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In the past ten years, multiphoton microscopes such as two-photon and three-photon have been widely used in in-vivo deep brain imaging to obtain the dynamic changes (morphology and function) of neurons under free motion.
The available excitation light wavelength of the two-photon microscope is approximately in the range of 690-1020nm, while the available excitation light of the three-photon microscope is in the range of 1000-1700nm.
Therefore, the attenuation of the excitation light of the three-photon microscope in the tissue will be weaker, and the imaging depth will be deeper.
In addition, the occurrence of three-photon absorption requires higher laser energy, and the background signal from the non-focal plane can be further weakened, which can increase the signal-to-background ratio and obtain higher-quality images.
Due to the needs of scientific research, multi-color, deep brain imaging is an inevitable trend.
Two-color three-photon imaging requires two different excitation wavelengths, the green fluorophore is excited at 1300 nm, and the red fluorophore is excited at 1700 nm.
However, dual-wavelength excitation requires a special excitation light source and complicated optical devices, which is expensive.
For the same number of fluorescent signals (such as green and red), using two separate laser wavelengths for excitation will inevitably increase the total excitation power and increase the damage to the tissue.
On March 17, 2021, Chris Xu, School of Applied and Engineering Physics, Cornell University, developed a technology that uses a single excitation light source to achieve multi-color three-photon imaging without complicated optical equipment.
Chris Xu is a well-known scientist in the field of three-photon microscopy deep brain imaging research.
He has been deeply involved in this field for more than 20 years.The researchers used single-photon, two-photon, and three-photon imaging to image Texas Red, SR 101, Alexa Fluor 546, DsRed, tdTomato, mCherry, and other red fluorescent dyes under different wavelength excitations, and found that the 1220～1360nm band The wavelength of the excitation light is the turning point from the lowest energy of the two-photon to the highest energy excited state of the three-photon.
More specifically, the signal-to-noise ratio of the image acquired under 1340nm excitation light is 4.
6 times, 15 times, and 82 times that at 1300 nm, 1260 nm, and 1220 nm, respectively.
The wavelength of the excitation light in the superficial cortex does not have a great influence on the signal-to-noise ratio of the images obtained by the Texas Red-labeled cerebral blood vessels on the two-photon and three-photon microscopes.
But in deep brain imaging (1050um), this difference is obvious: the two-photon microscope has an image signal-to-noise ratio of 0.
77 at 1220nm (the lowest energy excited state), while the three-photon microscope is excited by 1260, 1300, 1340, and 1650.
The image signal-to-noise ratios are 1.
4, 12, 47, and 16, respectively.
Whether it is imaging a variety of different red fluorescent dyes or brain regions at different depths, the image signal-to-noise ratio obtained at the excitation wavelength at 1340nm is the best.
PrismPlus mouse is a kind of rainbow transgenic tool mouse.
It expresses cyan fluorescent protein that marks oligodendrocytes, green fluorescent protein that marks microglia, yellow fluorescent protein that marks neurons, and astrocytes.
Red fluorescent protein.
Researchers can simultaneously excite a variety of fluorescence at a laser wavelength of 1340nm, realize four-color fluorescence imaging at different depths under four different photomultiplier tubes, and can observe the dynamic changes of these four cells at the same time.
With the passage of laser excitation time, the intensity of fluorescence excited by fluorescent substances gradually weakens, which is naked photobleaching toxicity.
In order to further confirm the photobleaching properties of the 1340nm wavelength, the researchers excited the same fluorescent substance marking astrocytes at 1340nm and 1650nm respectively, and found that the 1650nm wavelength excitation photobleaching is more toxic.
In general, this article has developed a multi-color three-photon imaging technology that uses a single wavelength to realize the real-time observation of the interactions between various cells in the brain in the deep brain regions of the living body.
[References] 1.
Hontani et al.
, Sci.
Adv.
2021; 7: eabf3531, Multicolor three-photon fluorescence imaging with single-wavelength excitation deep inmouse brain The pictures in the article are all from the references
In the past ten years, multiphoton microscopes such as two-photon and three-photon have been widely used in in-vivo deep brain imaging to obtain the dynamic changes (morphology and function) of neurons under free motion.
The available excitation light wavelength of the two-photon microscope is approximately in the range of 690-1020nm, while the available excitation light of the three-photon microscope is in the range of 1000-1700nm.
Therefore, the attenuation of the excitation light of the three-photon microscope in the tissue will be weaker, and the imaging depth will be deeper.
In addition, the occurrence of three-photon absorption requires higher laser energy, and the background signal from the non-focal plane can be further weakened, which can increase the signal-to-background ratio and obtain higher-quality images.
Due to the needs of scientific research, multi-color, deep brain imaging is an inevitable trend.
Two-color three-photon imaging requires two different excitation wavelengths, the green fluorophore is excited at 1300 nm, and the red fluorophore is excited at 1700 nm.
However, dual-wavelength excitation requires a special excitation light source and complicated optical devices, which is expensive.
For the same number of fluorescent signals (such as green and red), using two separate laser wavelengths for excitation will inevitably increase the total excitation power and increase the damage to the tissue.
On March 17, 2021, Chris Xu, School of Applied and Engineering Physics, Cornell University, developed a technology that uses a single excitation light source to achieve multi-color three-photon imaging without complicated optical equipment.
Chris Xu is a well-known scientist in the field of three-photon microscopy deep brain imaging research.
He has been deeply involved in this field for more than 20 years.The researchers used single-photon, two-photon, and three-photon imaging to image Texas Red, SR 101, Alexa Fluor 546, DsRed, tdTomato, mCherry, and other red fluorescent dyes under different wavelength excitations, and found that the 1220～1360nm band The wavelength of the excitation light is the turning point from the lowest energy of the two-photon to the highest energy excited state of the three-photon.
More specifically, the signal-to-noise ratio of the image acquired under 1340nm excitation light is 4.
6 times, 15 times, and 82 times that at 1300 nm, 1260 nm, and 1220 nm, respectively.
The wavelength of the excitation light in the superficial cortex does not have a great influence on the signal-to-noise ratio of the images obtained by the Texas Red-labeled cerebral blood vessels on the two-photon and three-photon microscopes.
But in deep brain imaging (1050um), this difference is obvious: the two-photon microscope has an image signal-to-noise ratio of 0.
77 at 1220nm (the lowest energy excited state), while the three-photon microscope is excited by 1260, 1300, 1340, and 1650.
The image signal-to-noise ratios are 1.
4, 12, 47, and 16, respectively.
Whether it is imaging a variety of different red fluorescent dyes or brain regions at different depths, the image signal-to-noise ratio obtained at the excitation wavelength at 1340nm is the best.
PrismPlus mouse is a kind of rainbow transgenic tool mouse.
It expresses cyan fluorescent protein that marks oligodendrocytes, green fluorescent protein that marks microglia, yellow fluorescent protein that marks neurons, and astrocytes.
Red fluorescent protein.
Researchers can simultaneously excite a variety of fluorescence at a laser wavelength of 1340nm, realize four-color fluorescence imaging at different depths under four different photomultiplier tubes, and can observe the dynamic changes of these four cells at the same time.
With the passage of laser excitation time, the intensity of fluorescence excited by fluorescent substances gradually weakens, which is naked photobleaching toxicity.
In order to further confirm the photobleaching properties of the 1340nm wavelength, the researchers excited the same fluorescent substance marking astrocytes at 1340nm and 1650nm respectively, and found that the 1650nm wavelength excitation photobleaching is more toxic.
In general, this article has developed a multi-color three-photon imaging technology that uses a single wavelength to realize the real-time observation of the interactions between various cells in the brain in the deep brain regions of the living body.
[References] 1.
Hontani et al.
, Sci.
Adv.
2021; 7: eabf3531, Multicolor three-photon fluorescence imaging with single-wavelength excitation deep inmouse brain The pictures in the article are all from the references
