by the secondary combining Shu Pinjie sub-beam path, then are combined with the combined beam laser transmitted by the secondary polarization beam combination prism, finally pass through the secondary lens together, and a complete rectangular scanning field distributed in a 4 multiplied by 4 array formed by 16 sub-scanning areas is formed on the back focal plane of the secondary lens.
7. The ellipsometric large-field high-flux two-photon microscope of claim 6, wherein said photomultiplier tube detection array is an array of 4 x 4 photomultiplier tubes, each corresponding to fluorescence detection of the imaging field of view of 1 scanning module.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| PCT/CN2021/133173 WO2023010718A1 (en) | 2021-08-05 | 2021-11-25 | Elliptical hemispherical curved surface large-field-of-view high-throughput two-photon microscope |
| DE112021008078.4T DE112021008078T5 (en) | 2021-08-05 | 2021-11-25 | Ellipsoidal hemispherical two-photon microscope with large field of view and high throughput |
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| WO2023010718A1 (en) | 2023-02-09 |
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