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For a long time, wide field of view, high temporal and spatial resolution, and deep tissue imaging technology are urgently needed in the field of neuroscience
.
In the early days, two-photon microscopy imaging for dynamic observation of neuronal structure or calcium ion changes in vivo was mainly fixed on the operating table after anesthesia, which greatly limited its use
.
Scientists at home and abroad have been exploring whether it is possible to apply two-photon microscopy to free-motion scenes
.
In 2001, the Winfried Denk research team of the Max-Planck Institute for Neuroscience in Heidelberg, Germany (Professor Winfried Denk was the inventor of the two-photon microscope and invented it during his postdoctoral period in 1990) first developed a miniature head-mounted two-photon microscope.
The first is The two-photon laser light is extracted from the mode-locked laser, and then the laser light is transmitted through a single-mode fiber into a microscopic microscope
.
Its specific parameters are that the microscope is 7.
5 cm long and weighs 25 grams (equivalent to the weight of an adult mouse)
.
The lens with a magnification of 55 times and a numerical aperture of 0.
8 can observe blood vessels in a free motion state, but it has disadvantages such as low resolution and severe motion artifacts.
This is the prototype of the earliest miniature two-photon microscope
.
In 2017, the team of Academician Cheng Heping released the Micro Two-Photon Microscope 1.
0, breaking the above limitations.
The research results were published in Nature Methods
.
The first author is Zong Weijian, who "shrinks" the huge two-photon microscope device to a probe of about 2.
2g, creating a precedent for two-photon in vivo observation in a free state
.
On the other hand, the technique of imaging calcium ions in free-motion state using single-photon microscopic microscopes has developed rapidly and has been widely used in neuroscience research
.
However, its main drawback is that Z-axis information cannot be obtained
.
In 2021, the team of Academician Cheng Heping will upgrade the micro two-photon microscope to version 2.
0, the imaging field of view will be expanded to 420 × 420 square microns, and the imaging depth will be 180um, but there are still some defects: its weight (about 5 grams) and the stiffness of the fiber will Interfere with the movement of mice, which is not conducive to the experimental requirements of long-term free movement
.
On March 18, 2022, Chong Weijian (currently in the laboratory of Edvard I.
Moser, winner of the Nobel Prize in Physiology or Medicine) once again upgraded the miniature two-photon microscope to version 3.
0, which can achieve large-field calcium ion in vivo imaging.
The research results were published in the journal Cell
.
Figure 1: Microscopes of different weights affect the voluntary movements of mice First, they tested the free movement ability of mice without the microscopic microscope, wearing a 5g microscope and wearing a 3g microscope, and found that wearing a 3g microscope hardly affected the mice's movement.
In the state of exercise, the 5g weight obviously restricts its autonomous movement
.
The second-generation fiber bundle of the micro two-photon microscope has a diameter of 1.
5mm, a Z-axis scanning module with a collection depth of 180um, and a weight of 1.
8g.
Together with the Grins lens, housing, base and other materials, the total weight is about 5g
.
In the third-generation micro-microscope, the fiber bundle diameter is 0.
7mm, the acquisition depth of the Z-axis scanning module is 240um, and the weight is 0.
06g.
Together with the Grins lens, shell, base and other materials, the total weight is about 3g.
The state of motion in the natural state
.
In order to obtain a faster imaging speed and a larger imaging field, the researchers developed two versions of the third generation, namely the miniature two-photon microscope MINI2P-L (imaging speed is 15Hz, imaging field is 500 × 500 square microns) , MINI2P-F (imaging speed is 40Hz, imaging field is 420×420 square microns)
.
Figure 2: Calcium Imaging in Different Regions by the Third Generation Microscope By in vivo calcium imaging of hippocampal CA1, visual cortex, and entorhinal cortex, about 400-600 neurons can be collected
.
In order to acquire more neurons, researchers can simultaneously acquire calcium ion changes in thousands of neurons by increasing the imaging field of view, multiple plane imaging, and increasing the adjacent imaging field of view
.
In general, this paper upgrades and iterates on the miniature two-photon microscope: the overall weight is reduced without affecting the motor ability of mice; the imaging speed and imaging field of view are improved, and the calcium ion changes of thousands of neurons are obtained
.
【References】1.
Zong, W.
, Wu, R.
, Chen, S.
, Wu, J.
, Wang, H.
, Zhao, Z.
, Chen, G.
, Tu, R.
, Wu, D.
, Hu, Y.
, et al.
(2021).
Miniature two-photon microscopy for enlarged field-ofview, multi-plane and long-term brain imaging.
Nat.
Methods 18, 46–49.
https://doi.
org /10.
1038/s41592-020-01024-z2.
Zong, W.
, Wu, R.
, Li, M.
, Hu, Y.
, Li, Y.
, Li, J.
, Rong, H.
, Wu, H.
, Xu, Y.
, Lu, Y.
, et al.
(2017).
Fast high-resolution miniature two-photon microscopy for brain imaging in freely behaving mice.
Nat.
Methods 14, 713–719.
https://doi.
org/10.
1038/nmeth.
4305.
3.
Zong et al.
, Large-scale two-photon calcium imaging in freely moving mice, Cell (2022), https://doi.
org/10.
1016/j.
cell.
2022.
02.
017 Image in text from reference
For a long time, wide field of view, high temporal and spatial resolution, and deep tissue imaging technology are urgently needed in the field of neuroscience
.
In the early days, two-photon microscopy imaging for dynamic observation of neuronal structure or calcium ion changes in vivo was mainly fixed on the operating table after anesthesia, which greatly limited its use
.
Scientists at home and abroad have been exploring whether it is possible to apply two-photon microscopy to free-motion scenes
.
In 2001, the Winfried Denk research team of the Max-Planck Institute for Neuroscience in Heidelberg, Germany (Professor Winfried Denk was the inventor of the two-photon microscope and invented it during his postdoctoral period in 1990) first developed a miniature head-mounted two-photon microscope.
The first is The two-photon laser light is extracted from the mode-locked laser, and then the laser light is transmitted through a single-mode fiber into a microscopic microscope
.
Its specific parameters are that the microscope is 7.
5 cm long and weighs 25 grams (equivalent to the weight of an adult mouse)
.
The lens with a magnification of 55 times and a numerical aperture of 0.
8 can observe blood vessels in a free motion state, but it has disadvantages such as low resolution and severe motion artifacts.
This is the prototype of the earliest miniature two-photon microscope
.
In 2017, the team of Academician Cheng Heping released the Micro Two-Photon Microscope 1.
0, breaking the above limitations.
The research results were published in Nature Methods
.
The first author is Zong Weijian, who "shrinks" the huge two-photon microscope device to a probe of about 2.
2g, creating a precedent for two-photon in vivo observation in a free state
.
On the other hand, the technique of imaging calcium ions in free-motion state using single-photon microscopic microscopes has developed rapidly and has been widely used in neuroscience research
.
However, its main drawback is that Z-axis information cannot be obtained
.
In 2021, the team of Academician Cheng Heping will upgrade the micro two-photon microscope to version 2.
0, the imaging field of view will be expanded to 420 × 420 square microns, and the imaging depth will be 180um, but there are still some defects: its weight (about 5 grams) and the stiffness of the fiber will Interfere with the movement of mice, which is not conducive to the experimental requirements of long-term free movement
.
On March 18, 2022, Chong Weijian (currently in the laboratory of Edvard I.
Moser, winner of the Nobel Prize in Physiology or Medicine) once again upgraded the miniature two-photon microscope to version 3.
0, which can achieve large-field calcium ion in vivo imaging.
The research results were published in the journal Cell
.
Figure 1: Microscopes of different weights affect the voluntary movements of mice First, they tested the free movement ability of mice without the microscopic microscope, wearing a 5g microscope and wearing a 3g microscope, and found that wearing a 3g microscope hardly affected the mice's movement.
In the state of exercise, the 5g weight obviously restricts its autonomous movement
.
The second-generation fiber bundle of the micro two-photon microscope has a diameter of 1.
5mm, a Z-axis scanning module with a collection depth of 180um, and a weight of 1.
8g.
Together with the Grins lens, housing, base and other materials, the total weight is about 5g
.
In the third-generation micro-microscope, the fiber bundle diameter is 0.
7mm, the acquisition depth of the Z-axis scanning module is 240um, and the weight is 0.
06g.
Together with the Grins lens, shell, base and other materials, the total weight is about 3g.
The state of motion in the natural state
.
In order to obtain a faster imaging speed and a larger imaging field, the researchers developed two versions of the third generation, namely the miniature two-photon microscope MINI2P-L (imaging speed is 15Hz, imaging field is 500 × 500 square microns) , MINI2P-F (imaging speed is 40Hz, imaging field is 420×420 square microns)
.
Figure 2: Calcium Imaging in Different Regions by the Third Generation Microscope By in vivo calcium imaging of hippocampal CA1, visual cortex, and entorhinal cortex, about 400-600 neurons can be collected
.
In order to acquire more neurons, researchers can simultaneously acquire calcium ion changes in thousands of neurons by increasing the imaging field of view, multiple plane imaging, and increasing the adjacent imaging field of view
.
In general, this paper upgrades and iterates on the miniature two-photon microscope: the overall weight is reduced without affecting the motor ability of mice; the imaging speed and imaging field of view are improved, and the calcium ion changes of thousands of neurons are obtained
.
【References】1.
Zong, W.
, Wu, R.
, Chen, S.
, Wu, J.
, Wang, H.
, Zhao, Z.
, Chen, G.
, Tu, R.
, Wu, D.
, Hu, Y.
, et al.
(2021).
Miniature two-photon microscopy for enlarged field-ofview, multi-plane and long-term brain imaging.
Nat.
Methods 18, 46–49.
https://doi.
org /10.
1038/s41592-020-01024-z2.
Zong, W.
, Wu, R.
, Li, M.
, Hu, Y.
, Li, Y.
, Li, J.
, Rong, H.
, Wu, H.
, Xu, Y.
, Lu, Y.
, et al.
(2017).
Fast high-resolution miniature two-photon microscopy for brain imaging in freely behaving mice.
Nat.
Methods 14, 713–719.
https://doi.
org/10.
1038/nmeth.
4305.
3.
Zong et al.
, Large-scale two-photon calcium imaging in freely moving mice, Cell (2022), https://doi.
org/10.
1016/j.
cell.
2022.
02.
017 Image in text from reference
