CN215986702U - Large-visual-field high-flux two-photon microscope with elliptic hemispherical curved surface - Google Patents
Large-visual-field high-flux two-photon microscope with elliptic hemispherical curved surface Download PDFInfo
- Publication number
- CN215986702U CN215986702U CN202121819956.6U CN202121819956U CN215986702U CN 215986702 U CN215986702 U CN 215986702U CN 202121819956 U CN202121819956 U CN 202121819956U CN 215986702 U CN215986702 U CN 215986702U
- Authority
- CN
- China
- Prior art keywords
- sub
- laser
- scanning
- path
- splicing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Microscoopes, Condenser (AREA)
Abstract
The utility model discloses a large-visual-field high-flux two-photon microscope with an elliptic hemispherical curved surface, which comprises: the device comprises a near-infrared femtosecond pulse laser unit group, a beam splitting delay module group, a scanning unit group, a beam combining and splicing module, an ellipsoid curved surface large-visual-field reflective objective lens, a dichroic sheet and a photomultiplier detection array. The utility model realizes the large view field of the elliptic curved surface by 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 a multi-path laser parallel scanning and multi-path fluorescence parallel detection mode, non-adjacent areas are divided into a group by utilizing a four-color principle, and the energy efficiency of multi-time polarization beam combination is maximized by utilizing an area gap and a combined half-wave plate; time delay is introduced between the lasers in different groups, and through the scheme that the regions at the same time point are not adjacent and the adjacent regions are not synchronous, the source regions of the fluorescence signals can be effectively distinguished, and signal crosstalk caused by fluorescence scattering is greatly avoided.
Description
Technical Field
The utility model relates to the field of microscopic imaging instruments, in particular to an elliptic hemispherical curved surface large-field high-flux two-photon microscope.
Background
The two-photon microscope is well applied to the nerve tissue functional structure imaging of animal cerebral cortex at present because of the deep biological tissue imaging depth and high spatial resolution, and promotes the development of neuroscience (brain science).
Scientists desire to simultaneously observe and record the functional signals of a wider range of more neurons, and new demands are made on the two-photon imaging technology. Firstly, the resolution of a neuron needs to be achieved, the optical resolution is required to be 1-2 microns, and the corresponding numerical aperture is 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 in a single time is larger.
The maximum visual field diameter of the current international two-photon imaging is about 5mm, but the imaging speed of the full visual field is less than 1 frame per second, and the requirement of functional signal detection cannot be met. Under the conditions of real-time imaging and single cell resolution, the visual field area of two-photon imaging is only 1mm multiplied by 1mm, and only a single brain functional area is covered for a mouse. The mouse has a total cerebral cortex area of about 200mm2, and the surface of the brain is uneven and close to an elliptical hemisphere, which is a great challenge for optical microscopy imaging.
The method is characterized in that the animal can realize the large imaging visual field area and simultaneously make the visual field bent to match with the curvature of the cerebral cortex, so that the difference of the visual field design concept of the animal in the single neuron resolution dynamic large visual field imaging of the body cerebral cortex is huge compared with the visual field design concept of the conventional flat field objective; secondly, the large-field high-resolution image requires a great number of pixels and ensures the time resolution, so that the required imaging data flux is huge, and the laser scanning and fluorescence detection fluxes need to be improved by orders of magnitude.
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 independently scanned respectively, and fluorescence signals of all the areas are detected by combining a multi-channel detector or an area array detector, so that the imaging flux can be obviously improved. However, the current parallel scanning scheme has large laser energy loss when the field of view is spliced by combining beams for multiple times, and the laser energy is reduced by half every time the beam passes through the depolarization beam combining prism; the multi-channel parallel detection has signal crosstalk caused by fluorescence scattering, specifically, the fluorescence scattering area is large, and fluorescence falls in several adjacent areas at the same time, so that the signal crosstalk among channels is caused.
Therefore, a more reliable solution is now needed.
SUMMERY OF THE UTILITY MODEL
The utility model aims to solve the technical problem of providing an elliptic hemispherical curved surface large-visual-field high-flux two-photon microscope aiming at the defects in the prior art.
In order to solve the technical problems, the utility model adopts the technical scheme that: an elliptic hemispherical curved surface large-field high-flux two-photon microscope, comprising: the system comprises a near-infrared femtosecond pulse laser unit group, a beam splitting delay module group, a scanning unit group, a beam combining and splicing module, an ellipsoidal curved surface large-visual-field reflective objective lens, a dichroic sheet and a photomultiplier detection array;
laser emitted by the near-infrared femtosecond pulse laser unit is transmitted through the dichroic sheet after sequentially passing through the beam splitting delay module unit, the scanning unit and the beam combination splicing module, and then is irradiated onto a sample through the ellipsoidal curved surface large-visual-field reflective objective lens, and fluorescence generated by exciting the sample is reflected to the photomultiplier detection array through the dichroic sheet after being collected through the ellipsoidal curved surface large-visual-field reflective objective lens;
the ellipsoidal curved surface large-visual-field reflective objective lens comprises a primary mirror, a secondary mirror and a tertiary mirror which are sequentially arranged along a light path, wherein the optical surface of the primary mirror is a hyperboloid, the optical surface of the secondary mirror is a flat ellipsoid, the optical surfaces of the tertiary mirrors are flat ellipsoids, and laser entering the ellipsoidal curved surface large-visual-field reflective objective lens is sequentially reflected by the primary mirror, the secondary mirror and the tertiary mirror and then irradiates a sample;
the near-infrared femtosecond pulse laser unit group comprises 4 near-infrared femtosecond pulse laser units;
the beam splitting delay module group comprises 4 beam splitting delay modules, and each beam splitting delay module comprises a delay light path and a beam splitting light path;
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 lasers emitted by the 4 near-infrared femtosecond pulse lasers form 4 paths of delay lasers with time intervals of T/4 in sequence after passing through the delay light path, each path of delay laser is equally divided into 4 paths of sub-lasers after passing through the beam splitting light path, the 4 paths of sub-lasers from the same path of delay laser enter the same scanning unit, each path of sub-laser corresponds to 1 scanning module and is used for realizing the scanning of 1 sub-scanning area, and therefore the 16 paths of sub-lasers correspond to the 16 scanning modules one by one to realize the scanning of 16 sub-scanning areas; in a further preferred embodiment, T is 12.5ns, and the time delay between two adjacent groups is 3.125ns apart; the sub-scanning areas obtained by scanning of each scanning module in the same scanning unit are located at the same time point, and the sub-scanning areas obtained by scanning of the scanning modules in different scanning units are located at different time points;
the beam combination splicing module is used for realizing beam combination splicing of 16 paths of sub-lasers emitted by the 16 scanning modules, 16 sub-scanning areas are combined and spliced to form a rectangular scanning field distributed in a 4x4 array, boundaries of the 4 sub-scanning areas located at the same time point are not adjacent, and any 4 areas adjacent to the boundaries are located at different time points in time.
Preferably, the optical surface of the primary mirror is an eight-order hyperboloid, the optical surface of the secondary mirror is a secondary flat ellipsoid, and the optical surface of the three mirrors is a six-order flat ellipsoid.
More preferably, the numerical aperture range of the ellipsoidal curved surface large-field reflection type objective lens is 0.3-0.5. The imaging visual field of the reflective objective lens is an ellipsoid curved surface, the curvature radius of a long axis is 9-12 mm, the curvature radius of a short axis is 6-9 mm, and the plane projection size of the visual field is 6mm multiplied by 6 mm.
Preferably, the delay optical path comprises a first reflecting mirror and a second reflecting mirror, the laser emitted by the near-infrared femtosecond pulse laser is reflected by the first reflecting mirror and the second reflecting mirror in sequence 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 includes first beam splitting component, second beam splitting component and third beam splitting component, the time delay laser of second mirror output advances equally divide into two the tunnel behind the first beam splitting component, transmits all the way reach behind the first beam splitting component second beam splitting component, and quilt the second beam splitting component equally divides into two the tunnel, another way quilt arrive after the reflection of first beam splitting component third beam splitting component, and quilt the third beam splitting component equally divides into two the tunnel to equally divide time delay laser into 4 ways.
Preferably, the first light splitting element, the second light splitting element and the third light splitting element are all depolarizing beam splitters or 50/50 beam splitters.
Preferably, the scanning module comprises a fast axis resonant scanning mirror and a slow axis galvanometer mirror.
Preferably, the beam combining and splicing module includes 4 first-stage beam combining and splicing modules and 1 second-stage beam combining and splicing module, each first-stage beam combining and splicing module corresponds to 1 scanning unit respectively, so as to combine and splice 4 paths of sub-lasers emitted by 4 scanning modules in 1 scanning unit;
the secondary beam combination splicing module is used for carrying out beam combination splicing on 4 groups of laser emitted by the 4 primary beam combination splicing modules again and then inputting the laser into the dichroic film.
Preferably, the first-stage beam combining and splicing module includes 2 first-stage beam combining and splicing sub-optical paths, a first-stage polarization beam combining prism, a first primary lens, a first-stage combined half-wave plate and a second first-stage lens, laser light emitted from the 2 first-stage beam combining and splicing sub-optical paths passes through the first-stage polarization beam combining prism and then sequentially passes through the first primary lens, the first-stage combined half-wave plate and the second first-stage lens and is output, and the first-stage combined half-wave plate includes 4 different half-wave plates;
the 2 first-stage beam combination splicing sub-optical paths have the same structure and respectively comprise a first-stage sub-half-wave plate, a second first-stage sub-half-wave plate, a first-stage polarization beam combination prism, a first primary sub-lens, a first-stage sub-combined half-wave plate and a second first-stage sub-lens, wherein the first-stage sub-combined half-wave plate comprises two different half-wave plates, and the 2 first-stage beam combination splicing sub-optical paths are respectively marked as a first-stage beam combination splicing sub-optical path and a second-stage beam combination splicing sub-optical path;
the 4 paths of sub-lasers emitted by 4 scanning modules in the same scanning unit are respectively marked 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 transmit the first-order polarization beam combining prism after being combined through the first-order beam combining and splicing 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 first-stage 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 first-stage sub polarization beam combining prism, then is combined with the first path of sub laser which transmits the first-stage sub polarization beam combining prism, and then passes through the first primary sub lens together, 2 sub scanning areas are formed on two different half-wave plates of the first-stage sub combined half-wave plate on the back focal plane of the first primary sub lens, and the 2 sub scanning areas are separated by the distance of one sub scanning area; the combined two paths of sub-lasers pass through the second primary sub-lens and then are converted into collimated lasers, and then the collimated lasers transmit the primary polarization beam combining prism, wherein the polarization states of the 2 paths of lasers passing through the primary sub-combined half-wave plate are the same;
the third path of sub laser and the fourth path of sub laser are reflected by the first-stage polarization beam combining prism after being combined by the second first-stage beam combining and splicing sub optical path, and then are combined with the combined beam laser which transmits the first-stage polarization beam combining prism; and then the light beams pass through the first primary lens together, 4 sub-scanning areas are formed on four different half-wave plates of the first-stage combined half-wave plate on the back focal plane of the first primary lens, the 4 sub-scanning areas are separated by the distance of one sub-scanning area, and finally the light beams pass through the second primary lens and are converted back into collimated laser beams to be output to the second-stage beam combining and splicing module, wherein the polarization states of the 4 paths of laser beams passing through the first-stage combined half-wave plate are the same.
Preferably, the secondary beam combining and splicing module comprises 2 paths of secondary beam combining and splicing sub-optical paths, a secondary polarization beam combining prism and a secondary lens, and laser emitted from the 2 secondary beam combining and splicing sub-optical paths passes through the secondary lens after being combined by the secondary polarization beam combining prism and then is incident on the dichroic sheet;
the 2 secondary beam combining and splicing sub-optical paths have the same structure and respectively comprise a secondary polarization beam combining prism, a first secondary sub-lens and a second secondary sub-lens, and the 2 secondary beam combining and splicing sub-optical paths are respectively marked as a first secondary beam combining and splicing sub-optical path and a second secondary beam combining and splicing sub-optical path;
the combined beam laser of 4 paths of sub-laser emitted by the 4 first-stage combined beam splicing modules is respectively recorded as: 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 path of sub-beam combination laser and the second path of sub-beam combination laser are transmitted through the second-level polarization beam combination prism after being combined by the first second-level beam combination splicing sub-optical path, and the method specifically comprises the following steps:
after transmitting the secondary sub-polarization beam combining prism, a first path of sub-combined beam laser is combined with a second path of sub-combined beam laser reflected by the secondary sub-polarization beam combining prism, then the first path of sub-combined beam laser and the second path of sub-combined beam laser 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 two equally-divided scanning areas formed by splicing 4 sub-scanning areas of the first path of sub-combined beam laser and the second path of sub-combined beam laser, the 4 sub-scanning areas in each scanning area are adjacent in sequence, and the two scanning areas are separated by the distance of one scanning area;
and the third path of sub-beam-combining laser and the fourth path of sub-beam-combining laser are reflected by the secondary polarization beam-combining prism after being combined by the second secondary beam-combining splicing sub-optical path, then are combined with the combined beam laser which transmits the secondary polarization beam-combining prism, and finally pass through the secondary lens together, so that a complete rectangular scanning field which is formed by 16 sub-scanning areas and distributed in a 4x4 array is formed on the back focal plane of the secondary lens.
Preferably, the photomultiplier tube detection array is an array consisting of 4 × 4 photomultiplier tubes, each corresponding to the fluorescence detection of the imaging field of view of 1 scanning module.
The utility model realizes the large view field of the elliptic curved surface by 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 hemisphere, thereby being adapted to the curved shape of the animal cerebral cortex.
The utility model can realize high flux scanning by adopting a multi-path laser parallel scanning and multi-path fluorescence channel parallel detection mode, and divides the sub-visual field areas of the 16-path 4X4 array into 4 groups by utilizing a four-color principle in the process of splicing and combining beams of a plurality of sub-visual field areas, and the boundaries of the 4 sub-areas in the same group are not adjacent. The beam combination splicing of 4 subregions in the same group is firstly carried out, the scanning visual field can fall on half-wave plates with different crystal directions by utilizing the characteristic that the boundaries of the subregions are not adjacent, 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 the multi-time polarization beam combination, the efficiency of each beam combination is better than 90 percent, and the laser energy is reduced by half by passing through a depolarization beam combination prism in the traditional method. The beam combination splicing among different groups adopts a traditional beam combination mode.
In each beam combination process, the relative position between the scanning sub-fields of view is controlled by adjusting the angle of incidence to the polarization beam splitter prism or the depolarization beam splitter prism, so that the designed position distribution effect after splicing is achieved.
Through the delay module, the laser beams corresponding to the 4 sub-regions in the same group are at the same time point in time, the laser beams in different groups have difference in time, the delay between two groups adjacent in time is 3.125ns apart, which is equivalent to equally dividing the pulse period of 12.5ns into 4 parts, and each group of laser is at one time point.
The photomultiplier detection array is an array consisting of 4 multiplied by 4 photomultiplier tubes, and each array element photomultiplier tube corresponds to the fluorescence detection of the imaging field of view of 1 scanning unit. Due to the fact that the fluorescence is scattered, 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. Due to the fact that the scanning laser of adjacent regions has delay difference, the generated fluorescence has the same time difference, the source regions of the fluorescence can be distinguished in time, and therefore signal crosstalk caused by fluorescence scattering is reduced.
In general, the utility model can effectively distinguish the source areas of the fluorescence signals in time and space by the thought that the areas of the same time point of the scanning area are not adjacent and the adjacent areas are not same, thereby greatly avoiding the signal crosstalk between the areas.
The utility model has the beneficial effects that:
the large-view-field high-flux two-photon microscope with the elliptic hemispherical curved surface provided by the utility model realizes the large view field of the elliptic curved surface through the design of the reflective objective lens, and can convert a planar rectangular laser scanning field into a laser scanning field with an elliptic hemispherical surface, so that the two-photon microscope can be adapted to the curved shape of an animal cerebral cortex;
the utility model adopts a multi-path laser parallel scanning and multi-path fluorescence parallel detection mode to realize high-flux scanning, in the process of splicing and combining beams of a plurality of sub-visual field areas, non-adjacent areas are divided into a group by utilizing a four-color principle, and the energy efficiency of multiple polarization combining beams is maximized by utilizing area gaps and a combined half-wave plate; time delay is introduced between the lasers, and the source regions of the fluorescence signals can be effectively distinguished in time and space through the scheme that the regions at the same time point are not adjacent and the adjacent regions are not synchronous, so that signal crosstalk between the regions caused by fluorescence scattering can be greatly avoided.
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 in an embodiment of the utility model.
Fig. 2 is a light path diagram of the beam splitting delay module.
Fig. 3 is a schematic diagram of time distribution of each laser beam on a time axis after passing through a delay optical path.
Fig. 4 is a schematic view of the distribution of the scanning field of view of each laser after scanning and beam combination splicing.
Fig. 5 is an optical path diagram of the first-stage 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 at the photomultiplier tube detection array.
Description of reference numerals:
1-near infrared femtosecond pulse laser combination; 1.1-1.4-near infrared femtosecond pulse laser;
2, combining beam splitting delay modules; 2.1-2.4-beam splitting delay module; 2.11 — first mirror; 2.12 — second mirror; 2.13 — first light splitting element; 2.14 — second light splitting element; 2.15 — third light splitting element;
3, scanning unit grouping; 3.11-3.14-scanning module;
4, combining and splicing the modules; 4.1-4.4-first-stage beam combination splicing module; 4.5-a second-stage beam combination splicing module; 4.1.01-first stage beam combining and splicing sub-optical path 4.1.01; 4.1.02-second-stage beam-combining splicing sub-optical path; 4.1.1, 4.1.7 — first order sub-half-wave plate; 4.1.2, 4.1.8-the second first-order sub-half-wave plate; 4.1.3, 4.1.9-first order sub-polarization beam-combining prism; 4.1.4, 4.1.10 — first order sub-lens; 4.1.5, 4.1.11-first order sub-merged half-wave plate; 4.1.6, 4.1.12 — second order sub-lens; 4.1.13-first order polarization beam-combining prism; 4.1.14 — first primary lens; 4.1.15-first order merged half-wave plate; 4.1.16 — second stage lens;
4.5.01-first two-stage beam combining and splicing sub-optical path 4.5.01; 4.5.02-second two-stage beam combining and splicing sub-optical path 4.5.02; 4.5.1, 4.5.4 — secondary sub-polarization beam-combining prism; 4.5.2, 4.5.5 — first order sub-lens; 4.5.3, 4.5.6 — second order sub-lens; 4.5.7-secondary polarization beam-combining prism; baffle-4.5.8; 4.5.9-secondary lens;
5-ellipsoidal curved surface large-field-of-view reflective objective lens; 5.1 — primary mirror; 5.2-secondary mirror; 5.3-three mirrors;
6-dichroic sheet;
7-photomultiplier detection array;
A. b, C, D-laser; a1-first path laser; a2-second path of sub-laser; a3 — third path of sub-laser; a4 — fourth path sub laser.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the utility model with reference 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, the large-field high-flux two-photon microscope with the elliptic hemispherical curved surface of the present embodiment includes: the system comprises a near-infrared femtosecond pulse laser unit group 1, a beam splitting delay module group 2, a scanning unit group 3, a beam combination splicing module 4, an ellipsoid curved surface large-visual-field reflection type objective lens 5, a dichroic sheet 6 and a photomultiplier detection array 7;
laser emitted by the near-infrared femtosecond pulse laser unit combination 1 sequentially passes through the beam splitting delay module combination 2, the scanning unit combination 3 and the beam combination splicing module 4 to be transmitted through the dichroic sheet 6, then passes through the ellipsoidal curved surface large-visual-field reflective objective lens 5 to be irradiated onto a sample, and fluorescence generated by the sample after being excited is collected by the ellipsoidal curved surface large-visual-field reflective objective lens 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 unit group 1 includes 4 near-infrared femtosecond pulse laser units;
the beam splitting delay module group 2 comprises 4 beam splitting delay modules, and each beam splitting delay module comprises a delay light path and a beam splitting light path;
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 4 near-infrared femtosecond pulse lasers forms 4 paths of delay laser with time intervals of T/4 in sequence after passing through a delay optical path, each path of delay laser is equally divided into 4 paths of sub-lasers after passing through a beam splitting optical path, the 4 paths of sub-lasers from the same path of delay laser enter the same scanning unit, each path of sub-laser corresponds to 1 scanning module and is used for realizing the scanning of 1 sub-scanning area, and therefore the scanning of 16 sub-scanning areas is realized by the one-to-one correspondence of 16 paths of 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 of each scanning module in the same scanning unit are located at the same time point, and the sub-scanning areas obtained by scanning of the scanning modules in different scanning units are located at different time points;
the beam combination splicing module 4 is used for realizing beam combination splicing of 16 paths of sub-lasers emitted by the 16 scanning modules, 16 sub-scanning areas are combined and spliced to form a rectangular scanning field distributed in a 4x4 array, boundaries of the 4 sub-scanning areas located at the same time point are not adjacent, and any 4 areas adjacent to the boundaries are located at different time points in time.
The present invention is further described with reference to the following more specific embodiments and accompanying drawings.
Referring to fig. 2-8, in the present embodiment, the near-infrared femtosecond pulse laser unit 11 includes four near-infrared femtosecond pulse laser units 1.1, 1.2, 1.3, and 1.4, which have the same type and respectively emit femtosecond laser A, B, C, D to enter the respective beam splitting delay modules 2.1 to 2.4.
In this embodiment, the beam splitting delay module group 2 includes 4 beam splitting delay modules 2.1 to 2.4, and each beam splitting delay module includes a delay light path and a beam splitting light path, as shown in fig. 2 (only 2.1 is shown, and 2.2 to 2.4 are the same); the delay optical path comprises a first reflector 2.11 and a second reflector 2.12, laser A emitted by the near-infrared femtosecond pulse laser is reflected by the first reflector 2.11 and the second reflector 2.12 in sequence to form delay laser output, and the output delay laser generates different delay amount L/c by adjusting the distance L between the first reflector 2.11 and the second reflector 2.12, wherein c is the light speed;
the beam splitting optical path comprises a first light splitting element 2.13, a second light splitting element 2.14 and a third light splitting element 2.15, the delay laser output by the second reflector 2.12 is divided into two paths after entering the first light splitting element 2.13, one path of the delay laser transmits the first light splitting element 2.13 and reaches the second light splitting element 2.14, and is divided into two paths by the second light splitting element 2.14, the other path of the delay laser is reflected by the first light splitting element 2.13 and reaches the third light splitting element 2.15, and is divided into two paths by the third light splitting element 2.15, and therefore the delay laser is divided into 4 paths. In a preferred embodiment, the first beam splitter 2.13, the second beam splitter 2.14 and the third beam splitter 2.15 are all depolarizing beam splitters or 50/50 beam splitters.
In a preferred embodiment, the repetition rate of the femtosecond laser pulses is typically 80MHz, i.e., a period of 12.5 ns. The four groups of laser pulses from A to D are separated in time by adjusting respective delay amounts of the beam splitting delay modules 2.1 to 2.4, the delay between two adjacent paths is separated by 3.125ns, as shown in FIG. 3, which is equivalent to equally dividing a pulse period of 12.5ns into 4 parts, and each group of laser is at one time point.
The laser light of each laser is divided equally into 4 paths, thus 16 paths of laser light are divided out. Each laser beam further enters the parallel scanning unit group 33 and enters the corresponding scanning unit. The A1-A4 laser corresponds to the scanning units 3.11-3.14, the B1-B4 laser corresponds to the scanning units 3.21-3.24, the C1-C4 laser corresponds to the scanning units 3.31-3.34, and the D1-D4 laser 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 vibrating mirror, two-dimensional fast scanning of each independent sub-region is achieved, and the scanning view field surface of each sub-region is rectangular.
The 16 sub-region scan view planes need to be beam-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, which is a4 × 4 scan view array. The arrangement characteristics of the sub-areas formed by each path of laser scanning are as follows: the boundaries of the sub-areas of the same group of lasers are not adjacent, for example, A1-A4 are not adjacent; the sub-regions adjacent to the boundary are derived from different groups of lasers, such as A1, B1, C1 and D1 which are adjacent to each other and are derived from four groups of lasers; the details will be described below.
The beam combination splicing module 4 comprises 4 primary beam combination splicing modules 4.1-4.4 and 1 secondary beam combination splicing module 4.5, each primary beam combination splicing module corresponds to 1 scanning unit respectively, and 4 paths of sub-lasers emitted by 4 scanning modules in 1 scanning unit are combined and spliced;
the second-stage beam combination splicing module 4.5 is used for carrying out beam combination splicing on 4 groups of laser emitted by the 4 first-stage beam combination splicing modules again, and then inputting the laser into the dichroic sheet 6.
The 16 scanning branches are combined and field-of-view spliced by the beam combining and splicing module 44, and finally form a scanning field as shown in fig. 4. The four scanning branches of the same group of laser beams are firstly subjected to beam combination and splicing through the primary beam combination and splicing module, for example, A1-A4 are subjected to beam combination and splicing through the primary 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-stage beam combining and splicing module 4.1 is taken as an example to describe how to perform beam combining and splicing, and 4.2-4.4 are the same.
Referring to fig. 5, the first-stage beam combining and splicing module includes 2 first-stage beam combining and splicing sub optical paths 4.1.01 and 4.1.02, a first-stage polarization beam combining prism 4.1.13, a first primary lens 4.1.14, a first-stage combined half-wave plate 4.1.15 and a second first-stage lens 4.1.16, laser light emitted from 4.10 of the 2 first-stage beam combining and splicing sub optical paths passes through the first-stage polarization beam combining prism 4.1.13, is then sequentially output after passing through the first-stage lens 4.1.14, the first-stage combined half-wave plate 4.1.15 and the second first-stage lens 4.1.16, and the first-stage combined half-wave plate 4.1.15 includes 4 different half-wave plates;
the 2 first-stage beam combining and splicing sub-optical paths 4.1.01 and 4.1.02 have the same structure and respectively comprise a first-stage half-wave plate 4.1.1, a second first-stage half-wave plate 4.1.2, a first-stage 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 first-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 beam combining and splicing sub-optical paths are respectively marked as a first-stage beam combining and splicing sub-optical path 4.1.01 and a second first-stage beam combining and splicing sub-optical path 4.1.02;
4 paths of sub-lasers emitted by 4 scanning modules 3.11-3.14 in the same scanning unit are respectively recorded as: a first sub laser A1, a second sub laser A2, a third sub laser A3, a fourth sub laser A4,
the first sub laser a1 and the second sub laser a2 are combined by the first one-to-one beam combining and splicing sub light path 4.1.01 and then transmit the first polarization beam combining prism 4.1.13, which specifically includes:
the first path of sub-laser A1 passes through a first-order sub-half-wave plate 4.1.1 and then transmits a first-order sub-polarization beam combining prism 4.1.3, the second path of sub-laser A2 passes through a second first-order sub-half-wave plate 4.1.2 and then is reflected by the first-order sub-polarization beam combining prism 4.1.3, then is combined with the first path of sub-laser A1 which transmits the first-order sub-polarization beam combining prism 4.1.3, then passes through a first-order sub-lens 4.1.4 together, 2 sub-scanning areas are formed on two different half-wave plates of a first-order sub-combined half-wave plate 4.1.5 on the back focal plane of the first-order sub-lens 4.1.4, and the distance of one sub-scanning area is separated 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 enable the beam combination efficiency of laser to be maximum, the angle of the laser incident to the first-stage sub-polarization beam combination prism 4.1.3 is adjusted to enable two sub-scanning areas to be in proper positions, namely the distance of one sub-scanning area is arranged between the 2 sub-scanning areas, scanning fields formed by A1 and A2 are not adjacent to each other to form a gap, so that the scanning fields can further fall on two different half-wave plates completely, and the two half-wave plates form a first-stage sub-combined half-wave plate 4.1.5 in a gluing mode;
the combined two paths of sub-lasers pass through a second primary sub-lens 4.1.6 and then are converted into collimated lasers, and then the collimated lasers transmit a primary polarization beam combining prism 4.1.13, wherein the 2 paths of lasers passing through a primary sub-combined half-wave plate 4.1.5 are the same in polarization state;
the polarization states of the A1 and the A2 after polarization combination are different and are just vertical to each other, and then the A1 and the A2 after polarization combination are separated again and lose the combination effect. The crystal orientations of the two half-wave plates glued in the first-order sub-combined half-wave plate 4.1.5 are properly selected to be different, so that the laser polarization states of the A1 and A2 areas are consistent after passing through 4.1.5, and are just adjusted to the optimal polarization state required by the next polarization combination.
Similarly, the third sub-laser A3 and the fourth sub-laser a4 are combined by the second primary beam combining sub-optical path 4.1.02, and then reflected by the primary polarization beam combining prism 4.1.13, so that the A3 and the a4 are combined with the combined beam laser a1 and the a2 which transmit the primary polarization beam combining prism 4.1.13, and then both pass through the first primary lens 4.1.14, 4 sub-scanning regions are formed on four different half-wave plates of the primary combined half-wave plate 4.1.15 on the back focal plane of the first primary lens 4.1.14, and the 4 sub-scanning regions are separated by a distance of one sub-scanning region, and finally pass through the second primary lens 4.1.16 and are changed back to collimated laser, and are output to the secondary beam combining module 4.5, wherein the polarization states of the 4 laser beams after passing through the primary combined half-wave plate 4.1.15 are the same.
The beam combining efficiency is maximized by the first order sub-combined half-wave plates 4.1.5, 4.1.11, and the mutual positions of the scanning fields of view of the four scanning subregions on the back focal plane of the first primary lens 4.1.14 are set to the designed proper positions by adjusting the incident angle of 4.1.13. The four sub-fields of view are still not adjacent in their boundaries and therefore fall completely on four different half-wave plates. The four half-wave plates are glued to form a first order merged half-wave plate 4.1.15. By controlling and selecting the crystal orientation of each half-wave plate, the polarization state of the four sub-fields passing through 4.1.15 is the same, and is the optimal polarization state required by the next polarization beam combination. The laser light is then changed back to collimated laser light by a second primary lens 4.1.16.
At this time, beam combination and visual field combination are completed by the A1-A4 through the primary beam combination and combination module, and the combination mode of B, C, D groups is the same and will not be described.
Referring to fig. 6, A, B, C, D four groups of lasers are beam-combined and spliced by a two-stage beam-combining and splicing module 4.5.
The secondary beam combining and splicing module 4.5 comprises 2 paths of secondary beam combining and splicing sub-optical paths 4.5.01 and 4.5.02, a secondary polarization beam combining prism 4.5.7 and a secondary lens 4.5.9, wherein laser emitted by the 2 secondary beam combining and splicing sub-optical paths 4.5.0 is combined by the secondary polarization beam combining prism 4.5.7, then passes through the secondary lens 4.5.9, and then is incident on the dichroic plate 6;
the 2 secondary beam combining and splicing sub-optical paths 4.5.01 and 4.5.02 have the same structure and respectively 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, and the 2 secondary beam combining and splicing sub-optical paths 4.5.0 are respectively marked as a first secondary beam combining and splicing sub-optical path 4.5.01 and a second secondary beam combining and splicing sub-optical path 4.5.02;
the combined beam laser of 4 paths of sub-lasers emitted by 4 first-stage combined beam splicing modules 4.1-4.4 is respectively recorded as: a first path of sub-beam combination laser A, a second path of sub-beam combination laser B, a third path of sub-beam combination laser C and a fourth path of sub-beam combination laser D;
the first path of sub-combined laser a and the second path of sub-combined laser B are combined by the first secondary combined sub-beam path 4.5.01 and then transmit the secondary polarization beam combining prism 4.5.7, which specifically includes:
after the first path of sub-beam-combined laser A transmits the second-level sub-polarization beam-combined prism 4.5.1, the first path of sub-beam-combined laser A and the second path of sub-beam-combined laser B reflected by the second-level sub-polarization beam-combined prism 4.5.1 are combined, then the combined laser A and the second path of sub-beam-combined laser B pass through the first second-level sub-lens 4.5.2 together, 8 sub-scanning areas are formed on the back focal plane of the first second-level sub-lens 4.5.2, the 8 sub-scanning areas are two equally divided scanning areas formed by splicing the respective 4 sub-scanning areas of the first path of sub-beam-combined laser A and the second path of sub-beam-combined laser B, the 4 sub-scanning areas in each row of scanning areas are adjacent in sequence, and the distance of one row of scanning areas is arranged between the two rows of scanning areas;
the third path of sub-combined laser C and the fourth path of sub-combined laser D are combined by the second secondary beam combining sub-optical path 4.5.02, reflected by the secondary polarization beam combining prism 4.5.7, then combined with the combined laser transmitted through the secondary polarization beam combining prism 4.5.7, and finally passed through the secondary lens 4.5.9, so as to form a complete rectangular scanning field formed by 16 sub-scanning regions and distributed in a4 × 4 array on the back focal plane of the secondary lens 4.5.9, as shown in fig. 4.
The group A and the group B lasers are combined through the secondary polarization beam combining prism 4.5.7, and the polarization states of the group A and the group B lasers are adjusted to be optimal through the combined half-wave plate in the respective primary beam combining splicing modules, so that the beam combining efficiency is guaranteed. The angle of incidence to secondary polarization beam-combining prism 4.5.7 is adjusted so that the respective fields of the two groups of 8 sub-regions formed on the back focal plane of first secondary sub-lens 4.5.2 are at the appropriate positions as designed. And the beam combination and splicing of the lasers in the group C and the group D are the same.
With continued reference to fig. 6, in a preferred embodiment, the second combined beam splicing module 4.5 further comprises a baffle 4.5.8, and half of the laser light lost by the depolarized combined beam is blocked by the baffle 4.5.8. In a further preferred embodiment, the baffle 4.5.8 can also be replaced by a camera, which is used to monitor the current splicing situation of each view in real time, and if the view position of a certain sub-region deviates, the maintenance personnel can find and adjust the position in time.
In a preferred embodiment, secondary polarization beam combining prism 4.5.7 is replaced with a 50/50 beam splitting plate.
The dichroic sheet 6 can efficiently transmit the laser band, while the visible fluorescence band will reflect and have high reflectivity. The laser after the scanning visual fields are combined reaches the reflective objective lens after passing through the dichroic sheet 6, an excitation scanning field is formed in the sample, and the excited fluorescence in the sample reaches the dichroic sheet 6 through the reflective objective lens and is reflected to the photomultiplier detection array to perform photoelectric signal conversion.
Referring to fig. 7, the planar scan field (fig. 4) obtained by splicing a plurality of sub-scan regions is converted into a curved scan field of an elliptical hemisphere by an ellipsoidal curved large-field-of-view reflective objective lens 55.
In this embodiment, the ellipsoidal curved surface large-visual-field reflective objective lens 5 includes a primary mirror 5.1, a secondary mirror 5.2, and a tertiary mirror 5.3 that are sequentially arranged along a light path, an optical surface of the primary mirror 5.1 is an eight-order hyperboloid, an optical surface of the secondary mirror 5.2 is a secondary flat ellipsoid, an optical surface of the tertiary mirror 5.3 is a six-order flat ellipsoid, and laser entering the ellipsoidal curved surface large-visual-field reflective objective lens 5 is sequentially reflected by the primary mirror 5.1, the secondary mirror 5.2, and the tertiary mirror 5.3 and then irradiates a sample. The numerical aperture range of the ellipsoidal curved surface large-visual-field reflective objective lens 5 is 0.3-0.5. The imaging visual field of the reflective objective lens is an ellipsoid curved surface, the curvature radius of a long axis is 9-12 mm, the curvature radius of a short axis is 6-9 mm, and the plane projection size of the visual field is 6mm multiplied by 6 mm.
A 16-way scanning laser beam will excite the sample, producing corresponding fluorescence. AA 1-AA 4 are respectively fluorescence excited by A1-A4 laser, BB 1-BB 4 are respectively fluorescence excited by B1-B4 laser, CC 1-CC 4 are respectively fluorescence excited by C1-C4 laser, and DD 1-DD 4 are respectively fluorescence excited by D1-D4 laser.
The photomultiplier detection array 7 is an array consisting of 4 × 4 photomultipliers, and each array element photomultiplier corresponds to fluorescence detection of the imaging field of view of 1 scanning unit, as shown in fig. 8. The fluorescence from the curved field of view is converted back to a planar matrix distribution in the detection array 7 after the field of view of the objective 5 has been scanned.
Due to the fact that the fluorescence is scattered, 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. Due to the fact that the scanning laser of adjacent regions has delay difference, the generated fluorescence has the same time difference, the source regions of the fluorescence can be distinguished in time, and therefore signal crosstalk caused by fluorescence scattering is reduced.
In general, the utility model can effectively distinguish the source areas of the fluorescence signals in time and space by the thought that the areas at the same time point are not adjacent and the adjacent areas are not at the same time, thereby greatly avoiding the signal crosstalk between the areas.
The embodiment of the utility model describes a scanning and detecting method of a 16-path 4x4 array, and according to the four-color principle, for any number of areas distributed in a plane, the areas can be always divided into four types, and the areas of the same type are not adjacent, so that the energy transmittance of combined beams is improved through the unique beam combining and splicing method provided by the utility model, and the crosstalk of fluorescent signals is reduced through the time-space division method provided by the utility model, therefore, the embodiment of the utility model can be popularized to any number of parallel scanning and detecting.
While embodiments of the utility model have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the utility model, and further modifications may readily be effected by those skilled in the art, so that the utility model is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.
Claims (9)
1. A large-field high-flux two-photon microscope with an elliptic hemispherical curved surface is characterized by comprising: the system comprises a near-infrared femtosecond pulse laser unit group, a beam splitting delay module group, a scanning unit group, a beam combining and splicing module, an ellipsoidal curved surface large-visual-field reflective objective lens, a dichroic sheet and a photomultiplier detection array;
laser emitted by the near-infrared femtosecond pulse laser unit is transmitted through the dichroic sheet after sequentially passing through the beam splitting delay module unit, the scanning unit and the beam combination splicing module, and then is irradiated onto a sample through the ellipsoidal curved surface large-visual-field reflective objective lens, and fluorescence generated by exciting the sample is reflected to the photomultiplier detection array through the dichroic sheet after being collected through the ellipsoidal curved surface large-visual-field reflective objective lens;
the ellipsoidal curved surface large-visual-field reflective objective lens comprises a primary mirror, a secondary mirror and a tertiary mirror which are sequentially arranged along a light path, wherein the optical surface of the primary mirror is a hyperboloid, the optical surface of the secondary mirror is a flat ellipsoid, the optical surfaces of the tertiary mirrors are flat ellipsoids, and laser entering the ellipsoidal curved surface large-visual-field reflective objective lens is sequentially reflected by the primary mirror, the secondary mirror and the tertiary mirror and then irradiates a sample;
the near-infrared femtosecond pulse laser unit group comprises 4 near-infrared femtosecond pulse laser units;
the beam splitting delay module group comprises 4 beam splitting delay modules, and each beam splitting delay module comprises a delay light path and a beam splitting light path;
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 lasers emitted by the 4 near-infrared femtosecond pulse lasers form 4 paths of delay lasers with time intervals of T/4 in sequence after passing through the delay light path, each path of delay laser is equally divided into 4 paths of sub-lasers after passing through the beam splitting light path, the 4 paths of sub-lasers from the same path of delay laser enter the same scanning unit, each path of sub-laser corresponds to 1 scanning module and is used for realizing the scanning of 1 sub-scanning area, and therefore the 16 paths of sub-lasers correspond to the 16 scanning modules one by one to realize the scanning of 16 sub-scanning areas; wherein T is the pulse period of the laser emitted by the near-infrared femtosecond pulse laser; the sub-scanning areas obtained by scanning of each scanning module in the same scanning unit are located at the same time point, and the sub-scanning areas obtained by scanning of the scanning modules in different scanning units are located at different time points;
the beam combination splicing module is used for realizing beam combination splicing of 16 paths of sub-lasers emitted by the 16 scanning modules, 16 sub-scanning areas are combined and spliced to form a rectangular scanning field distributed in a 4x4 array, boundaries of the 4 sub-scanning areas located at the same time point are not adjacent, and any 4 areas adjacent to the boundaries are located at different time points in time.
2. The large-field-of-view high-throughput two-photon microscope with an elliptic hemispherical curved surface according to claim 1, wherein the optical surface of the primary mirror is an eight-order hyperboloid, the optical surface of the secondary mirror is a secondary flat ellipsoid, and the optical surface of the three mirrors is a six-order flat ellipsoid.
3. The large-view-field high-flux two-photon microscope with the elliptic hemispherical curved surface according to claim 1, wherein the delay optical path comprises a first reflecting mirror and a second reflecting mirror, laser emitted by the near-infrared femtosecond pulse laser sequentially reflects by the first reflecting mirror and the second reflecting mirror to form delayed laser output, and the distance between the first reflecting mirror and the second reflecting mirror is adjusted to enable the output delayed laser to generate different delay amounts;
the beam splitting light path includes first beam splitting component, second beam splitting component and third beam splitting component, the time delay laser of second mirror output advances equally divide into two the tunnel behind the first beam splitting component, transmits all the way reach behind the first beam splitting component second beam splitting component, and quilt the second beam splitting component equally divides into two the tunnel, another way quilt arrive after the reflection of first beam splitting component third beam splitting component, and quilt the third beam splitting component equally divides into two the tunnel to equally divide time delay laser into 4 ways.
4. The large-field high-flux two-photon microscope with the elliptic hemispherical curved surface according to claim 3, wherein 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.
5. The elliptical hemispherical curved large-field high-flux two-photon microscope of claim 1, wherein the scanning module comprises a fast axis resonant scanning mirror and a slow axis galvanometer mirror.
6. The large-field-of-view high-flux two-photon microscope with the elliptic hemispherical curved surface according to claim 3, wherein the beam combination splicing module comprises 4 primary beam combination splicing modules and 1 secondary beam combination splicing module, and each primary beam combination splicing module corresponds to 1 scanning unit respectively so as to combine and splice 4 paths of sub-laser emitted by 4 scanning modules in 1 scanning unit;
the secondary beam combination splicing module is used for carrying out beam combination splicing on 4 groups of laser emitted by the 4 primary beam combination splicing modules again and then inputting the laser into the dichroic film.
7. The elliptic hemispherical curved surface large-field high-flux two-photon microscope according to claim 6, wherein the first-stage beam combining and splicing module comprises 2 first-stage beam combining and splicing sub-optical paths, a first-stage polarization beam combining prism, a first primary lens, a first-stage combined half-wave plate and a second primary lens, laser emitted from the 2 first-stage beam combining and splicing sub-optical paths is combined by the first-stage polarization beam combining prism and then sequentially passes through the first primary lens, the first-stage combined half-wave plate and the second primary lens to be output, and the first-stage combined half-wave plate comprises 4 different half-wave plates;
the 2 first-stage beam combination splicing sub-optical paths have the same structure and respectively comprise a first-stage sub-half-wave plate, a second first-stage sub-half-wave plate, a first-stage polarization beam combination prism, a first primary sub-lens, a first-stage sub-combined half-wave plate and a second first-stage sub-lens, wherein the first-stage sub-combined half-wave plate comprises two different half-wave plates, and the 2 first-stage beam combination splicing sub-optical paths are respectively marked as a first-stage beam combination splicing sub-optical path and a second-stage beam combination splicing sub-optical path;
the 4 paths of sub-lasers emitted by 4 scanning modules in the same scanning unit are respectively marked 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 transmit the first-order polarization beam combining prism after being combined through the first-order beam combining and splicing 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 first-stage 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 first-stage sub polarization beam combining prism, then is combined with the first path of sub laser which transmits the first-stage sub polarization beam combining prism, and then passes through the first primary sub lens together, 2 sub scanning areas are formed on two different half-wave plates of the first-stage sub combined half-wave plate on the back focal plane of the first primary sub lens, and the 2 sub scanning areas are separated by the distance of one sub scanning area; the combined two paths of sub-lasers pass through the second primary sub-lens and then are converted into collimated lasers, and then the collimated lasers transmit the primary polarization beam combining prism, wherein the polarization states of the 2 paths of lasers passing through the primary sub-combined half-wave plate are the same;
the third path of sub laser and the fourth path of sub laser are reflected by the first-stage polarization beam combining prism after being combined by the second first-stage beam combining and splicing sub optical path, and then are combined with the combined beam laser which transmits the first-stage polarization beam combining prism; and then the light beams pass through the first primary lens together, 4 sub-scanning areas are formed on four different half-wave plates of the first-stage combined half-wave plate on the back focal plane of the first primary lens, the 4 sub-scanning areas are separated by the distance of one sub-scanning area, and finally the light beams pass through the second primary lens and are converted back into collimated laser beams to be output to the second-stage beam combining and splicing module, wherein the polarization states of the 4 paths of laser beams passing through the first-stage combined half-wave plate are the same.
8. The large-view-field high-flux two-photon microscope with the elliptic hemispherical curved surface according to claim 7, wherein the secondary beam combining and splicing module comprises 2 secondary beam combining and splicing sub-optical paths, a secondary polarization beam combining prism and a secondary lens, and laser light emitted from the 2 secondary beam combining and splicing sub-optical paths passes through the secondary lens after being combined by the secondary polarization beam combining prism and then is incident on the dichroic sheet;
the 2 secondary beam combining and splicing sub-optical paths have the same structure and respectively comprise a secondary polarization beam combining prism, a first secondary sub-lens and a second secondary sub-lens, and the 2 secondary beam combining and splicing sub-optical paths are respectively marked as a first secondary beam combining and splicing sub-optical path and a second secondary beam combining and splicing sub-optical path;
the combined beam laser of 4 paths of sub-laser emitted by the 4 first-stage combined beam splicing modules is respectively recorded as: 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 path of sub-beam combination laser and the second path of sub-beam combination laser are transmitted through the second-level polarization beam combination prism after being combined by the first second-level beam combination splicing sub-optical path, and the method specifically comprises the following steps:
after transmitting the secondary sub-polarization beam combining prism, a first path of sub-combined beam laser is combined with a second path of sub-combined beam laser reflected by the secondary sub-polarization beam combining prism, then the first path of sub-combined beam laser and the second path of sub-combined beam laser 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 two equally-divided scanning areas formed by splicing 4 sub-scanning areas of the first path of sub-combined beam laser and the second path of sub-combined beam laser, the 4 sub-scanning areas in each scanning area are adjacent in sequence, and the two scanning areas are separated by the distance of one scanning area;
and the third path of sub-beam-combining laser and the fourth path of sub-beam-combining laser are reflected by the secondary polarization beam-combining prism after being combined by the second secondary beam-combining splicing sub-optical path, then are combined with the combined beam laser which transmits the secondary polarization beam-combining prism, and finally pass through the secondary lens together, so that a complete rectangular scanning field which is formed by 16 sub-scanning areas and distributed in a 4x4 array is formed on the back focal plane of the secondary lens.
9. The large field of view high flux two-photon microscope according to claim 8, wherein the photomultiplier tube detection array is an array of 4x4 photomultiplier tubes, each corresponding to fluorescence detection of the imaging field of view of 1 scanning module.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202121819956.6U CN215986702U (en) | 2021-08-05 | 2021-08-05 | Large-visual-field high-flux two-photon microscope with elliptic hemispherical curved surface |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202121819956.6U CN215986702U (en) | 2021-08-05 | 2021-08-05 | Large-visual-field high-flux two-photon microscope with elliptic hemispherical curved surface |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN215986702U true CN215986702U (en) | 2022-03-08 |
Family
ID=80575112
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202121819956.6U Active CN215986702U (en) | 2021-08-05 | 2021-08-05 | Large-visual-field high-flux two-photon microscope with elliptic hemispherical curved surface |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN215986702U (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113655026A (en) * | 2021-08-05 | 2021-11-16 | 中国科学院苏州生物医学工程技术研究所 | Large-visual-field high-flux two-photon microscope with elliptic hemispherical curved surface |
-
2021
- 2021-08-05 CN CN202121819956.6U patent/CN215986702U/en active Active
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113655026A (en) * | 2021-08-05 | 2021-11-16 | 中国科学院苏州生物医学工程技术研究所 | Large-visual-field high-flux two-photon microscope with elliptic hemispherical curved surface |
| WO2023010718A1 (en) * | 2021-08-05 | 2023-02-09 | 中国科学院苏州生物医学工程技术研究所 | Elliptical hemispherical curved surface large-field-of-view high-throughput two-photon microscope |
| CN113655026B (en) * | 2021-08-05 | 2024-01-23 | 中国科学院苏州生物医学工程技术研究所 | Elliptic hemispherical surface large-view high-flux two-photon microscope |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US5243619A (en) | Process wherein several radiation sources, arranged in one row or several rows, are imaged, and apparatus for this purpose | |
| WO2023197734A1 (en) | Multi-channel super-resolution gene detector and detection method thereof | |
| JP6189839B2 (en) | Laser scanning microscope with illumination array | |
| JPH10311950A (en) | Scanning microscope | |
| US6987259B2 (en) | Imaging system with an integrated source and detector array | |
| US11835442B2 (en) | Flow imaging system based on matrix laser scanning | |
| CN113258415B (en) | Pulse laser spectrum time sequence synthesis system and method | |
| JP7350936B2 (en) | High-throughput hyperspectral imaging system | |
| JP2016516219A (en) | Random access stimulated emission suppression (STED) microscope | |
| CN215986702U (en) | Large-visual-field high-flux two-photon microscope with elliptic hemispherical curved surface | |
| CN217639718U (en) | Receiving system and microscope | |
| US20160302740A1 (en) | Compact scanner assembly with a resonant scanner and two galvanometer scanners for multi region of interest (mroi) imaging and targeting of intact tissue | |
| CN211086784U (en) | Microscope array | |
| CN113655026A (en) | Large-visual-field high-flux two-photon microscope with elliptic hemispherical curved surface | |
| US7212347B2 (en) | Reflective illumination system | |
| KR20030065844A (en) | Collimating lens, collimating system and image displaying apparatus employing the same | |
| CN112445005B (en) | Laser light source and laser light source system | |
| US20200355897A1 (en) | Reverberation microscopy systems and methods | |
| WO2022120595A1 (en) | Super-resolution measurement system and super-resolution measurement method | |
| JP2023528970A (en) | LIDAR system with coarse angle control | |
| US20230266577A1 (en) | Apparatuses and methods for high-speed laser scanning | |
| CN111061054A (en) | Multi-MEMS micro-mirror chip optical path system | |
| CN109211854B (en) | Multi-beam multi-photon rescanning microscopic imaging device | |
| CN214623044U (en) | Imaging device | |
| US20240035978A1 (en) | Multi-spot confocal imaging system with spectral multiplexing |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| GR01 | Patent grant | ||
| GR01 | Patent grant |