WO2018145659A1 - 一种飞秒脉冲激光调制器及微型双光子显微成像装置 - Google Patents

一种飞秒脉冲激光调制器及微型双光子显微成像装置 Download PDF

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WO2018145659A1
WO2018145659A1 PCT/CN2018/076305 CN2018076305W WO2018145659A1 WO 2018145659 A1 WO2018145659 A1 WO 2018145659A1 CN 2018076305 W CN2018076305 W CN 2018076305W WO 2018145659 A1 WO2018145659 A1 WO 2018145659A1
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laser
fiber
modulator
optical path
sample
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PCT/CN2018/076305
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English (en)
French (fr)
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陈良怡
宗伟健
程和平
吴润龙
李明立
张云峰
王爱民
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北京大学
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Priority to US16/479,913 priority Critical patent/US20190380585A1/en
Priority to JP2019541739A priority patent/JP2020509402A/ja
Priority to EP18751055.7A priority patent/EP3582342A4/en
Publication of WO2018145659A1 publication Critical patent/WO2018145659A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/0033—Features or image-related aspects of imaging apparatus classified in A61B5/00, e.g. for MRI, optical tomography or impedance tomography apparatus; arrangements of imaging apparatus in a room
    • A61B5/004—Features or image-related aspects of imaging apparatus classified in A61B5/00, e.g. for MRI, optical tomography or impedance tomography apparatus; arrangements of imaging apparatus in a room adapted for image acquisition of a particular organ or body part
    • A61B5/0042—Features or image-related aspects of imaging apparatus classified in A61B5/00, e.g. for MRI, optical tomography or impedance tomography apparatus; arrangements of imaging apparatus in a room adapted for image acquisition of a particular organ or body part for the brain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
    • A61B5/0062—Arrangements for scanning
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
    • A61B5/0071—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence by measuring fluorescence emission
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/4833—Physical analysis of biological material of solid biological material, e.g. tissue samples, cell cultures
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0032—Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0036—Scanning details, e.g. scanning stages
    • G02B21/0048—Scanning details, e.g. scanning stages scanning mirrors, e.g. rotating or galvanomirrors, MEMS mirrors
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/008—Details of detection or image processing, including general computer control
    • G02B21/0084—Details of detection or image processing, including general computer control time-scale detection, e.g. strobed, ultra-fast, heterodyne detection
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01S—DEVICES USING THE PROCESS OF LIGHT AMPLIFICATION BY STIMULATED EMISSION OF RADIATION [LASER] TO AMPLIFY OR GENERATE LIGHT; DEVICES USING STIMULATED EMISSION OF ELECTROMAGNETIC RADIATION IN WAVE RANGES OTHER THAN OPTICAL
    • H01S3/00—Lasers, i.e. devices using stimulated emission of electromagnetic radiation in the infrared, visible or ultraviolet wave range
    • H01S3/005—Optical devices external to the laser cavity, specially adapted for lasers, e.g. for homogenisation of the beam or for manipulating laser pulses, e.g. pulse shaping
    • H01S3/0057—Temporal shaping, e.g. pulse compression, frequency chirping
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01S—DEVICES USING THE PROCESS OF LIGHT AMPLIFICATION BY STIMULATED EMISSION OF RADIATION [LASER] TO AMPLIFY OR GENERATE LIGHT; DEVICES USING STIMULATED EMISSION OF ELECTROMAGNETIC RADIATION IN WAVE RANGES OTHER THAN OPTICAL
    • H01S3/00—Lasers, i.e. devices using stimulated emission of electromagnetic radiation in the infrared, visible or ultraviolet wave range
    • H01S3/005—Optical devices external to the laser cavity, specially adapted for lasers, e.g. for homogenisation of the beam or for manipulating laser pulses, e.g. pulse shaping
    • H01S3/0085—Modulating the output, i.e. the laser beam is modulated outside the laser cavity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B2503/00—Evaluating a particular growth phase or type of persons or animals
    • A61B2503/40—Animals
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B2207/00—Coding scheme for general features or characteristics of optical elements and systems of subclass G02B, but not including elements and systems which would be classified in G02B6/00 and subgroups
    • G02B2207/114—Two photon or multiphoton effect
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B26/00—Optical devices or arrangements for the control of light using movable or deformable optical elements
    • G02B26/08—Optical devices or arrangements for the control of light using movable or deformable optical elements for controlling the direction of light
    • G02B26/10—Scanning systems
    • G02B26/101—Scanning systems with both horizontal and vertical deflecting means, e.g. raster or XY scanners

Definitions

  • the present invention relates to the field of optical imaging technology, and in particular to a femtosecond pulse laser modulator and a miniature two-photon microscopy imaging device.
  • One of the ultimate goals of neuroscience is to understand the basic principles of subcellular, cellular, loop, and higher-level neuronal information processing in free-living animals.
  • optical microscopy has become a basic research tool in this task because it allows direct observation of neuronal activity over multiple temporal and spatial scales.
  • a single synapse is the basic unit of information transfer, processing, and storage, and is essential for understanding brain function and disease mechanisms.
  • Postsynaptic structures - Dendritic spines are submicron structures that are deeply buried in the brain and move at milliseconds.
  • Multiphoton microscopy has been the technology of choice for non-invasive optical brain imaging in the past two decades due to its inherent optical sectioning and deep tissue penetration.
  • TPM Tu-Photon Microscopy
  • Observing the complex dendritic spine activity of the living sample head in awake state can be achieved by a state-of-the-art benchtop multiphoton microscope equipped with fast image acquisition (acquisition frequency greater than 15 Hz), high excitation and photodetection efficiency.
  • acquisition frequency greater than 15 Hz
  • photodetection efficiency the head of the living sample was fixed all the time, under physical constraints and emotional stress throughout the experiment, and there was no prior verification that the response of the neuron to the outside world was equivalent under virtual reality and free exploration. More importantly, many social behaviors, such as parent-child care, mating, and fighting, cannot be studied with head-fixed experiments.
  • mTPMs 920 nm femtosecond laser pulses to ensure no distortion of the pulse to the sample.
  • mTPMs often exhibit lower than their theoretical resolution in in vivo experiments, possibly due to the low sampling rate and the image of the miniature graded index (English name “GradedIndex Lenses”, hereinafter referred to as "GRIN"). Poor, micro optics defects and motion-induced imaging noise and so on.
  • GRIN Genetic name “GradedIndex Lenses”
  • the activity of neuronal structures with dendritic spine resolution cannot be well solved by mTPMs, although its theoretical lateral resolution is about 1 ⁇ m.
  • the present invention provides a femtosecond pulse laser modulator comprising a negative dispersion optical path and a positive dispersion optical path; wherein the negative dispersion optical path comprises a laser input optical fiber, and the laser input optical fiber is used for transmitting pre-compensation compensation
  • the laser is disposed between the femtosecond pulse laser and the negative dispersion optical path for pre-compensating for the negative dispersion generated by the laser in the laser input fiber.
  • the invention also provides a miniature two-photon microscopic imaging device comprising:
  • a femtosecond pulsed laser for generating a laser with a center wavelength of 920 nm
  • the femtosecond pulse laser modulator for receiving the laser light output by the femtosecond pulse laser, performing pulse amplification, and then outputting to the micro probe;
  • micro probe comprising:
  • a fluorescent output fiber for outputting the fluorescent signal.
  • the invention also provides a living body sample behavior imaging system, comprising:
  • a miniature two-photon microscopic imaging apparatus according to any of the above;
  • the laser input fiber and the fluorescent output fiber being mounted on the case by the line mounting assembly in a freely rotatable manner relative to the case;
  • a data acquisition component for collecting the fluorescent signal output by the fluorescent output fiber.
  • the invention also provides a miniature two-photon microscopic imaging method, which comprises:
  • Selecting an area to be imaged fixing a living body sample, and selecting a region to be imaged on the living body sample by using a bench-top two-photon microscope;
  • the micro probe of the micro two-photon microscopic imaging device is mounted on the living body sample, and the living body sample is released to collect the fluorescent signal outputted by the area to be imaged.
  • the femtosecond laser modulator provided by the invention provides distortion-free transmission for a femtosecond pulsed laser with a center wavelength of 920 nm, and effectively excites commonly used biological indicators.
  • the miniature two-photon microscopy imaging device (hereinafter referred to as "FIRM-TPM") including the femtosecond laser modulator is fast and high-resolution, and can be used to solve the imaging of individual dendritic spines in free-living animals. problem.
  • the microscopic microscope of the present invention can be used for somatic cells of the GCaMP6f-labeled cortical neurons, trees Bursting and dendritic spines are observed.
  • the FIRM-TPM represents the next generation of microscopic microscopes that meet the needs of high-resolution brain imaging in free-living animals.
  • FIG. 1 is a schematic structural view of a preferred embodiment of a FIRM-TPM provided by the present invention.
  • Fig. 2 is a schematic view showing the state in which the microprobe of Fig. 1 is mounted on a mouse.
  • FIG. 3 is a schematic diagram of the optical path of the microprobe of FIG. 1.
  • FIG. 4 is a schematic structural view of a preferred embodiment of a femtosecond pulse laser modulator provided by the present invention.
  • Figure 5a is a schematic view showing the structure of a living sample behavior imaging system using the miniature two-photon microscopic imaging apparatus of Figure 1 of the present invention.
  • Figure 5b is an exploded schematic view of the data collection assembly and line mounting assembly of Figure 5a.
  • Figure 6 is a schematic diagram of the optical path of the integrated platform of the desktop TPM, miniature wide field fluorescence microscope and FIRM-TPM in benchtop two-photon mode, which shows the structure of the desktop TPM.
  • FIG. 7 is a schematic diagram of the optical path of the integrated platform of FIG. 6 in a wide field imaging mode.
  • FIG. 8 is a schematic view of the optical path of the integrated platform of FIG. 6 in the FIRM-TPM imaging mode, which is illustrated in the optical path between the objective lens and the object surface (live sample or living sample) in the desktop TPM of FIG. Figure 1 of the FIRM-TPM.
  • Figures 9a-9f compare the two-photon excitation efficiencies of GCaMP-6f at 800 nm, 920 nm, and 1030 nm excitation.
  • Figure 10a shows the transmission loss and dispersion parameters of a custom-designed HC-920 fiber.
  • the illustration on the upper left shows the cross-section of the HC-920.
  • the illustration on the upper right shows the HC-920 and is 3mm.
  • Figure 10b is an autocorrelation distribution of the 920-nm femtosecond laser pulse width at the exit of a 1 meter HC-920 at different laser powers.
  • the left image shows the frequency response of the x (light grey) and y (black) axes of the MEMS scanner.
  • Right side plate The mechanical tilt angle is a function of the drive voltage of the x (black) and y (light gray) axes of the MEMS scanner.
  • Figures 11a-11d are illustrations of dispersion compensation in the femtosecond pulsed laser modulator of Figure 4.
  • Figures 12a-12e compare the dendritic activity in the cortex of the living sample V-1 in a dark environment under head fixation and free movement conditions.
  • Figures 13a-13c are imaging visual cortical activity of a living sample in a different behavioral paradigm using the in vivo sample behavioral imaging system of Figure 5.
  • Figures 14a-14d are performance comparisons of FIRM-TPM with benchtop TPM, miniature wide field microscopes.
  • a miniature two-photon microscopic imaging device (referred to as “Fast high resolution miniature two photon microscope” in the present invention; hereinafter referred to as “FIRM-TPM”) includes a femtosecond pulse laser. (shown in Figure 1), a femtosecond pulsed laser modulator 1 and a microprobe 2, wherein:
  • the femtosecond pulsed laser is used to generate a laser having a center wavelength of 920 nm.
  • the femtosecond pulsed laser is not shown in the figure. It can use a coherent company chameleon solid-state laser as a light source with a center wavelength of 920 nm and an output power of 100 ⁇ 1 mW.
  • lasers with a wavelength of 920 nm can effectively excite commonly used biological indicators (such as Thy1-GFP and GCaMP-6f, among which GCaMP is the most commonly used calcium ion indicator). Agent) produces fluorescence.
  • Figure 9a is an imaging of the same set of neuronal cell bodies cell1, cell2, and cell3 in AAV-transfected live samples expressing GCaMP-6f in the V-1 cortex at different excitation wavelengths, from left
  • the right-to-right order is a fluorescence image obtained using lasers of 800 nm, 920 nm, and 1030 nm, respectively.
  • Figure 9b is the frequency of neuronal cell bodies of the Ca 2+ signal of 10 glial cells.
  • FIG. 9c c is the amplitude of the neurons 10 Ca 2+ signal glial cells, d of Figure 9c is a mean duration of neurons 10 Ca 2+ signal glial cells.
  • Figure 9d as in Figure 9a, the neuron dendrites dendrite 1, dendrite 2 and dendrite 3 are imaged at different focal planes, with left-to-right sequential fluorescence images obtained using 800 nm, 920 nm and 1030 nm lasers, respectively.
  • Figure 9e is the frequency of the ten dendritic Ca 2+ signals shown in Figure 9d.
  • g in Figure 9f is the amplitude of the ten dendritic Ca 2+ signals from Figure 9d
  • h in Figure 9f is the average time course from the ten dendritic Ca 2+ signals shown in Figure 9d.
  • the femtosecond laser pulse centered at 800 nm can not excite the GCaMP6 signal from the neuron or dendrites at all, and the 1030 nm excitation can only record the dendritic calcium ion activity compared with the 920 nm excitation. /10, while the amplitude of the recorded fluorescent signal is significantly reduced.
  • the experimental data further demonstrates that the use of a laser with a wavelength of 920 nm can more efficiently excite GCaMP-6f to produce fluorescence.
  • the femtosecond pulse laser modulator 1 is configured to receive the laser output from the femtosecond pulse laser, amplify the transmission power of the laser to a preset value, and compensate the pulse width of the laser by pre-compensation, and then output.
  • a 85fs, 920nm centered laser pulse after being amplified by the femtosecond pulsed laser modulator 1, is modulated into a laser pulse of approximately 100fs, and the transmission power can be from 5mW to 200mW (this data is experimentally applied to the laser input fiber 11 The measurement at the exit is obtained).
  • the micro probe 2 includes a scanning imaging portion and a fluorescent output fiber 21, wherein: the scanning imaging portion is configured to receive laser light output by the femtosecond pulse laser modulator 1, the laser scanning a tissue inside the living sample to excite the living body The sample produces a fluorescent signal.
  • the fluorescent output fiber 21 is configured to receive the fluorescent signal output by the scanning imaging portion and output the same.
  • the fluorescent output fiber 21 is selected from a novel flexible fiber bundle (SFB).
  • the fluorescent output fiber 21 in the present embodiment is formed by welding 700-900 glass fibers while maintaining The glass fibers are loose and separated between the two, which facilitates lightweight microscope probes carried by free-moving living samples and reduces motion artifacts, thereby minimizing torque and tension caused by animal motion without reducing photon collection efficiency.
  • the ends of all the glass fiber splices are 1.5 mm diameter cylinders for easy mounting on the microprobe 2.
  • Table 1 below is a performance parameter of the fluorescent output fiber 21 in the present embodiment, which has higher collection efficiency than the multimode fiber, and is more flexible than the previously used plastic fiber and the conventional fusion type fiber bundle. Multi-wavelength emission detection.
  • the fluorescent output fiber 21 transmits the fluorescent signal from the objective lens in the microprobe 2 to the distal GaAsP PMT (10770P-40, Hamamatsu, Japan), and its total collection efficiency is about 80% at 532 nm.
  • the microprobe 2 is integrated on the fixed bracket 28.
  • the microprobe 2 is attached to the head of a living body sample (such as a living sample such as a mouse shown in Fig. 2) in a detachable manner (screw connection).
  • the total weight of the microprobe 2 and the fixed bracket 28 is approximately 2.15 g and the volume is no more than 1 cm 3 .
  • the system is small enough and compact and light enough to be carried by the living sample.
  • the fixing bracket 28 is made of an aluminum material such as an aluminum frame.
  • the shape of the fixing bracket 28 can be designed in the shape of a helmet so that it can be easily mounted and disassembled, and the microprobe 2 can be stably mounted on the head of a freely movable living sample, thereby facilitating imaging for several hours in the same animal. And the field of view of the miniature two-photon microscopic imaging device (hereinafter referred to as "FOV") is maintained without drift in the presence of strong body and head movements.
  • FOV miniature two-photon microscopic imaging device
  • the living body sample is pre-trained to accommodate the microprobe mounted on its skull and 1.5% low melting point agarose is added to fill the brain tissue of the objective lens and the living sample of the microprobe 2 Between these, these operations significantly reduce the relative motion between the microprobe 2 and the brain. These measures improve the short-term and long-term stability of the experiment, allowing for high detection of behavior in animals that involve a large number of free movements of the body and head. Resolution imaging.
  • the femtosecond pulse laser modulator 1 has a negative dispersion optical path and a positive dispersion optical path, wherein: the negative dispersion optical path includes a laser input optical fiber 11 for using a pulse broadening pre- A compensated laser is transmitted to the scanned imaging portion.
  • the positive dispersion light path is located between the femtosecond pulse laser and the negative dispersion light path for compensating for a negative dispersion caused by the laser input fiber 11 during laser transmission.
  • the laser input optical fiber 11 used in this embodiment is a hollow photonic crystal fiber (hereinafter referred to as "HC-PCF"), that is, HC-920 appearing in the figure or in the text, and most of the laser propagation is through the intermediate of HC-920. Inflated core, which effectively minimizes pulse broadening due to nonlinear effects.
  • the laser input fiber 11 of the present embodiment is used to transmit a 920 nm femtosecond laser pulse of several hundred milliwatts, and its nonlinear pulse broadening is negligible (Fig. 10a and Table 2).
  • the laser input fiber 11 of the present embodiment provides distortion-free transmission for the 920 nano femtosecond laser pulse, an improvement that makes it possible to effectively activate commonly used biological indicators such as Thy1-GFP and GCaMP-6f.
  • the positive dispersion optical path includes a dispersion compensating element 12 and an acousto-optic modulator 13, wherein: the acousto-optic modulator 13 is configured to receive the laser light compensated by the dispersion compensating element 12, and adjust the laser intensity, and then output The scanned portion is imaged.
  • the acousto-optic modulator 13 can be implemented using existing products.
  • a dispersion compensating element 12 is disposed adjacent to the femtosecond pulse laser for compensating for the negative dispersion caused by the laser input fiber 11 during the transmission of the laser.
  • the negative dispersion in the laser input fiber 11 is relatively low, and the dispersion compensating element 12 can be accurately compensated by a pre-twisting method using a commercial material having a positive dispersion of a specific length.
  • Commercial material herein refers to a versatile material that finds a suitable positive dispersion compensation, i.e., dispersion compensating element 12, by selecting material and length after determining the negative dispersion of the laser input fiber relative to the custom laser input fiber 11.
  • the 85fs, 920nm centered laser pulse is transmitted through the 1m long laser input fiber 11 to become a pulse of about 100fs, and the transmission power can be from 5mW to 200mW (the data is experimentally applied to the laser input fiber). The measurement at the exit of 11 is obtained) (Fig. 10b).
  • the dispersion compensating element 12 is an H-ZF62 glass tube having a positive dispersion of 458 ps/nm/km in a laser of 920 nm wavelength.
  • Figure 11a is a schematic diagram of the compensation of HC-920 dispersion using H-ZF62 glass tube
  • Figure 11b is the dispersion parameter of glass H-ZF62
  • Figure 11c is a 920 nm femtosecond laser output from a 1-meter HC-920 without dispersion compensation Pulse width
  • Figure 11d is the pulse width of a 920 nm femtosecond laser before and after a 1 meter HC-920 with and without dispersion compensation.
  • the dispersion of the material In the laser input fiber 11, most of the laser propagates through the air core, so the nonlinear effect is minimal.
  • the optical density at the center of the optical member such as the lens and the mirror In the optical path of the microprobe 2, the optical density at the center of the optical member such as the lens and the mirror is relatively low, since the beam size is large (the diameter is not more than 2 mm), and thus almost no nonlinearity is caused. Since the front part of the microscope and the micro-components in the sample are relatively short in length, they are also negligible. Therefore, the main expansion effect originates from the dispersion of materials caused by the benchtop optics and the laser input fiber. By simplification, only the second-order dispersion is considered in the system of the present embodiment. Therefore, the pulse broadening of the entire system is:
  • n the number of optical devices in the system
  • Dn( ⁇ ) is the dispersion parameter caused by the nth component (optical device)
  • Ln represents the optical path length of the nth component (optical device);
  • ⁇ and ⁇ are the center wavelength and spectral width of the laser;
  • represents positive dispersion from the lens (negative D( ⁇ )) and abnormal dispersion from the laser input fiber (positive D) ( ⁇ ))
  • the final pulse width is expressed as:
  • Pinput and Poutput represent the input and output pulse widths of the laser.
  • the HC-920 used in the system provided in this embodiment has a length of 1 m (Lhc-920), measured by the manufacturer and shown in Figure 10a. Therefore, the extension of HC-920 is:
  • GVD represents the group velocity dispersion in the medium
  • c is the speed of light
  • n is the refractive index of the medium.
  • the negative dispersion caused by HC-920 is pre-compensated by inserting an H-ZF62 glass tube (the dispersion compensating element 12 in this embodiment is an H-ZF62 glass tube) into an optical path having positive dispersion.
  • This embodiment selects the H-ZF62 glass tube because it has a large positive dispersion (a dispersion of about 458 ps/nm/km at a wavelength of 920 nm), calculated by the refractive index information provided by Scott and equation (1.4). As shown in Figure 11b).
  • the required length of the H-ZF62 glass tube is calculated as follows:
  • this example uses a 12 cm long, 12.7 mm diameter H-ZF62 glass tube, which is less than the calculated value.
  • the laser pulse width from the HC-920 is compressed to approximately 100 fs over the entire range of test illumination power (Fig. 11d and Fig. 10b).
  • the FIRM-TPM of this embodiment can experimentally image commonly used biological indicators by using a self-designed HC-PCF to transmit a 920 nm femtosecond laser pulse (named HC-920) with negligible nonlinear pulse broadening. And achieved comparable performance with the desktop TPM.
  • the positive dispersion optical path further includes a laser orientation adjustment component disposed between the dispersion compensation component 12 and the acousto-optic modulator 13, the laser orientation adjustment component including the first half wave a sheet 14, a first mirror 15 and a second mirror 16, wherein:
  • the first half wave plate 14 is for receiving the laser light compensated by the dispersion compensating element 12, and adjusting the laser polarization direction so that the modulation efficiency of the acousto-optic modulator 13 is the highest.
  • the first mirror 15 is for receiving the laser light passing through the first half-wave plate 14, and reflects the laser light to adjust the position at which the laser light is incident on the acousto-optic modulator 13.
  • the second mirror 16 is for receiving the laser light passing through the first mirror 15, and reflects the laser light to adjust the angle at which the laser light enters the acousto-optic modulator 13 and transmits it to the acousto-optic modulator 13.
  • the positive-dispersion optical path further includes a beam splitting component disposed adjacent to the laser input fiber 11 for receiving the laser light adjusted in intensity by the acousto-optic modulator 13 and dividing the laser into at least two beams, And transmitted to the laser input fiber 11.
  • the number of laser input fibers 11 is at least two, including a first laser input fiber and a second laser input fiber.
  • the beam splitting assembly includes a polarization beam splitter 17, a second half wave plate 18, a third mirror 19, a first collimating lens 110, a third half wave plate 111, a fourth mirror 112, and a second collimating lens 113. And a fourth half wave plate 114, wherein:
  • the polarization beam splitter 17 is for receiving the laser light whose intensity is adjusted via the acousto-optic modulator 13 and dividing the laser light into at least two beams, which are respectively transmitted to the first laser input fiber and the second laser input fiber.
  • the second half-wave plate 18 is disposed between the polarization beam splitter 17 and the acousto-optic modulator 13 for changing the splitting ratio of the polarization beam splitter 17.
  • the third mirror 19 is disposed between the polarization beam splitter 17 and the second half-wave plate 18 for reflecting the laser light to adjust the position of the laser light and project onto the incident surface of the polarization beam splitter 17.
  • the first collimating lens 110 is disposed between the first exit surface of the polarization beam splitter 17 and the first laser input fiber 11 for receiving the laser light output from the polarization beam splitter 17 and coupling the laser light into the first laser input fiber 11 .
  • the third half wave plate 111 is disposed between the first exit surface of the polarization beam splitter 17 and the first collimating lens 110 for receiving the laser light output by the polarization beam splitter 17, and adjusting the polarization direction of the laser so that the laser is coupled
  • the first laser input fiber 11 is the most efficient.
  • the fourth mirror 112 is disposed between the second exit surface of the polarization beam splitter 17 and the second laser input fiber for receiving laser light output by the polarization beam splitter 17 for reflecting the laser to adjust the position of the laser And projected into the second laser input fiber.
  • a second collimating lens 113 is disposed between the fourth mirror 112 and the second laser input fiber for receiving the laser light output by the polarization beam splitter 17 and coupling the laser light into the second laser input fiber.
  • the fourth half wave plate 114 is disposed between the fourth mirror 112 and the second collimating lens 113 for receiving the laser light reflected by the fourth mirror 112 and adjusting the polarization direction of the laser so that the laser is coupled into the second laser
  • the input fiber is the most efficient.
  • the stray light shutter 115 prevents unwanted light from being emitted.
  • the scanned imaging portion includes a microelectromechanical scanner 22, an objective lens 23, a scanning lens 24, a collimator 25, a dichroic mirror 26, and a collection lens 27, wherein:
  • a microelectromechanical scanner 22 is used to two-dimensionally scan a tissue plane inside the living sample by rotating the angle of the incident angle of the laser.
  • the microelectromechanical scanner 22 has a diameter of 0.8 mm, a package size of 9 ⁇ 9 mm 2 , a first resonant frequency of no more than 6 kHz, a maximum optical scanning angle of ⁇ 10 degrees, and a support frame size of 256 ⁇ 256 and maximum.
  • the field of view is 40 Hz imaging at 130 x 130 ⁇ m 2 to achieve video rate image acquisition.
  • the microelectromechanical scanner 22 Compared to other scanning methods, such as the use of a spiral of a piezoelectric actuator or a fiber scanning of a Lissajous pattern, the microelectromechanical scanner 22 provided by the present embodiment scans for its high speed, uniform excitation and largeness over the entire field of view. Both the scanning angle and the large field of view are advantageous.
  • the x and y control signals during the scanning of the MEMS scanner 22 are generated by an FPGA (Field Programmable Gate Array) card (PXI-7853R), which is also used to drive the acousto-optic modulator to adjust the laser intensity and trigger other devices ( For example, infrared camera acquisition).
  • FPGA Field Programmable Gate Array
  • the signal from the photodetector (H10770P-40, Hamamatsu Japan) is amplified by a high speed preamplifier (DHPCA-100, FEMTO Gmbh, Berlin, Germany) and then connected to a 120MS/s digitizer adapter module (NI5734, National Instruments Inc) ., Austin, TX, USA) and FlexRIO FPGA (PXIe-7961R, National Instruments Inc., Austin, TX, USA) for high speed data acquisition.
  • the entire system is controlled by custom development software based on the LabVIEW platform.
  • the objective lens 23 is used to concentrate the laser light from the microelectromechanical scanner 22 into the living body sample to excite the living body sample to generate the fluorescent signal and to output the fluorescent signal.
  • the numerical aperture of the objective lens 23 (hereinafter abbreviated as "NA") is 0.8, the NA is higher, and the optical layer is thinner, which is advantageous for achieving submicron imaging resolution, and thus can be distinguished in freely moving samples. Changes in the structure and function of dendrites and dendritic spines.
  • the objective lens 23 is a high-resolution water immersion small objective lens (GT-MO-080-018-AC900-450) of GRINTECH GmbH (Jena, Germany), which uses a magnification of 4.76 ⁇ , 0.80.
  • the object side NA and the image side NA of 0.173 are subject to limited correction.
  • the objective lens 23 has a length of 6 mm, a diameter of 1.4 mm, and a working distance of 200 ⁇ m.
  • the objective lens 23 specifically includes a thin Fresnel lens 231, a plano-convex lens 232, a relay lens 233, and a focus lens 234, wherein the thin Fresnel lens 231 and the plano-convex lens 232 are all miniature graded index (GRIN) lenses.
  • GRIN graded index
  • the diffraction component (Fresnel lens) incident on the objective lens 23 of the present embodiment effectively corrects a large chromatic aberration between the excitation and emission wavelengths during two-photon imaging, which makes A good beam focuses on quality and improves signal collection efficiency, which facilitates sub-micron imaging resolution, which in turn provides advantages for distinguishing between structural and functional changes in dendrites and dendritic spines in freely moving samples.
  • the scanning lens 24 is disposed on the optical path between the microelectromechanical scanner 22 and the objective lens 23 for converting the angularly varying laser light generated by the two-dimensional scanning of the microelectromechanical scanner 22 into a positionally varying laser light.
  • the scanning lens 24 uses an aspherical lens (#355160B, Lightpath Technologies, Orlando, FL, USA; diameter: 3 mm; equivalent focal length: 2.7 mm) as a scanning lens to reduce spherical aberration.
  • a collimator 25 is disposed between the laser input fiber 11 and the microelectromechanical scanner 22 for collimating the laser light output from the laser input fiber 11 and reducing chromatic aberration between lasers of different frequencies to match the objective lens 23 with the scanning lens 24.
  • the collimator 25 of the present embodiment uses an achromatic collimating lens (#65-286, Edmund Optics Inc., Barrington, NJ, USA; diameter: 2 mm, equivalent focal length: 3 mm, dedicated near-infrared light), capable of Collimating the output laser and reducing the chromatic aberration between the different frequency components of the femtosecond laser helps to improve transmission efficiency (up to 50% from laser source to sample), beam focusing and excitation efficiency.
  • a dichroic mirror 26 is provided between the scanning lens 24 and the objective lens 23 for separating the laser light and the fluorescent signal and outputting the fluorescent signal.
  • the dichroic mirror 26 is manufactured by China Sunlight Co., Ltd., having a size of 3 ⁇ 3 ⁇ 0.2 mm 3 , a reflection band of 750-1100 nm, and a transmission band of 400-650 nm to reflect the laser beam from the scanning lens 24 to Objective lens 23.
  • Acquisition lens 27 is used to efficiently collect fluorescent signals.
  • the present invention uses optical design software (ZEMAX) to simulate and optimize all optical components and the distance between them, and geometric design using commercial CAD software (Solidworks).
  • ZEMAX optical design software
  • CAD software Solidworks
  • the effective excitation NA (NAex) is determined by the entire optical path of the excitation.
  • the diameter D 1 of the laser beam after the collimator 25 is:
  • NA hc-920 is the NA of HC-920 (about 0.15), and EFLc is the equivalent focal length (3 mm) of collimator 25.
  • the size of the microelectromechanical scanner 22 is considered to be affected by its size. The limit is given by:
  • NA ex (1/2 ⁇ D m /EFL s ) ⁇ NA o /NA i (2.2)
  • EFL s is the equivalent focal length (2.7 mm) of the scanning lens 24.
  • NA o , NA i respectively represent the object side NA and the image side NA of the objective lens, which are 0.8 and 0.173, respectively.
  • the diffraction limit transverse direction ( ⁇ xy ) and the axial ( ⁇ z ) 1/e radius of the point spread function of the two-photon excitation (IPSF2) are calculated using the equation in Reference 2, and this embodiment has:
  • the immersion medium (physiological saline) has a refractive index of not more than 1.34, and the laser has a wavelength of 920 nm.
  • the resolution is usually defined as the full width at half maximum (FWHM) of PSF2.
  • This embodiment has:
  • the image was measured by using 100 nm fluorescent beads embedded in agarose.
  • the micro two-photon microscopic imaging device provided by the present invention has a lateral resolution of 0.64 ⁇ 0.02 ⁇ m and an axial resolution of 3.35 ⁇ 0.36 ⁇ m.
  • the experimental samples were prepared by testing the resolution of the present invention by first diluting 2 ⁇ L of TetraSpeck 100-nm fluorescent beads (T7270, Life Technologies, Oregon USA) into 100 ⁇ L of physiological saline, and then with 900 ⁇ L of 1.5. % low melting point agarose mixed. Agarose droplets containing microsphere fluorescent beads (4 ⁇ M) were then added to the coverslips and allowed to stand for 10 minutes.
  • the equivalent lateral pixel size is 61 nm and the axial scanning interval is 80 nm, both of which are greater than 5 times the Nyquist sampling theorem requirement, which is sufficient to measure the true optical resolution of the microscope.
  • Table 3 is a comparison table between the FIRM-TPM provided by the present invention and the resolution of the mTPM in the prior art, and the second column is a description of the resolution and parameters of the FIRM-TPM provided by the present invention, and columns 3-6 are existing.
  • SNR signal-to-noise ratio
  • the resolution of the FIRM-TPM provided by the present invention is about twice the highest resolution of the mTPM in the prior art, and this high resolution is to solve a single tree in a free-living animal.
  • the imaging problem of the spine provides favorable conditions.
  • this example establishes an integrated platform that allows benchtop TPM mode on the same sample ( Figure 6) Switching between the wide field imaging mode (Fig. 7) and the FIRM-TPM imaging mode (Fig. 8).
  • the integrated platform is equipped with miniature and conventional objectives, multiple illumination sources (lasers for TPM and high-power mercury lamps for wide fields) and detection devices (GaAsP for TPM and In the wide field sCMOS camera).
  • Different imaging modes have the same focal plane and concentric field of view, total frame acquisition time and imaging NA, while the average illumination power and detector sensitivity are also carefully selected and matched.
  • the above integrated platform is described below with reference to FIG. 6 to FIG. composition.
  • the above integrated platform images the same focal plane of the same specimen in the desktop TPM mode, the wide field imaging mode, and the FIRM-TPM imaging mode, respectively.
  • Laser (Chameleon Vision-S, Coherent, USA; center wavelength: 920 nm), passed through an H-ZF62 glass tube followed by an acousto-optic modulator (MT110-B50A1.5-IR-Hk, AA Sa, Orsay Cede, France).
  • the laser with a center wavelength of 920 nm is then split into two beams, one coupled to one HC-920 used in the FIRM-TPM and the other coupled to another HC-920 used in the benchtop TPM.
  • the collimated laser from HC-920 is scanned by a pair of galvanometer scanning mirrors A1 (6215H, Cambridge Technology, MA, USA) and then passed through scanning lens A2 (LMS05- BB, Thorlabs Inc, New Jersey), tube lens A3 (Olympus Japan) and first dichroic mirror A4 (DM1, DMLP650R, Thorlabs Inc, New Jersey, USA), and finally delivered to the back focal plane of the objective lens A5 (BFP) (CFI Apo 40XW NIR, Nikon, Japan; 40 x NA 0.8, working distance: 3.5 mm).
  • the fluorescent emission signal from the objective lens A5 is reflected by the first dichroic mirror A4, through the collecting lens A6 (LA1213-A, Thorlabs Inc, New Jersey, USA), the second dichroic mirror A7 (DM2, #87-284, Edmund) Optics Inc., Barrington, USA, reflection bands: 375-393, 466-490 and 546-565 nm; transmission bands: 420-460, 510-531, 590-624 and 677-725 nm), by concentrating lens A8 (ACL 25416U-A, Thorlabs Inc, New Jersey, USA), finally detected by photomultiplier tube A9 (GaAsP PMT 7422P-40, Hamamatsu, Japan).
  • the beam size of the excitation laser beam at the back focal plane (BFP) of the objective lens A5 is set to:
  • 200 mm is the EFL for the matching tube lens A3 of the objective lens A5, and 40 is the magnification of the objective lens A5.
  • the microprobe 2 provided by the present invention is connected to a custom micro triaxial manual micromanipulator (Sigma Koki, Tokyo, JAPAN).
  • the object mirror plane in the micro probe 2 is a focal plane of an air objective lens (Plan Apo NIR 5X, Mitutoyo, Japan; 5 ⁇ , NA is 0.14, working distance is 37.5 mm).
  • a 4-port objective dial (OT1, Thorlabs Inc, New Jersey, USA) with high precision bidirectional repeatability ( ⁇ 2.5 ⁇ m) was used to switch between the microprobe 2 and the 40 x Nikon objective.
  • the present invention can provide that the micro probe 2 and the Nikon objective lens have the same focal plane.
  • a fluorescent illuminator (X--) with a blue light filter (MF475-35, Thorlabs Inc, New Jersey, USA; center wavelength 475 nm, bandwidth 40 nm) was used.
  • the first dichroic mirror A4 in the benchtop TPM construction was changed to dichroic mirror B2 (DM3, #87-284, Edmund Optics Inc., Barrington, NJ, USA; reflection bands: 375-393, 466-490 and 546-565 nm)
  • the transmission band includes: 420-460, 510-531, 590-624, and 677-725 nm) to reflect illumination light to the microprobe 2, and to transmit the fluorescent emission signal from the microprobe 2 to the scientific CMOS camera B3 (8050M-GE, Thorlabs) Inc, New Jersey, USA). Imaging in the desktop TPM and FIRM-TPM configurations uses a similar average power after the target, typically 10-25 mW.
  • the average power used in single-photon imaging is typically much lower than the average power in two-photon imaging.
  • the present invention uses 100-500 [mu]W illumination (after the micro objective) in a wide field fluorescence microscope configuration, similar to the 170-600 [mu]W illumination used in previous reports.
  • the left image of Figure 14a is a 3D morphological image of neuronal dendrites and dendritic spines in a Thy1-GFP transgenic live sample brain, total exposure at 1 s (average of 8 Hz for 8 Hz for FIRM-TPM and micro-wide field of view microscopy) The value, and the average of the 2 Hz 2 frames of the desktop TPM, yields an image at approximately the same focal plane.
  • the right curve of Figure 14a is the transverse of two pairs of adjacent dendritic spines in the cropped region shown in the left image. Section profile.
  • Figure 14b is an image of a plane (-130 [mu]m) enriched in neuronal cell bodies in PFC expressing a biosample of GCaMP-6f, upper panel: 30 second average image of the same ROI.
  • the miniature wide field image is shown as a normalized ⁇ F/F (see online method); the lower wavy line: the time course of Ca 2+ in the three selected neurons (duration: 100 seconds) (used in the above image) Digital mark).
  • the left image of Figure 14c is the Ca 2+ signal from the dendrites and spinous processes (focal plane - 120 ⁇ m) of the same living sample in ( Figure 14b), due to the lack of discernible dendritic signals, not shown in miniature width
  • the image recorded by the field of view microscope the upper right image of Figure 14c is imaged with a dendritic axis (D1) and dendritic spines (S1, S2, S3) and a Ca 2+ signal captured by a benchtop TPM (duration: 100 s)
  • the lower right image of Figure 14c is an imaging and Ca 2+ signal (duration: 100 s) with a dendritic axis (D1) and dendritic spines (S1, S2, S3) (left) captured by FIRM.
  • the present invention images the V1 region (130 ⁇ 130 ⁇ m 2 , from the surface to 60 ⁇ m or less) in the fixed brain of the Thy1-GFP transgenic living sample to obtain 3D image data (Fig. 14a).
  • FIRM-TPM and benchtop TPM exhibited the same contrast and resolution in dendritic imaging (Fig. 14a).
  • structural details are essentially indistinguishable, primarily because of strong background signals from out-of-focus tissue.
  • the present invention uses the method of adeno-associated virus infection to express the Ca 2+ indicator agent GCaMP-6f in the prefrontal cortical neurons of the living sample, and performs calcium imaging on the head-fixed conscious living body sample (Fig. 14b). - Figure 14d). Two focal planes were chosen to visualize activity from the cell body (130 ⁇ m below the surface) or dendrites and dendritic spines (120 ⁇ m below the surface).
  • the fast microelectromechanical scanner 22 used in the FIRM-TPM has a more linear voltage-tilt response and controllability than the resonant scanner used in the high speed benchtop TPM. It therefore gives it greater flexibility in imaging, enabling fast raster scan imaging, random scan imaging of any given area (ROI), and ultra-fast line scan imaging in a single system.
  • 128 Hz random scan imaging helps to resolve the ultrastructure of the Ca 2+ signal over time and can confer mTPM the ability to perform precise optogenetic manipulations in freely moving animals.
  • the 10 kHz line scan imaging capability is critical for the rapid observation of action potentials using genetically encoded voltage indicators.
  • Dendritic spine activity is a fundamental event in neuronal information processing. Studies using bench-top TPMs on head-fixed animals have shown that different dendritic spines of individual nerve cells can be activated by visual stimuli of different orientations or by sound stimulation of different intensity frequencies. Although the overall activity of hundreds of neurons obtained by micro-wide field microscopy can help detect neural network coding characteristics under different behavioral conditions, the FIRM-TPM of the present invention provides an even more powerful tool to choose from. The temporal and spatial characteristics of the more basic neurocoding units are observed in the behavioral paradigm that is not achievable in head-fixed animals.
  • the newly developed FIRM-TPM has achieved high spatial and temporal resolution, good mechanical stability, and effective excitation of the most widely used indicators.
  • the present invention achieves the goal that scientists have dreamed of for long-term imaging at single synaptic levels in freely moving animals. It is foreseeable that there will be more improvements and extensions in the future.
  • the FOV and penetration depth can be further extended with a properly designed miniature high NA objective. Deep brain imaging can be achieved by inserting a miniature objective directly into the brain, or by embedding a GRIN lens in the brain, similar to previously reported methods.
  • FIRM-TPMs For larger animals, such as rats or marmosets, multiple FIRM-TPMs can be mounted on the skull to explore issues related to remote structural and functional connections in the brain.
  • the present invention contemplates that FIRM-TPM will prove to be an important tool for neuroscientists and biomedical scientists to explore health and disease mechanisms in animals.
  • the present invention further provides a living sample behavior imaging system, the living body sample behavior imaging system comprising a box 3, a femtosecond pulse laser, a femtosecond pulse laser modulator 1, a micro probe 2, and a data collection component. 7 and line installation components, where:
  • a femtosecond pulsed laser is used to generate a laser with a wavelength of 920 nm.
  • the femtosecond pulse laser modulator 1 is configured to receive the laser output from the femtosecond pulse laser, amplify the transmission power of the laser to its preset value, and compensate the pulse width of the laser by pre-compensation, and then output.
  • the micro probe 2 is mounted on the living body sample, and the input end is connected to the output end of the femtosecond pulse laser through a laser input fiber in the femtosecond pulse laser modulator for receiving the femtosecond pulse laser modulator
  • An output laser that scans tissue inside the living sample to excite the living sample to generate a fluorescent signal; and receives the fluorescent signal output from the scanned imaging portion and outputs the fluorescent signal.
  • the femtosecond pulse laser, the femtosecond pulse laser modulator 1 and the micro probe 2 have been described in detail above and will not be described again here.
  • the box 3 provides a defined space for the free movement of the living sample, which is much larger than the size of the living sample, and can provide a sufficient space for the free movement of the living sample.
  • the data collection assembly 7 is mounted on the housing 3, and the input end of the data collection assembly 7 is coupled to the output of the microprobe 2 via a fluorescent output fiber 21 for collecting the fluorescent signal output by the microprobe 2.
  • the laser input fiber 11 and the fluorescent output fiber 21 are mounted on the casing 3 by the line mounting assembly so as to be rotatable relative to the 3 casing. By the wire mounting assembly, the fluorescent output fiber 21, the laser input fiber 11 and the other power supply wires 8 can be prevented from intertwining with each other, providing favorable conditions for the free movement of the living sample.
  • the data collection assembly 7 provides a probe optical path that includes a coaxially disposed photomultiplier tube 71, a concentrator 72, an emission filter 73, a short pass filter 74, and a collection lens 75
  • the photomultiplier tube 71 is used to convert the optical signal into an electrical signal and output it.
  • a concentrator 72 is used to concentrate the fluorescent signal transmitted by the fluorescent output fiber 21.
  • the emission filter 73 is used to filter out a laser having a wavelength of 920 nm.
  • the short pass filter 74 is used to filter out stray light other than the signal light.
  • the fluorescent output fiber 21 is coaxial with the collecting lens 75, and the fluorescent output fiber 21 is located on the focal plane of the collecting lens 75, and the collecting lens 75 is used to collect the fluorescent signal more fully into the photomultiplier tube 71.
  • the line mounting assembly comprises an electric rotating head 6 and a bracket 10, wherein: the electric rotating head 6 is disposed at the top of the box 3, and one end is connected to an external power source, and the other end is connected to the power supply line 8 to make the outside
  • the power source and the power supply line 8 can be electrically connected.
  • the bracket 10 is disposed outside the electric rotating head 6 located at the top of the casing 3, and the laser input fiber 11 and the fluorescent output fiber 21 pass through the outer casing of the electric rotating head 6, and the fluorescent output fiber 21 and the collecting lens 75 in the data collecting unit 7 are light. Signal connection.
  • the fluorescent output fiber 21 is connected to the data collecting unit 7 via a bearing 9, so that the electric rotating head 6 and the bearing 9 allow the fluorescent output fiber 21 and the power supply line 8 to be independently rotated while maintaining the detection optical path unchanged.
  • This design prevents the living body from being invigorated.
  • the entanglement of the connecting line caused by the free exploration of the environment greatly improves the stability of the microprobe 2, even if the living sample undergoes strong physical activity, so that the line is twisted and entangled during free exploration. minimize.
  • the freely moving living sample behavior imaging device further includes a power supply line 8 for powering the microelectromechanical scanner 22.
  • the power supply line 8 is mounted on the box through the line mounting assembly so as to be rotatable relative to the box, thereby ensuring that the power supply line 8 can be powered normally, and also avoiding the power supply line 8 and the laser input fiber 11 and The fluorescent output fibers 21 are intertwined with each other to minimize distortion and entanglement of the wires during free exploration.
  • the freely moving living sample behavior imaging device further includes a plurality of cameras 4, both of which are mounted on the inner wall of the case 3, such as the side and top mounted on the case 3 shown in FIG. Face, the behavior of the free moving process of the living sample is taken and recorded at different angles.
  • the freely moving living sample behavior imaging device further includes an illumination lamp 5 mounted on an inner wall of the case 3 for illuminating the inner cavity of the case 3.
  • Figure 12a is an image of 50 average FIRM-TPM (micro-two-photon microscopy imaging devices of the present invention) of V-1 cortical L2/3 neurons, circled 1, 2 in the image. 3 dendrites.
  • Figure 12b is a two-dimensional trajectory (duration: 100 seconds) of a living sample in the arena, the trajectory is calculated from the video recording captured by the camera at the top, and the moving speed is color coded.
  • Figures 12c and 12d are the time course (duration: 100 s) of the Ca 2+ signal from three dendrites (circled in Figure 12a) under head fixation (12c) and free movement conditions (12d), respectively. Time-dependent changes in the speed of the living sample are also shown at the bottom.
  • Figure 13a is an imaging of neuronal activity in the visual cortex within three different behavioral paradigms: hangover test, platform, social behavior.
  • Leftmost first column image a snapshot of different behaviors taken by live samples taken by two cameras on the side of the behavioral device; middle second column image: GCaMP6f labeled neuronal cell body, dendrites and dendritic spines FIRM-TPM imaging; image of the third column on the right: corresponding calcium activity profile (duration: 100 s).
  • the fourth column image on the far right the Ca 2+ signal of the three ROIs indicated in the FIRM-TPM image (duration: 50 seconds).
  • Figure 13b shows the short-term and long-term stability of FIRM-TPM imaging.
  • the left curve of Figure 13b is the lateral displacement distribution based on the inter-frame cross-correlation analysis in each behavioral example, where FM (free moving) means free movement; (tail suspension) means a tailing; SD (stepping down from an elevated stage) means a platform; SI (social interaction) means a social behavior.
  • the curve on the right side of Figure 13b is the FOV drift during the 4 hour test.
  • the present invention performs 40 Hz imaging on a 130 ⁇ 130 ⁇ m 2 FOV in the primary visual cortex of a living sample to observe the activity of the neuron, which is in three behaviors.
  • the samples were tested in turn, including tail-hanging experiments, platform jumping, and social behavior. None of these examples can be implemented in an experimental strategy for head fixation, and the entire test process lasts about 4 hours.
  • the present invention analyzes the lateral displacement by frame-by-frame cross-correlation and drift of the field of view over a period of time (Fig. 13b).
  • the present inventors have found that within 2000 consecutive frames, the average displacement between frames is the largest in the example of the hopping behavior (0.19 ⁇ 0.09 ⁇ m; 75% below 0.24 ⁇ m), but still less than half the pixel size.
  • the overall drift of the FOV of the FIRM-TPM was ⁇ 10 ⁇ m, and most of the drift occurred during the first hour of strenuous exercise in the tail suspension experiment.
  • the present invention further analyzes the relationship between global and local activities by tracking Ca 2+ activity on dendritic spines and their parent dendritic axes. .
  • the present invention provides a miniature two-photon microscope that enables stable observation of dendritic and dendritic spine activity in freely moving animals in a natural physiological environment.
  • the invention also provides a miniature two-photon micro imaging method, the micro two-photon micro imaging method comprising:
  • Step 1 Select an area to be imaged: fix the living body sample, and select a region to be imaged on the living body sample by using a desktop TPM.
  • step 1 it is necessary to adopt the imaging platform as shown in FIG. 6 and in the desktop TPM mode, that is, to select the area to be imaged on the living sample by using the desktop TMP, the specific operation is: fixing the living sample by using the body fixer, The area of the virus infection at the head of the living sample is confirmed by the desktop TPM.
  • Step 2 collecting a fluorescent signal: mounting a micro probe connected with a laser input fiber on the living body sample, and releasing the living body sample to detect the fluorescence signal outputted in the image to be imaged selected in step 1 to complete the internal of the living sample. Imaging of the tissue plane. Step 2 is as follows:
  • the microprobe 2 provided by the present invention is adhered to a stent of a skull of a living sample head which has been fixed on the skull of the head of the living sample during the preparation of the craniotomy, and then the microprobe 2 is reinforced with dental cement. Go to the stent of the skull of the living sample head.
  • the process of labeling neurons is: artificial cerebrospinal fluid (Sigma-Aldrich, Shanghai, China; mM, 125NaCl, 4.5KCl, 26NaHCO 3 , 1.25NaH 2 PO 4 , 2CaCl) 2 , 1MgCl 2 , 20 glucose, pH 7.4 when filled with 95% O 2 and 5% CO 2 ) covers the exposed brain area. 50 ⁇ g of Cal-520AM (AAT Bioquest.
  • the living body sample is released from the holder and placed in the box 3 of the small animal imaging system of the above embodiment, and the living sample is subjected to a suspension test or a slip test test using the micro probe 2 (cylindrical flat, 5 cm). 2 , 5cm high) and social behavior (30 ⁇ 30cm 2 square event venue).
  • Step 3 processing the fluorescent signal to obtain a three-dimensional image of a freely moving living sample.
  • step 4 is further included to prepare a living sample, which specifically includes:
  • a live sample is selected: C57BL/6 wild type and Thy-1-GFP transgenic live samples (8-16 weeks after birth) are used in the experiment. All procedures are in accordance with the standards of the Peking University Animal Use and Care Committee and the Laboratory Animal Care Assessment and Certification Association, including animal breeding and experimental operations. Thy-1-GFP transgenic live samples were anesthetized and perfused with 0.9% saline, then perfused with 4% paraformaldehyde, and the brain removed. Fix overnight in 4% paraformaldehyde in PBS at 4 °C.
  • Step 42 osteotomy for in vivo cortical imaging: C57BL/6 wild type in vivo samples were used in these experiments. Briefly, live samples were anesthetized by inhalation of 1-1.5% isoflurane in pure O 2 and placed on a stereotactic frame (68025, RWD, Shenzhen, China). At the same time, the warming plate (37.5-38 ° C) was used to maintain the normal temperature of the living sample. After topical application of xylocaine and removal of skin and muscle, a small square head window (0.5 x 0.5 mm 2 ) was drilled in the middle of the target skin with a high speed cranial drill (tip diameter 0.5 mm).
  • GCaMP-6f was expressed with recombinant AAV under the calmodulin-dependent kinase II (CaMK II) promoter (serotype 2/9; >2 x 1013 genomic copies/ml, produced by the University of Pennsylvania gene therapy program vector core).
  • CaMK II calmodulin-dependent kinase II
  • a total of 0.5-0.8 ⁇ l of AAV was slowly injected into the 2/3 layer of the targeted cortex of the living sample in 20 minutes using a syringe (Nanoliter 2010, World Precision Instruments, Sarasota, USA) and a glass microelectrode (depth is 130-400 ⁇ m).
  • the live samples were anesthetized with isoflurane, and the self-made stent was attached to the skull with cyanoacrylate glue and reinforced with dental cement.
  • a small square head window (2.5 x 2.5 mm 2 ) was drilled on the target cortex with a cranial drill. Carefully remove the dura mater and place a small glass coverslip (3.5 x 2.5 mm 2 ) on the craniotomy.
  • the live samples were trained daily to accommodate head fixation for 30 minutes for 3 days. After the awake live sample is adapted, steps 1 and 2 are performed.

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Abstract

一种飞秒脉冲激光调制器、微型双光子显微成像装置、活体样本行为成像系统及微型双光子显微成像方法，该飞秒脉冲激光调制器包括负色散光路和正色散光路，负色散光路包括激光输入光纤，用于传输预啁啾补偿后的激光，正色散光路位于飞秒脉冲激光器和负色散光路之间，用于通过预啁啾补偿激光在激光输入光纤中产生的负色散。

Description

一种飞秒脉冲激光调制器及微型双光子显微成像装置 技术领域
本发明涉及光学成像技术领域，特别是涉及一种飞秒脉冲激光调制器及微型双光子显微成像装置。
背景技术
神经科学的最终目标之一是在自由活动的动物上了解亚细胞、细胞、环路和更高层次的神经元信息处理的基本原理。结合荧光指示剂，光学显微镜已经成为这一任务中的基本研究工具，因为它允许在多个时间和空间尺度上直接观测神经元活动。单个突触是信息传递、处理和存储的基本单位，对于理解脑功能和疾病机理至关重要。突触后结构——树突棘是亚微米结构，深埋在脑内，并以毫秒级的速度活动。由于其固有的光学切片和深层组织穿透能力，多光子显微镜一直是过去二十年内体内无创光学脑成像的首选技术。使用台式双光子显微镜(英文全称为“Two-Photon Microscopy”，下文均简称为“TPM”)，已能够在活体内观察到树突棘的形态变化，比如学习和记忆的神经元的活动。观察在清醒状态下活体样本头部复杂的树突棘活动可以通过配备有快速图像采集(采集频率大于15Hz)、高激发和光电探测效率的最先进的台式多光子显微镜实现。然而，活体样本的头部一直被固定，整个实验期间都处在物理约束和情绪压力下，而且没有先验证据表明神经元对外界的响应在虚拟现实和自由探索下是等价的。更重要的是，许多社会行为，比如亲子护理、交配和战斗，都不能用头部固定的实验来研究。
为了应对这些挑战，一个理想的解决方案是开发能够长时间观察活体样本在自由活动过程中的树突棘的结构和功能动态的微型化显微镜。Denk和他的同事在2001年建立了基于光纤尖端扫描的第一个微型双光子显微镜(英文全称为“micro Two-PhotonMicroscopy”，下文均简称为“mTPM”)，然后接下来的十年里，其他许多课题组采取不同方法继续尝试。然而，这些mTPM没有一个被用于后续的生物应用，主要是由于两个主要的限制：第一，没人能够成像应用最广泛的荧光探针，如GCaMPs，原因是缺乏适当的光纤用来传输920纳米飞秒激光 脉冲，以保证脉冲到样品没有失真。第二，mTPMs在体内实验中经常表现出低于它们的理论分辨率，原因可能是采样率低，微型渐变折射率(英文全称为“GradedIndex Lenses”，下文均简称为“GRIN”)透镜的像差，微型光学器件的缺陷和运动引起的成像噪声等等。目前，树突棘分辨率的神经元结构的活动不能被mTPMs很好的解决，尽管其理论横向分辨率为大约1μm。
另一方面，微型单光子宽场显微镜，例如由Ghosh及其同事开发的显微镜，已经实现了快速采集和大视场(英文全称为“Field of Vision”，下文均简称为“FOV”)以及解决了运动引起的噪声问题(在神经元分辨率)。然而，当前的无创微型宽场显微镜仅能获得细胞分辨率，并且图像对比度受到累积的焦外背景信号的影响。到目前为止，可以提供很好的成像能力和实验方案，而且足够强大到可以满足神经科学家的日常实验的新型的mTPM仍有待完成。
因此，希望有一种技术方案来克服或至少减轻现有技术的上述缺陷中的至少一个。
发明内容
发明的目的在于提供一种飞秒脉冲激光调制器及微型双光子显微成像装置来克服或至少减轻现有技术的上述缺陷中的至少一个。
为实现上述目的，本发明提供一种飞秒脉冲激光调制器，包括负色散光路和正色散光路；其中，所述负色散光路包括激光输入光纤，所述激光输入光纤用于传输预啁啾补偿后的激光；所述正色散光路位于飞秒脉冲激光器和所述负色散光路之间，用于通过预啁啾补偿所述激光在所述激光输入光纤中产生的负色散。
本发明还提供一种微型双光子显微成像装置，包括：
飞秒脉冲激光器，用于产生中心波长为920纳米的激光；
上述任一项所述的飞秒脉冲激光调制器，用于接收所述飞秒脉冲激光器输出的激光，并进行脉冲放大，然后输出给微型探头；
以及微型探头，所述微型探头包括：
扫描成像部分，用于接收所述飞秒脉冲激光调制器输出的激光，并将所述激光对活体样本内部的组织进行扫描，以激发所述活体样本内部的生物指示剂产生荧光信号；以及，
荧光输出光纤，其用于输出所述荧光信号。
本发明还提供一种活体样本行为成像系统，包括：
上述任一项所述的微型双光子显微成像装置；
箱体，用于为活体样本的自由移动提供限定空间；
线路安装组件，所述激光输入光纤和荧光输出光纤通过所述线路安装组件以相对于箱体随意转动的方式安装在箱体上；以及，
数据采集组件，用于收集所述荧光输出光纤输出的所述荧光信号。
本发明还提供一种微型双光子显微成像方法，其特征在于，包括：
选取待成像区域：固定活体样本，并利用台式双光子显微镜在所述活体样本上选取待成像区域；
采集荧光信号：将上述任一项所述的微型双光子显微成像装置的微型探头安装在所述活体样本上，释放活体样本，以采集所述待成像区域输出的荧光信号。
本发明提供的飞秒激光调制器为中心波长920纳米的飞秒脉冲激光提供了无畸变传输，进行有效激发常用的生物指示剂。包括该飞秒激光调制器的微型双光子显微成像装置(下文简称为“FIRM-TPM”)测试和应用的速度快、分辨率高，能够用于解决自由活动动物中单个树突棘的成像问题。在涉及到不规则的、频繁的身体和头部运动的行为范例中(例如，尾悬挂，跳台，和社交行为)，本发明的微型显微镜都可以对GCaMP6f标记的皮层神经元的体细胞，树突和树突棘进行观测。综合起来，FIRM-TPM代表了下一代微型显微镜，它满足在自由活动动物中进行高分辨率脑成像的需求。
附图说明
图1是本发明所提供的FIRM-TPM一优选实施方式的结构示意图。
图2是图1中的微型探头安装在小老鼠上的状态示意图。
图3是图1中的微型探头的光路原理示意图。
图4是本发明所提供的飞秒脉冲激光调制器一优选实施方式的结构示意图。
图5a是本发明利用图1的微型双光子显微成像装置的活体样本行为成像系统的结构示意图。
图5b是图5a中的数据收集组件和线路安装组件的分解示意图。
图6是台式TPM、微型宽场荧光显微镜和FIRM-TPM各项性能的集成平 台在台式双光子模式下的光路示意图，该图示意出了台式TPM的结构示意图。
图7是图6的集成平台在宽场成像模式下的光路示意图。
图8是图6的集成平台在FIRM-TPM成像模式下的光路示意图，该图示意出了在图6的台式TPM中的物镜与物面(活体样本或活体样本)之间的光路上设置有图1的FIRM-TPM。
图9a-9f是比较GCaMP-6f在800nm、920nm和1030nm激发下的双光子激发效率。
图10a是定制设计的HC-920光纤的传输损耗和色散参数，其中，上方左侧的插图显示了HC-920的截面照片，上方右侧的插图显示了由HC-920射出并由具有3mm的焦距的双合透镜准直之后的920nm激光的图像。
图10b是在不同激光功率下1米HC-920出口处920-nm飞秒激光脉冲宽度的自相关分布。
图10c中，左侧图：MEMS扫描器的x(浅灰色)和y(黑色)轴的频率响应。右侧板：机械倾斜角作为MEMS扫描器的x(黑色)和y(浅灰色)轴的驱动电压的函数。
图11a-11d是图4的飞秒脉冲激光调制器中色散补偿说明。
图12a-12e是比较在头部固定和自由移动条件下在黑暗环境下活体样本V-1皮质中的树突活性。
图13a-13c是利用图5中的活体样本行为成像系统在不同的行为范例中活体样本的成像视觉皮层活动。
图14a-14d是FIRM-TPM与台式TPM、微型宽视场显微镜的性能比较。
具体实施方式
在附图中，使用相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面结合附图对本发明的实施例进行详细说明。
在本发明的描述中，术语“中心”、“纵向”、“横向”、“前”、“后”、“左”、“右”、“竖直”、“水平”、“顶”、“底”“内”、“外”等指示的方位或位置关系为基于附图所示的方位或位置关系，仅是为了便于描述本发明和简化描述，而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作，因此不能理解为对本发明保护范围的限制。
如图1所示，本实施例所提供的一种微型双光子显微成像装置(英文全称为 “Fast high resolution miniature two photon microscope”；下文均简称为“FIRM-TPM”)包括飞秒脉冲激光器(图1中为示出)、飞秒脉冲激光调制器1和微型探头2，其中：
所述飞秒脉冲激光器用于产生中心波长为920纳米的激光。所述飞秒脉冲激光器在图中并未示意出来，其可以使用相干公司变色龙固体激光器作为光源，中心波长为920nm，输出功率100±1mW。相比于现有的波长为1030nm和800nm的激光，波长为920纳米的激光能够有效地激发常用的生物指示剂(例如Thy1-GFP和GCaMP-6f，其中的GCaMP是最普遍使用的钙离子指示剂)产生荧光。下面是比较GCaMP-6f分别在800nm、920nm和1030nm激光激发下的双光子激发效率，利用可调谐的钛宝石飞秒激光器(Coherent，USA，Vision-S，680-1080nm，85-110fs)中选择不同波长进行激发。
如图9a-9f所示，图9a是在不同激发波长下在V-1皮质中表达GCaMP-6f的AAV转染活体样本中成像相同组的神经元胞体cell1、cell2和cell3，图中从左至右依序分别是使用800nm、920nm和1030nm激光获得的荧光图像。图9b是10个胶质细胞的Ca 2+信号的神经元胞体的频率。图9c中的c是10个胶质细胞的Ca 2+信号的神经元胞体的幅度，图9c中的d是10个胶质细胞的Ca 2+信号的神经元胞体的平均时程。图9d如在图9a中，在不同的焦平面成像神经元树突dendrite1、dendrite2和dendrite3，图中从左至右依序分别是使用800nm、920nm和1030nm激光获得的荧光图像。图9e是来自图9d中所示的十个树突Ca 2+信号的频率。图9f中的g是来自图9d中所示的十个树突Ca 2+信号的幅度，图9f中的h是来自图9d中所示的十个树突Ca 2+信号的平均时程。从这些实验数据可以看出：以800nm为中心的飞秒激光脉冲完全不能激发来自神经元胞体或树突的GCaMP6信号，1030nm激发与920nm激发相比，仅能记录到树突钙离子活性的1/10，同时所记录的荧光信号的振幅显著降低。实验数据进一步证明使用波长为920nm的激光可以更加有效地激发GCaMP-6f产生荧光。
再次参阅图1，飞秒脉冲激光调制器1用于接收所述飞秒脉冲激光器输出的激光，将激光的传输功率放大至预设值，并通过预啁啾补偿激光的脉冲展宽，然后输出。例如，85fs、920nm为中心的激光脉冲，在经由飞秒脉冲激光调制器1放大之后，调制成大约为100fs的激光脉冲，传输功率可以从5mW到200mW(该数据是通过实验在激光输入光纤11的出口处测量获得)。
微型探头2包括扫描成像部分和荧光输出光纤21，其中：所述扫描成像部 分用于接收飞秒脉冲激光调制器1输出的激光，该激光对活体样本内部的组织进行扫描，以激发所述活体样本产生荧光信号。荧光输出光纤21用于接收所述扫描成像部分输出的所述荧光信号，并进行输出。
如图1和图2所示，荧光输出光纤21选择的是一种新型的柔性光纤束(SFB)，本实施方式中的荧光输出光纤21由700-900根玻璃光纤熔接而成，同时，保持各玻璃光纤松散并在两者之间分开，这样便于由自由活动的活体样本携带并减少运动伪影的轻质显微镜探头，进而最小化动物运动引起的扭矩和张力，而不降低光子收集效率。所有的玻璃光纤熔接的两个端部呈1.5mm直径的圆柱体，以方便安装在微型探头2上。下表1是本实施例中的荧光输出光纤21的性能参数，其与多模光纤相比具有更高的收集效率，并且比之前使用的塑料光纤和传统的融合型光纤束更灵活，能够进行多波长发射检测。荧光输出光纤21将来自微型探头2中的物镜的荧光信号传递到远端的GaAsP PMT(10770P-40，Hamamatsu，日本)中，其总收集效率在532nm处为大约80％。
表1
光纤数量 800
单个光纤直径(μm) 30
数值孔径(NA) 0.65
收集角度(度) 80
传输效率(％/m) 90
占空比(％) 90
整体传输效率(％) 80
如图1和图2所示，微型探头2中集成在固定支架28上。通过固定支架28，微型探头2以可拆卸的方式(螺钉连接)安装在活体样本(如图2中示出的小老鼠等活体样本)的头上。微型探头2和固定支架28的总重量大约位2.15g，体积不超过1cm 3，系统足够小而紧凑和足够轻以便活体样本携带。固定支架28采用铝材料制成，比如铝框架。固定支架28的形状可以设计成一个头盔形状，这样易于安装和拆卸，主要是可以稳固地将微型探头2安装在自由活动的活体样本的头部，因此有利于在同一个动物中进行数小时成像，并保持微型双光子 显微成像装置的视场(下文简称为“FOV”)在存在强烈的身体和头部运动的情况下不产生漂移。除此之外，利用本发明进行成像之前，会预先训练活体样本适应安装在其头骨上的微型探头并滴加1.5％低熔点琼脂糖使其充满在微型探头2的物镜和活体样本的脑组织之间，这些操作都显著降低了微型探头2与大脑之间的相对运动，这些措施改善了实验短期和长期的稳定性，允许在动物进行包含大量身体和头部自由活动的行为检测中进行高分辨率成像。
在一个实施例中，如图4所示，飞秒脉冲激光调制器1具有负色散光路和正色散光路，其中：所述负色散光路包括激光输入光纤11，激光输入光纤11用于将脉冲展宽预啁啾补偿好的激光传输给所述扫描成像部分。所述正色散光路位于所述飞秒脉冲激光器和所述负色散光路之间，用于补偿由所述激光输入光纤11在传输激光过程中引起的负色散。
由于材料色散和非线性效应，单模光纤会将飞秒激光脉冲展宽。而本实施例中使用的激光输入光纤11为空心光子晶体光纤(下文简称为“HC-PCF”)，即图中或文中出现的HC-920，其中大多数激光传播是通过HC-920中间的充气芯，这有效地最小化了由于非线性效应产生的脉冲展宽。本实施例的激光输入光纤11用于传输数百毫瓦的920纳米飞秒激光脉冲，其非线性脉冲展宽可忽略不计(图10a和表2)。
表2
Figure PCTCN2018076305-appb-000001
Figure PCTCN2018076305-appb-000002
本实施例的激光输入光纤11为920纳米飞秒激光脉冲提供了无畸变传输，这种改进让有效激发常用的生物指示剂成为可能，例如Thy1-GFP和GCaMP-6f。
在一个实施例中，所述正色散光路包括色散补偿元件12和声光调制器13，其中：声光调制器13用于接收经由色散补偿元件12补偿后的激光，并调节激光强度，然后输出给所述扫描成像部分。声光调制器13可以采用现有产品实现。
色散补偿元件12临近所述飞秒脉冲激光器设置，用于补偿由激光输入光纤11在传输激光过程中引起的负色散。
激光输入光纤11中的负色散相对较低，色散补偿元件12只要使用具有特定长度的正色散的商业材料通过预啁啾方法就可以精确地补偿。这里的商业材料是指通用材料，相对于定制的激光输入光纤11，即在确定激光输入光纤的负色散后，通过选取材料和长度找到合适的正色散补偿，即色散补偿元件12。在预啁啾补偿之后，85fs、920nm为中心的激光脉冲经过1m长的激光输入光纤11传输后变成大约为100fs的脉冲，传输功率可以从5mW到200mW(该数据是通过实验在激光输入光纤11的出口处测量获得)(图10b)。
优选地，色散补偿元件12采用的是H-ZF62玻璃管，其在920nm波长的激光中具有458ps/nm/km的正色散。其中：图11a是使用H-ZF62玻璃管补偿HC-920色散示意图；图11b是玻璃H-ZF62的色散参数；图11c是从没有色散补偿的1米的HC-920输出的920nm飞秒激光的脉冲宽度；图11d是在具有和没有色散补偿的1米的HC-920之前和之后的920nm飞秒激光器的脉冲宽度。
下面是对飞秒脉冲激光调制器1实现对85fs、920nm为中心的激光脉冲预啁啾补偿之后，变成大约为100fs的激光脉冲，传输功率可以从5mW到200mW 的详细说明。
在超快激光脉冲的传输期间，两个因素有导致脉冲展宽：材料的色散以及高激光峰值功率的非线性效应。在激光输入光纤11中，大多数激光传播通过空气芯，因此非线性效应最小。在微型探头2的光路中，诸如透镜和反射镜的光学部件的中心处的光密度相对较低，因为光束尺寸较大(直径为不超过2mm)，因此几乎不引起非线性。由于显微镜的前部件和样本中的微型部件的长度相对较短，因此也可忽略。因此，主要的扩展效应起源于由台式光学器件和激光输入光纤引起的材料色散。通过简化，在本实施方式的系统中仅考虑二阶色散。因此，整个系统的脉冲展宽为：
Bsystem＝|∑n{Ln∫λ,ΔλDn(λ)dλ}|(1.1)其中：n表示系统中的光学器件的数量；Dn(λ)由第n个分量(光学器件)引起的色散参数；Ln表示第n分量(光学器件)的光路长度；λ和Δλ是激光的中心波长和光谱宽度；||表示来自透镜的正色散(负D(λ))和来自激光输入光纤异常色散(正D(λ))总和的绝对值。因此，当Δλ<<λ，在本发明的系统中(λ＝920nm，Δλ不超过15nm)，方程简化为：
Bsystem＝|∑n{Ln·Dn(λ)·Δλ}|(1.2)
最终脉冲宽度表示为：
Poutput＝Pinput+|Bsystem|(1.3)
其中Pinput和Poutput表示激光器的输入和输出脉冲宽度。
在本实施例提供的系统中使用的HC-920的长度为1m(Lhc-920)，由制造商测量并在图10a中示出。因此，HC-920的扩展是：
Lhc-920·Dhc-920(920nm)·Δλ～1(m)·75(ps/nm/km)·15(nm)＝1.125(ps)
容易确定诸如偏振分束器、透镜和声光调制器的长度，并且通过它们使用的镜片折射率信息来计算，其表示为：
Figure PCTCN2018076305-appb-000003
和
Figure PCTCN2018076305-appb-000004
其中，GVD表示介质中群速度色散，c是光速，n是介质的折射率。
加在一起，这些分量引起约-200fs的展宽，因此从85fs变换限制脉冲的HC-920输出的计算脉冲宽度约为：
(0.085+1.125-0.2)(ps)＝1.01(ps)
其与实验测量的脉冲宽度(不超过1ps)(图11c)匹配，并且对于有效的双光子激发太弱。通过将H-ZF62玻璃管(本实施例中的色散补偿元件12均采用H-ZF62玻璃管)插入具有正色散的光路中，本实施例预补偿了由HC-920引起的负色散。本实施例选择H-ZF62玻璃管，因为它具有大的正色散(在波长为920nm中的色散大约为458ps/nm/km)，由Scott公司提供的折射率信息和等式(1.4)计算，如图11b所示)。所需的H-ZF62玻璃管的长度计算如下：
(1.125-0.2)(ps)/458(ps/nm/km)/15(nm)＝13.5(cm)
考虑到由显微镜前端和样品引起的小的正色散，本实施例使用一个12厘米长、12.7毫米直径的H-ZF62玻璃管，小于计算值。在H-ZF62玻璃管(图11b和图6)的这种“预线性调频”之后，来自HC-920的激光脉冲宽度在测试照明功率的整个范围内被压缩到大约为100fs(图11d和图10b)。本实施例的FIRM-TPM通过使用自行设计的HC-PCF来传输具有可忽略的非线性脉冲展宽的920纳米飞秒激光脉冲(名为HC-920)，能够实验对常用的生物指示剂进行成像，并取得了和台式TPM相当的性能。
在一个实施例中，所述正色散光路还包括激光方位调整组件，所述激光方位调整组件设置在色散补偿元件12与声光调制器13之间，所述激光方位调整组件包括第一半波片14、第一反射镜15和第二反射镜16，其中：
第一半波片14用于接收经由色散补偿元件12补偿后的激光，并调整激光偏振方向，以使得声光调制器13的调制效率最高。第一反射镜15用于接收经由第一半波片14的激光，并反射激光，以调整激光射入声光调制器13的位置。第二反射镜16用于接收经由第一反射镜15的激光，并反射激光，以调整激光射入声光调制器13的角度，并传输给声光调制器13。
在一个实施例中，所述正色散光路还包括分光组件，所述分光组件临近激光输入光纤11设置，用于接收经由声光调制器13调节好强度的激光以及将该激光分成至少两束，并传输给激光输入光纤11。
在一个实施例中，激光输入光纤11的数量至少为两根，包括第一激光输入光纤和第二激光输入光纤。
所述分光组件包括偏振分束器17、第二半波片18、第三反射镜19、第一准直透镜110、第三半波片111、第四反射镜112、第二准直透镜113和第四半波片114，其中：
偏振分束器17用于接收经由声光调制器13调节好强度的激光以及将该激光分成至少两束，分别传输给所述第一激光输入光纤和第二激光输入光纤。第二半波片18布置在偏振分束器17和声光调制器13之间，用于改变偏振分束器17的分光比。第三反射镜19布置在偏振分束器17和第二半波片18之间，用于反射激光，以调整激光的位置并投射到所述偏振分束器17的入射面上。第一准直透镜110布置在偏振分束器17的第一出射面与第一激光输入光纤11之间，用于接收偏振分束器17输出的激光以及将激光耦合进第一激光输入光纤11。第三半波片111布置在偏振分束器17的第一出射面与第一准直透镜110之间，用于接收偏振分束器17输出的激光，并调整激光偏振方向，使得激光耦合进第一激光输入光纤11的效率最高。第四反射镜112布置在偏振分束器17的第二出射面与所述第二激光输入光纤之间，用于接收偏振分束器17输出的激光，用于反射激光，以调整激光的位置并投射到所述第二激光输入光纤中。第二准直透镜113布置在第四反射镜112与第二激光输入光纤之间，用于接收偏振分束器17输出的激光以及将激光耦合进所述第二激光输入光纤。第四半波片114布置在第四反射镜112与第二准直透镜113之间，用于接收第四反射镜112反射的激光，并调整激光偏振方向，使得激光耦合进所述第二激光输入光纤的效率最高。可选地，杂散光遮挡器115，避免不需要的光射出。
如图1和图3所示，在一个实施例中，所述扫描成像部分包括微机电扫描仪22、物镜23、扫描透镜24、准直器25、双色镜26和采集透镜27，其中：
微机电扫描仪22(MEMS)用于通过转动改变激光入射角角度的方式将激光对所述活体样本内部的组织平面进行二维扫描。优选地，微机电扫描仪22的直径为0.8mm，封装尺寸为9×9mm 2，第一谐振频率为不超过6kHz，其最大光学扫描角度为±10度,支持帧大小为256×256和最大视场为130×130μm 2的40Hz成像,以实现视频速率图像采集。与其它扫描方式相比，例如使用压电致动器的螺旋或Lissajous图案的光纤扫描相比，本实施例提供的微机电扫描仪22扫描对于其在整个视场上的高速，均匀激发和大扫描角、大视野都是有利的。微机电扫描仪22扫描过程中的x和y控制信号由FPGA(现场可编程门阵列)卡(PXI-7853R)生成，该卡也用于驱动声光调制器以调整激光强度和触发其它器件(例如，红外摄像机采集)。来自光电检测器(H10770P-40，Hamamatsu Japan)的信号通过高速前置放大器(DHPCA-100，FEMTO Gmbh，Berlin，Germany)放大，然后连接到120MS/s数字转换器适配器模块(NI5734，National Instruments Inc.， Austin，TX，USA)和FlexRIO FPGA(PXIe-7961R，National Instruments Inc.，Austin，TX，USA)，用于高速数据采集。整个系统由基于LabVIEW平台的定制开发软件控制。
物镜23用于将来自微机电扫描仪22的激光会聚到所述活体样本内部，以激发所述活体样本产生所述荧光信号以及用于输出所述荧光信号。优选地，物镜23的数值孔径(下文均简称为“NA”)为0.8，NA较高，光学层切较薄，这有利于实现亚微米成像分辨率，进而能够在自由活动的样本中分辨出树突和树突棘的结构和功能变化。
本实施例中，物镜23使用的是GRINTECH GmbH(Jena，Germany)的高分辨率水浸小型物镜(GT-MO-080-018-AC900-450)，这是用4.76×的放大率，0.80的物方NA和0.173的像方NA进行有限校正。物镜23的长度为6mm长，直径是1.4mm，并且具有200μm的工作距离。物镜23具体包括薄菲涅耳透镜231、平凸透镜232、中继透镜233和聚焦透镜234，其中，薄菲涅耳透镜231和平凸透镜232都是微型渐变折射率(GRIN)透镜。与现有技术中使用的物镜相比，本实施例入射到物镜23中的衍射分量(菲涅尔透镜)有效地校正了双光子成像期间激发和发射波长之间的大色差，这会使得更好的光束聚焦质量，且改善信号收集效率，从而有利于实现亚微米成像分辨率，进而为能够在自由活动的样本中分辨出树突和树突棘的结构和功能变化提供有利条件。
扫描透镜24布置在微机电扫描仪22和物镜23之间的光路上，用于将微机电扫描仪22二维扫描所产生的角度变化的激光转化成位置变化的激光。扫描透镜24使用非球面透镜(#355160B，Lightpath Technologies，Orlando，FL，USA；直径：3mm；等效焦距：2.7mm)作为扫描透镜，以减少球面像差。
准直器25布置在激光输入光纤11与微机电扫描仪22之间，用于准直来自激光输入光纤11输出的激光以及减少不同频率激光之间的色差，以与扫描透镜24共同匹配物镜23的像方NA。本实施例的准直器25使用的是消色差准直透镜(#65-286，Edmund Optics Inc.，Barrington，NJ，USA；直径：2mm，等效焦距：3mm，专用近红外光)，能够准直输出激光器并减少飞秒激光器的不同频率分量之间的色差，这样有利于提高传输效率(从激光源到样本高达50％)，光束聚焦和激发效率。
双色镜26设在扫描透镜24和物镜23之间，用于将激光和荧光信号分开以及输出所述荧光信号。双色镜26采用的是中国福建Sunlight有限公司生产制造 的产品，尺寸3×3×0.2mm 3，反射带是750-1100nm，透射带是400-650nm，以将来自扫描透镜24的激光束反射到物镜23。采集透镜27用于有效收集荧光信号。
本发明使用光学设计软件(ZEMAX)来模拟和优化所有光学元件和它们之间的距离，并使用商业CAD软件(Solidworks)进行几何设计。
本发明提供的微型探头2的分辨率的具体计算过程如下：
首先，有效激发NA(NAex)由激发的整个光路确定。准直器25之后的激光束的直径D 1为：
D 1＝2·NA hc-920·EFL c(2.1)
其中，NA hc-920是HC-920的NA(大约为0.15)，EFLc是准直器25的等效焦距(3mm)。
因为激光束的直径D 1(0.9mm)大于微机电扫描仪22的尺寸(对于x方向为0.8mm，对于y方向为大约为0.6mm)，考虑到微机电扫描仪22的倾斜度受到其尺寸的限制并由下式给出：
NA ex＝(1/2·D m/EFL s)·NA o/NA i(2.2)
其中，EFL s是扫描透镜24的等效焦距(2.7mm)。NA o,NA i分别表示物镜的物方NA和像方NA，分别为0.8和0.173。
然后，由微机电扫描仪22的x方向决定的最大激发NA为：
NA ex＝(1/2·0.8/2.7)·0.8/0.173≈0.685
使用参考文献2中的方程计算双光子激发(IPSF2)的点扩散函数的衍射极限横向(ω xy)和轴向(ω z)1/e半径，本实施例有：
Figure PCTCN2018076305-appb-000005
Figure PCTCN2018076305-appb-000006
其中，浸没介质(生理盐水)的折射率不超过1.34，激光的波长为920nm。NA ex为0.685，ω xy＝0.304μm和ω z＝1.840μm。
分辨率通常定义为PSF2的半高宽(FWHM)，本实施例有：
Figure PCTCN2018076305-appb-000007
Figure PCTCN2018076305-appb-000008
利用嵌入琼脂糖中的100nm荧光珠的图像测量得到：本发明提供的微型双光子显微成像装置的横向分辨率为0.64±0.02μm，轴向分辨率为3.35±0.36μ m。
在上述实施例中,测试本发明的分辨率时制作实验样本的方法为：首先将2μL的TetraSpeck 100-nm荧光珠(T7270，Life Technologies，Oregon USA)稀释到100μL生理盐水中，然后与900μL 1.5％低熔点琼脂糖混合。再将含有微球荧光珠(4μM)的琼脂糖小滴加到盖玻片上，10分钟后备用。等效横向像素尺寸为61nm，轴向扫描间隔为80nm，两者均大于奈奎斯特采样定理要求的5倍以上，进而足以测量显微镜的真实光学分辨率。
表3是本发明提供的FIRM-TPM与现有技术中mTPM的分辨率的对比表，第2列是本发明提供的FIRM-TPM的分辨率及其参数说明，第3-6列是现有技术中mTPM的分辨率及其参数说明。需要说明的是：表3中的*：数据来自文献。从现有的实验数据证明：现有技术中mTPM尽管在动物的体外得到了较高的空间分辨率，但是，大多数mTPM在动物的体内表现不佳，并未能得到高信噪比(SNR)的荧光图像。从表3中的对比分析可以得出：本发明提供的FIRM-TPM的分辨率大约为现有技术中的mTPM的最高分辨率的两倍，这种高分辨率为解决自由活动动物中单个树突棘的成像问题提供了有利条件。
表3
Figure PCTCN2018076305-appb-000009
Figure PCTCN2018076305-appb-000010
为了在同一基准上测试和比较现有技术中的台式TPM和微型宽场显微镜与FIRM-TPM之间的性能差异，本实施例建立了一个集成平台，允许在同一样品上进行台式TPM模式(图6)、宽场成像模式(图7)和FIRM-TPM成像模式(图8)之间的切换。如图6-8所示，该集成平台配备有微型和常规物镜、多个照明源(用于TPM的激光器和用于宽场的高功率汞灯)和检测装置(用于TPM的GaAsP和用于宽场的sCMOS相机)。不同的成像模式具有相同的焦平面和同心视场、总帧采集时间和成像NA，而平均照明功率和检测器灵敏度也进行了仔细选择和匹配，下面结合图6至图8说明上述集成平台的组成。
上述集成平台分别在台式TPM模式、宽场成像模式和FIRM-TPM成像模式下对同一标本的同一焦平面进行成像。激光(Chameleon Vision-S，Coherent，USA；中心波长：920nm)，通过H-ZF62玻璃管，随后用声光调制器(MT110-B50A1.5-IR-Hk，AA Sa，Orsay Cede，France)。然后将中心波长为920nm的激光分成两束，一束耦合到在FIRM-TPM中使用的一根HC-920中，另一束耦合到在台式TPM中使用的另一根HC-920中。
如图6所示，在台式TPM模式中，来自HC-920的准直激光器由一对检流计计扫描镜A1(6215H，Cambridge Technology，MA，USA)扫描，然后通过扫描透镜A2(LMS05-BB，Thorlabs Inc，New Jersey)、管透镜A3(Olympus Japan)和第一二向色镜A4(DM1，DMLP650R，Thorlabs Inc，New Jersey，USA)，然后最终传送到物镜A5的后焦平面(BFP)(CFI Apo 40XW NIR，Nikon，Japan；40×NA 0.8，工作距离：3.5mm)。来自物镜A5的荧光发射信号由第一二向色镜A4反射，通过收集透镜A6(LA1213-A，Thorlabs Inc，New Jersey，USA)、第二分色镜A7(DM2，#87-284，Edmund Optics Inc.，Barrington，USA，反射 带：375-393,466-490和546-565nm；透射带：420-460,510-531,590-624和677-725nm)，通过聚光透镜A8(ACL25416U-A，Thorlabs Inc，New Jersey，USA)，最后用光电倍增管A9(GaAsP PMT 7422P-40，Hamamatsu，Japan)检测。为了实现与FIRM-TPM(0.685)相同的激发NA，将物镜A5的后焦平面(BFP)处的激发激光束的光束尺寸设置为：
(2×0.685×200)(mm)/40＝6.85(mm)
其中，200mm是用于该物镜A5的匹配管透镜A3的EFL，40是物镜A5的放大率。
如图7和图8所示，在宽场成像模式和FIRM-TPM成像模式中，将本发明提供的微型探头2连接到定制的微小三轴手动显微操作器(Sigma Koki，Tokyo，JAPAN)上，微型探头2中物镜像平面是空气物镜(Plan Apo NIR 5X，Mitutoyo，Japan；5×，NA为0.14，工作距离为37.5mm)的焦平面。使用具有高精度的双向重复性(±2.5μm)的4端口物镜转盘(OT1，Thorlabs Inc，New Jersey，USA)在微型探头2和40×尼康物镜之间切换。通过调整尼康物镜和微型探头2之前的镜头镜筒的螺纹，本发明可以设置微型探头2和尼康物镜具有相同的焦平面。
如图7所示，在微型宽视场荧光结构中，使用具有蓝光滤光器(MF475-35，Thorlabs Inc，New Jersey，USA；中心波长为475nm，带宽为40nm)的荧光照明器(X-Cite 120Q，Excelitas Technologies，MA，USA)：35nm)作为光源B1。将台式TPM构造中的第一二向色镜A4改变为分色镜B2(DM3，#87-284，Edmund Optics Inc.，Barrington，NJ，USA；反射带：375-393,466-490和546-565nm；透射带包括：420-460,510-531,590-624和677-725nm),以将照明光反射到微型探头2,并将来自微型探头2的荧光发射信号传输到科学CMOS照相机B3(8050M-GE，Thorlabs Inc，New Jersey，USA)。在台式TPM和FIRM-TPM配置中的成像在目标之后使用类似的平均功率，通常为10-25mW。由于单光子和双光子激发的吸收截面的差异，单光子成像中使用的平均功率通常比双光子成像中的平均功率低得多。因此，本发明在宽场荧光显微镜配置中使用100-500μW照明(在微型物镜之后)，类似于之前报告中使用的170-600μW照度。
图14a的左侧图像是Thy1-GFP转基因活体样本脑的神经元树突和树突棘的3D形态成像，在1s的总曝光(FIRM-TPM和微型宽视场显微镜的8Hz的8帧的平均值，以及台式TPM的2Hz的2帧的平均值)下获得大致相同焦平面处的图像，图14a的右侧曲线是左侧图像中所示的裁剪区域中两对相邻树突棘的 横截面轮廓。图14b是在表达GCaMP-6f的活体样本的PFC中富含神经元胞体的平面(-130μm)的图像，上部图片：相同ROI的30秒平均图像。微型宽场图像显示为归一化的ΔF/F(参见在线方法)；下部波浪线：三个选择的神经元中Ca 2+的时程变化(持续时间：100秒)(在上图中用数字标记)。图14c的左侧图像是来自于(图14b)中相同活体样本的树突和棘突(焦平面-120μm)的Ca 2+信号，由于缺乏可辨别的树突信号，未示出用微型宽视场显微镜记录的图像，图14c的右侧上部图像用台式TPM捕获的一个带树突轴(D1)和树突棘(S1，S2，S3)的成像和Ca 2+信号(持续时间：100s)，图14c的右侧下部图像是用FIRM捕获的一个带树突轴(D1)和树突棘(S1，S2，S3)(左)的成像和Ca 2+信号(持续时间：100s)。图14d是在不同配置中成像的Ca 2+信号的平均频率和幅度，来自相同组的神经元胞体(n＝4)，统计：树突(n＝8)和树突棘(n＝6)的数据。
对于形态学成像，本发明在Thy1-GFP转基因活体样本的固定脑中的V1区域(130×130μm 2，从表面到60μm以下)进行成像，获取3D的图像数据(图14a)。FIRM-TPM和台式TPM在树突成像中表现出相同的对比度和分辨率(图14a)。然而，在微型宽场成像模式下，基本上不能分辨结构细节，主要是因为来自焦外组织的强背景信号。在功能成像的比较中，本发明利用腺相关病毒感染的方式在活体样本前额皮层神经元中表示Ca 2+指标剂GCaMP-6f，并在头部固定的清醒活体样本上进行钙成像(图14b-图14d)。选择两个焦平面以显现来自胞体(表面以下130μm)或树突和树突棘(表面以下120μm)的活动。在图像归一化和对比增强(ΔF/F)之后，微型宽场成像以相似的频率分辨体细胞Ca 2+信号，而它们的幅度(ΔF/F为5-10％)比用台式TPM或FIRM-TPM(ΔF/F大约为150％)测量的幅度低约一个数量级(图14d)。值得注意的是，大多数源自树突结构的Ca 2+信号在这种成像配置中未被检测到(图14b和图14d)。总之，这些基准的测试证明本发明的FIRM-TPM达到与台式TPM相同的性能，同时在分辨清醒动物中树突和树突棘的结构和功能活性方面优于微型宽场显微镜。
在FIRM-TPM中使用的快速微机电扫描仪22比在高速台式TPM中使用的共振扫描器具有更加线性的电压-倾斜响应和可控性。因此其赋予其成像更大的灵活性，使得在单个系统种可以实现快速栅格式扫描成像、任意指定的区域(ROI)的随机扫描成像和超快线扫描成像。特别是128Hz的随机扫描成像有助于分辨Ca 2+信号时间上的超微结构，并且可以赋予mTPM在自由活动的动物中进行精确光遗传学操作的能力。10kHz线扫描成像能力对于利用基因编码的电压指示 剂来快速观测动作电位至关重要。
树突棘活动是神经元信息处理的基本事件，利用台式TPMs在头固定的动物上的研究表明单个神经细胞的不同树突棘可以被不同朝向的视觉刺激或不同强度频率的声音刺激所激活。虽然通过微型宽场显微镜获得的数百个神经元的整体活动可以帮助探测在不同行为条件下的神经元网络编码特性，本发明的FIRM-TPM提供了一个可供选择的更加强大的工具，在头固定动物上不能实现的行为范式中对更加基本的神经编码单位的时空特性进行观测。
总之，新开发的FIRM-TPM已经实现了高时空分辨率，良好的机械稳定性，以及对应用范围最广的指示剂的有效激发。通过使用新一代的mTPM，本发明实现了在自由活动的动物中单突触水平的长时间成像这一科学家梦寐以求的目标。可以预见未来会有更多的改进和扩展。使用正确设计的微型高NA物镜，可以进一步扩展FOV和穿透深度。深脑成像可以通过直接将微型物镜插入脑中，或通过在大脑中嵌入GRIN透镜来实现，类似于以前报告的方法。对于较大的动物，例如大鼠或狨猴，可以在头骨上安装多个FIRM-TPM，以探索大脑中远程结构和功能连接的相关问题。本发明预计，FIRM-TPM将被证明是神经科学家以及生物医学科学家在动物体内探索健康和疾病机制的重要工具。
如图5a所示，本发明还提供一种活体样本行为成像系统，所述活体样本行为成像系统包括箱体3、飞秒脉冲激光器、飞秒脉冲激光调制器1、微型探头2、数据收集组件7和线路安装组件，其中：
飞秒脉冲激光器用于产生波长为920纳米的激光。飞秒脉冲激光调制器1用于接收所述飞秒脉冲激光器输出的激光，将激光的传输功率放大至其预设值，并通过预啁啾补偿激光的脉冲展宽，然后输出。微型探头2用于安装在活体样本身上，且输入端通过所述飞秒脉冲激光调制器中的激光输入光纤连接所述飞秒脉冲激光器的输出端，用于接收所述飞秒脉冲激光调制器输出的激光，该激光对活体样本内部的组织进行扫描，以激发所述活体样本产生荧光信号；以及用于接收所述扫描成像部分输出的所述荧光信号，并进行输出。飞秒脉冲激光器、飞秒脉冲激光调制器1和微型探头2在上文均已经详细介绍，此处不再赘述。
箱体3为活体样本的自由移动提供限定空间，该限定空间相比于活体样本的体型要大很多，可以为活体样本的自由活动提供足够大的空间。数据收集组件7安装在箱体3上，数据收集组件7的输入端通过荧光输出光纤21连接微型探头2的输出端，用于收集微型探头2输出的所述荧光信号。激光输入光纤11 和荧光输出光纤21通过所述线路安装组件以能够相对于3箱体随意转动的方式安装在箱体3上。通过线路安装组件，可以防止荧光输出光纤21、激光输入光纤11和其它供电线8彼此之间相互缠绕，为活体样本的自由活动提供有利条件。
在一个实施例中，数据收集组件7提供探测光路，该探测光路上包括同轴布置的光电倍增管71、聚光器72、发射滤光片73、短通滤光片74和收集透镜75，其中：光电倍增管71用于将光信号转化为电信号后输出。聚光器72用于汇聚由荧光输出光纤21传输的荧光信号。发射滤光片73用于滤掉波长为920纳米的激光。短通滤光片74用于过滤掉除信号光以外的杂散光。荧光输出光纤21与收集透镜75共轴，且荧光输出光纤21位于收集透镜75的焦平面上，收集透镜75用于将所述荧光信号更充分的收集到光电倍增管71中。
优选地，所述线路安装组件包括电动旋转头6和支架10，其中：电动旋转头6贯穿设置在箱体3的顶部，并且，一端连接外部电源，另一端连接供电线8，使所述外部电源与供电线8能够电连接。支架10罩设在位于箱体3顶部的电动旋转头6外，激光输入光纤11和荧光输出光纤21穿过电动旋转头6的外壳，荧光输出光纤21与数据收集组件7中的收集透镜75光信号连接。荧光输出光纤21通过轴承9连接到数据收集组件7，这样，电动旋转头6和轴承9允许荧光输出光纤21和供电线8进行独立地旋转，同时保持探测光路不变，这种设计防止了活体样本自由探索其环境时造成的连接线的缠绕，极大地提高了微型探头2的稳定性，即使活体样本进行强烈的身体活动也不会有影响，以使动物在自由探索期间线的扭曲和缠绕最小化。
在一个实施例中，所述自由移动活体样本行为成像装置还包括供电线8，为微机电扫描仪22供电。供电线8通过所述线路安装组件以能够相对于所述箱体随意转动的方式安装在所述箱体上，从而保证供电线8能够正常供电，也能够避免供电线8与激光输入光纤11和荧光输出光纤21彼此相互缠绕，以使动物在自由探索期间线的扭曲和缠绕最小化。
在一个实施例中，所述自由移动活体样本行为成像装置还包括多个相机4，相机4都安装在箱体3的内壁上，比如图2中示出的安装在箱体3的侧面以及顶面，分别以不同的角度拍摄和记录活体样本的自由移动过程的行为。
在一个实施例中，所述自由移动活体样本行为成像装置还包括照明灯5，照明灯5安装在箱体3的内壁上，用于照明箱体3的内腔。
如图12a-12e所示，图12a是V-1皮质L2/3神经元的50张平均FIRM-TPM(本 发明的微型双光子显微成像装置)图像，图像中圈出了1、2、3个树突。图12b是活体样本在竞技场内的二维轨迹(持续时间：100秒)，从其中顶部的相机捕获的视频记录计算轨迹，并且移动速度用颜色编码。图12c和图12d分别是在头部固定(12c)和自由移动条件(12d)下从三个树突(在图12a中圈出)的Ca 2+信号的时程(持续时间：100s)，活体样本速度的时间相关变化也显示在底部。图12e是在头部固定和自由移动条件下所有树突状Ca 2+瞬变的平均频率；数据表示为平均值±s.e.m.，n＝15个树突。***p<0.001，配对t检验。由以上实验数据，可以发现当活体样本在黑暗中自由地探索时(轨迹显示在图12a-12e中)，其视觉皮层的神经元树突活性相对于活体样本头部固定的情况有显著的增加(图12a-12e)。
如图13a-13c所示，图13a是在三种不同的行为范例内的视觉皮层中成像神经元活动：悬尾试验、跳台、社交行为。最左侧第一列图像：通过在行为装置侧面上的侧面的两个相机拍摄的活体样本参与不同行为的快照；中间第二列图像：GCaMP6f标记的神经元胞体，树突和树突棘的FIRM-TPM成像；右侧第三列图像：相应的钙活动分布图(持续时间：100s)。最右侧第四列图像：FIRM-TPM图像中指示的三个ROI的Ca 2+信号(持续时间：50秒)。图13b是FIRM-TPM成像的短期和长期稳定性，图13b左侧曲线是每个行为范例中基于帧间互相关分析基础上的横向位移分布，其中，FM(free moving)表示自由移动；TS(tail suspension)表示悬尾；SD(stepping down from an elevated stage)表示跳台；SI(social interaction)表示社交行为。图13b右侧曲线是在4小时的试验期间的FOV漂移。
这个结果与多位点电学探针记录到的运动可以增强动物视觉皮层神经元活动一致。接下来，为了证明FIRM-TPM的鲁棒性，本发明在活体样本的初级视觉皮层中的130×130μm 2的FOV上进行40Hz的成像来观察其神经元的活动，该活体样本在三种行为范例中依次测试，包括悬尾实验、跳台和社交行为。这些范例都不能在头部固定的实验策略中实现，整个测试过程持续约4小时。值得注意的是，即使当活体样本在悬尾试验中强烈挣扎，从高台跳下或与其同胞交流时，来自不同胞体、树突和树突棘的活性仍然可以被稳定的观测。不同的行为范式似乎与同一V1皮层的不同区域的神经活动相关联(图13a)。
为了量化本发明提供的微型双光子显微镜的短期和长期稳定性，本发明通过逐帧互相关和视场的一段时间到另一段时间的漂移来分析横向位移(图13b)。 本发明发现，在2000连续帧内，帧与帧之间的平均位移在跳台行为范例中(0.19±0.09μm；75％在0.24μm以下)是最大的，但仍然小于像素大小的一半。在整个4h测试中，FIRM-TPM的FOV的整体漂移<10μm，大多数漂移发生在悬尾实验中剧烈运动的第一个小时。值得注意的是，较高的图像采集速度降低了帧内的错位，这种水平的运动伪影并不妨碍本发明对树突棘结构和功能的成像。为了演示本发明提供的微型双光子显微镜对单突触活动的成像能力，通过跟踪树突棘及其父树突轴上的Ca 2+活动，本发明进一步分析了整体和局部活动之间的关系。总而言之，本发明提供的微型双光子显微镜能够在自然生理环境中对自由活动的动物的树突和树突棘活动进行稳定的观测。
本发明还提供一种微型双光子显微成像方法，所述微型双光子显微成像方法包括：
步骤1，选取待成像区域：固定活体样本，并利用台式TPM在所述活体样本上选取待成像区域。在步骤1中，需要采用如图6示出的成像平台，并在台式TPM模式下，也就是说利用台式TMP在活体样本上选取待成像区域，具体操作是：利用身体固定器固定活体样本，以通过台式TPM来确认活体样本头部的病毒感染的区域。
步骤2，采集荧光信号：将连接有激光输入光纤的微型探头安装在所述活体样本上，释放活体样本，以检测步骤1中选取的待成像区域输出的荧光信号，完成所述活体样本内部的组织平面的成像。步骤2具体操作如下：
首先，将本发明提供的微型探头2粘合到活体样本头部颅骨的支架上，该支架在开颅手术准备期间已经固定在活体样本头部的颅骨上，再用牙科水泥将微型探头2加固到活体样本头部颅骨的支架上。
然后，将1.5％低熔点琼脂糖覆盖到暴露的脑组织区域。该方法显著的抑制了探头和大脑之间的相对运动。为了实现随机存取中对大量神经元的染色，标记神经元的过程为：本发明用人工脑脊液(Sigma-Aldrich，中国上海；mM，125NaCl，4.5KCl，26NaHCO 3，1.25NaH 2PO 4，2CaCl 2，1MgCl 2，20葡萄糖，当用95％O 2和5％CO 2充盈时pH为7.4)覆盖暴露脑区。将50微克Cal-520AM(AAT Bioquest.CA，USA)溶解于4μlDMSO(含20％Pluronic F-127)中，并用人工稀释液(mM，150NaCl，2.5KCl，10Hepes,pH 7.432)稀释至500μM。使用2-3MΩ电阻的硼硅酸盐移液管，通过施加压力脉冲(1分钟，400毫巴)将Ca 2+染料注射到初级视觉皮层的层2/3中。1小时后，将SR-101滴加到皮质 表面以鉴定星形胶质细胞。将活体样本头部固定，并使用FIRM-TPM以随机存取模式进行成像。
最后，将活体样本从固定器上释放，并放入上述实施例的小动物成像系统的箱体3中，利用微型探头2对活体样本进行悬尾实验测试或滑落实验测试(圆柱形平，5cm 2，5cm高)和社交行为(30×30cm 2方形活动场地)。
步骤3，处理所述荧光信号，获取活体样本自由活动的三维图像。
在上述步骤之前,还包括如下步骤4,制作活体样本，具体包括：
步骤41，选取活体样本：在实验中使用C57BL/6野生型和Thy-1-GFP转基因活体样本(出生后8-16周)。所有程序均符合北京大学动物使用与护理委员会和实验动物护理评估和认证协会的标准，包括动物育种和实验操作。将Thy-1-GFP转基因活体样本麻醉并用0.9％盐水灌注，然后用4％多聚甲醛灌注，并取出脑。在4℃的4％多聚甲醛的PBS中固定过夜。
步骤42，用于活体脑皮质成像的切骨术：在这些实验中使用C57BL/6野生型活体样本。简言之，通过吸入纯O 2中的1-1.5％异氟烷将活体样本麻醉，并置于立体定位框架(68025，RWD，中国深圳)上。同时，使用加温板(37.5-38℃)保持活体样本的正常温度。在局部施用xylocaine并去除皮肤和肌肉之后，在目标皮层的中部用高速颅钻(尖端直径0.5mm)钻开小方形头窗(0.5×0.5mm 2)。GCaMP-6f用重组AAV在钙调素依赖性激酶II(CaMK II)启动子(血清型2/9；>2×1013个基因组拷贝/ml，由宾夕法尼亚大学基因治疗程序载体核心产生)下表达。使用注射器(Nanoliter 2010，World Precision Instruments，Sarasota，USA)和玻璃微电极，在20分钟内将总量为0.5-0.8μl的AAV缓慢注射到活体样本的靶向皮质的2/3层(深度为130-400μm)。在表达3周后，用异氟烷麻醉活体样本，用氰基丙烯酸胶将自制的支架连接到颅骨上并用牙齿水泥加固。用颅钻在目标皮质上钻出小的方形头窗(2.5×2.5mm 2)。小心地去除硬脑膜，将小型玻璃盖玻片(3.5×2.5mm 2)放在开颅上。恢复一周后，然后每天训练活体样本适应头部固定30分钟，持续3天。在清醒的活体样本适应之后，操作步骤1和步骤2。
最后需要指出的是：以上实施例仅用以说明本发明的技术方案，而非对其限制。本领域的普通技术人员应当理解：可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。

Claims (20)

  1. 一种飞秒脉冲激光调制器，其特征在于，包括：
    负色散光路和正色散光路；其中，
    所述负色散光路包括激光输入光纤，所述激光输入光纤用于传输预啁啾补偿后的激光；
    所述正色散光路位于飞秒脉冲激光器和所述负色散光路之间，用于通过预啁啾补偿所述激光在所述激光输入光纤中产生的负色散。
  2. 根据权利要求1所述的飞秒脉冲激光调制器，其特征在于，所述输入光纤为空心光子晶体光纤，为中心波长920纳米的飞秒脉冲激光提供无畸变传输。
  3. 根据权利要求2所述的飞秒脉冲激光调制器，其特征在于，所述空心光子晶体光纤的纤芯材料为无污染硅，纤芯直径为8-9μm。
  4. 根据权利要求1所述的飞秒脉冲激光调制器，其特征在于，所述正色散光路包括色散补偿元件，所述色散补偿元件为商业材料。
  5. 根据权利要求4所述的飞秒脉冲激光调制器，其特征在于，所述色散补偿元件为H-ZF62玻璃管。
  6. 根据权利要求4所述的飞秒脉冲激光调制器，其特征在于，所述正色散光路还包括声光调制器，所述声光调制器用于接收所述色散补偿元件补偿后的激光，并调节激光强度，然后输出给所述激光输入光纤。
  7. 根据权利要求6所述的飞秒脉冲激光调制器，其特征在于，所述正色散光路还包括激光方位调整组件，其设置在所述色散补偿元件与所述声光调制器之间，用于调整所述声光调制器的调制效率。
  8. 根据权利要求6所述的飞秒脉冲激光调制器，其特征在于，所述正色散光路还包括分光组件，用于将声光调制器调节后的激光分成至少两束，并输出给所述激光输入光纤。
  9. 一种微型双光子显微成像装置，其特征在于，包括：
    飞秒脉冲激光器，用于产生中心波长为920纳米的激光；
    如权利要求1至8中任一项所述的飞秒脉冲激光调制器，用于接收所述飞秒脉冲激光器输出的激光，并进行脉冲放大，然后输出给微型探头；
    以及微型探头，所述微型探头包括：
    扫描成像部分，用于接收所述飞秒脉冲激光调制器输出的激光，并将所述激光对活体样本内部的组织进行扫描，以激发所述活体样本内部的生物指示剂产生荧光信号；以及，
    荧光输出光纤，其用于输出所述荧光信号。
  10. 根据权利要求9所述的微型双光子显微成像装置，其特征在于，所述扫描成像部分包括：
    微机电扫描仪，用于通过转动改变激光入射角角度的方式对所述活体样本内部的组织进行二维扫描；
    物镜，用于将来自所述微机电扫描仪的激光会聚到所述活体样本内部；
    扫描透镜，其布置在所述微机电扫描仪和物镜之间的光路上，用于将所述微机电扫描仪二维扫描所产生的角度变化的激光转化成位置变化的激光；以及，
    双色镜，其设在所述扫描透镜和物镜之间，用于将所述激光和所述荧光信号分开。
  11. 根据权利要求10所述的微型双光子显微成像装置，其特征在于，所述物镜的数值孔径为0.8。
  12. 根据权利要求10所述的微型双光子显微成像装置，其特征在于，所述物镜与所述活体样本之间填充1.5％低熔点琼脂糖。
  13. 根据权利要求10所述的微型双光子显微成像装置，其特征在于，所述物镜包括薄菲涅尔透镜。
  14. 根据权利要求10所述的微型双光子显微成像装置，其特征在于，所述扫描成像部分还包括准直器，其布置在所述激光输入光纤与所述微机电扫描仪之间。
  15. 根据权利要求9所述的微型双光子显微成像装置，其特征在于，所述荧光输出光纤为柔性光纤束。
  16. 根据权利要求15所述的微型双光子显微成像装置，其特征在于，所述柔性光纤束由700-900根玻璃光纤熔接两端而成，同时各个玻璃光纤保持松散和分开。
  17. 一种活体样本行为成像系统，其特征在于，包括：
    如权利要求9至16中任一项所述的微型双光子显微成像装置；
    箱体，用于为活体样本的自由移动提供限定空间；
    线路安装组件，所述激光输入光纤和荧光输出光纤通过所述线路安装组件以相对于箱体随意转动的方式安装在箱体上；以及，
    数据采集组件，用于收集所述荧光输出光纤输出的所述荧光信号。
  18. 根据权利要求17所述的活体样本行为成像系统，其特征在于，所述线路安装包括：
    电动旋转头，其贯穿设置在所述箱体的顶部，所述激光输入光纤和荧光输出光纤穿过所述电动旋转头的外壳；
    支架，其罩设在所述电动旋转头的外壳，所述数据采集组件设置在所述支架上；以及，
    轴承，所述荧光输出光纤通过所述轴承连接到所述数据采集组件。
  19. 根据权利要求18所述的活体样本行为成像系统，其特征在于，所述电动旋转头在箱体外的一端连接外部电源，在箱体内的另一端提供电连接接口。
  20. 一种微型双光子显微成像方法，其特征在于，包括：
    选取待成像区域：固定活体样本，并利用台式双光子显微镜在所述活体样本上选取待成像区域；
    采集荧光信号：将如权利要求9至16中任一项所述的微型双光子显微成像装置的微型探头安装在所述活体样本上，释放活体样本，以采集所述待成像区域输出的荧光信号。
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