Fluorescence collection device, miniature two-photon microscope and two-photon imaging method

Abstract

An embodiment of the present invention provides a fluorescence collection device, a miniature two-photon microscope, and a two-photon imaging method, wherein the fluorescence collection device includes a first optical path and a second optical path, and the first optical path includes a first lens and a first optical filter in sequence , a dichroic mirror, an infrared filter and a coupling lens; the second optical path includes a coupling lens, an infrared filter, a dichroic mirror, a second filter and a second lens in sequence. The fluorescence collecting device, the miniature two-photon microscope and the two-photon imaging method provided by the embodiment of the present invention integrate two optical paths, by selecting the filter and the dichroic mirror, multiplexing the infrared filter and the coupling lens, it is realized without increasing the micro The weight and volume of the two-photon microscopic probe can realize simultaneous optogenetic manipulation and fluorescence signal collection in the brain of freely moving animals, and realize different forms of optogenetic stimulation, and then realize the fluorescence signal under different optogenetic stimulation. Synchronous collection, simple operation and convenient use.

Description

technical field [0001] The embodiments of the present invention relate to the technical field of two-photon microscopy, and in particular to a fluorescence collection device integrated with optogenetic manipulation, a miniature two-photon microscope, and a neuron two-photon imaging method based on the miniature two-photon microscope. Background technique [0002] Neuron is the structural and functional unit of the nervous system, which has the function of receiving, integrating and transmitting information. Neurons can be divided into three types according to their functions: afferent neurons (sensory neurons), interneurons (connection neurons) and efferent neurons (motor neurons). If classified according to the effect on subsequent neurons, it can be divided into excitatory neurons and inhibitory neurons. As an important part of the nervous system, neurons have always been an important research topic in the fields of medicine and biology. The study of neuron activity gene...

AI Technical Summary

Problems solved by technology

[0003] Using a single optical fiber to observe neurons in the animal brain is to directly introduce the laser optical fiber to a specific brain area of ​​the animal brain, and then turn on the laser light source to irradiate (control) a specific optogenetic protein to observe the brain neurons. The advantage is the structure Simple, but this method cannot accurately complete the optogenetic manipulation of individual neurons, and cannot image the corresponding neurons; when the traditional desktop two-photon microscope is used to observe and image the neurons of the animal brain, due to the traditional The desktop two-photon microscope is very bulky, and the optogenetic optical path is integrated in the main mirror body. The experimental animals must be firmly fixed under the microscope objective or placed under anesthesia for imaging and optogenetics manipulation. The traditional desktop two-photon microscope is used to study the free movement of mice under the control of optogenetics. At present, a treadmill can only be installed under the mouse, and a display screen is placed in front of the mouse to simulate the movement of the mouse through virtual reality.

However, this method of virtual reality has been questioned by many scientists, who believe that this method is very different from the real movement and behavior of animals, and cannot obtain accurate neuron activity information.

The miniature single-photon microscope can be worn on the head of the mouse to observe and image the neurons in the neural region of the mouse brain under the condition of free movement of the mouse. However, due to the poor contrast of the miniature single-photon microscope, it is impossible to clearly see To nerve dendrites, axons and even neuron cell bodies, and because it is worn on the head of the mouse, limited by its volume, only the fluorescence collection device for fluorescence signal collection is integrated in the miniature single-photon microscope, and the optogenetic inheritance cannot be completed synchronously. Manipulation and fluorescence signal collection

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