CN118010700A - Cross-modal spectroscopy and imaging method and device based on single beam detection - Google Patents
Cross-modal spectroscopy and imaging method and device based on single beam detection Download PDFInfo
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Abstract
Description
技术领域Technical Field
本发明属于光学成像和分子振动光谱成像领域,具体涉及一种基于单光束探测的跨模态光谱与成像的方法及装置。The present invention belongs to the field of optical imaging and molecular vibration spectrum imaging, and in particular relates to a method and device for cross-modal spectrum and imaging based on single-beam detection.
背景技术Background technique
跨模态成像的目标是通过协同地结合不同的显微成像模式,为各种目标提供独特和互补的见解,为全面的化学、物理和生物分析提供了分子动力学的整体视角。与单一成像方法相比,跨模态成像可以提供额外的分子空间信息。例如,引入荧光成像模式以补充数字全息术和干涉散射成像的特异性不足,展示了跨模态成像在研究细胞器相互作用和纳米级动态事件追踪方面的能力。面对标记小分子和代谢物的挑战,已经开发出无标记成像方法,通过捕捉固有的分子结构信息来补充荧光成像模态中的分子种类限制。值得注意的是,二次谐波产生(SHG)、瞬态吸收(TA)、受激拉曼散射(SRS)和三阶和频产生(TSFG)等非线性光学过程相互兼容,为细胞代谢和病理机制的研究提供了新的见解。此外,通过结合散射、光声、相位和荧光等多种成像模态,基于光吸收诱导的光热效应的泵浦探测技术展示了显著的协同效应,极大地提高了无标记振动成像的分辨率、灵敏度和通量。The goal of cross-modal imaging is to provide unique and complementary insights into various targets by synergistically combining different microscopic imaging modes, providing a holistic view of molecular dynamics for comprehensive chemical, physical, and biological analyses. Compared with single imaging methods, cross-modal imaging can provide additional molecular spatial information. For example, the introduction of fluorescence imaging modes to complement the lack of specificity of digital holography and interferometric scattering imaging has demonstrated the power of cross-modal imaging in studying organelle interactions and tracking nanoscale dynamic events. Faced with the challenges of labeling small molecules and metabolites, label-free imaging methods have been developed to complement the molecular species limitation in fluorescence imaging modalities by capturing inherent molecular structural information. Notably, nonlinear optical processes such as second harmonic generation (SHG), transient absorption (TA), stimulated Raman scattering (SRS), and third-order sum frequency generation (TSFG) are compatible with each other and provide new insights into cellular metabolism and pathological mechanisms. In addition, by combining multiple imaging modalities such as scattering, photoacoustic, phase, and fluorescence, pump-probe techniques based on the photothermal effect induced by light absorption have demonstrated significant synergistic effects, greatly improving the resolution, sensitivity, and throughput of label-free vibrational imaging.
为了非侵入性地探测分子振动的内禀指纹信息,红外和拉曼光谱在各个领域中已经得到了丰富的应用,尤其是红外光谱,其吸收截面(约为10-18cm2)远高于常规的拉曼光谱(10-28cm2)。然而,单一的光谱模态难以确定完整的分子结构信息,因为具有拉曼活性的振动模式不一定具有红外活性,反之亦然,红外光谱对不对称振动非常敏感,而拉曼光谱则对对称振动敏感,这导致光谱信息不完整。因此,必须联合红外光谱和拉曼光谱进行同时探测,以提供互补和全面的振动光谱信息,从而更深入地理解复杂的过程。例如,通过集成红外光谱和拉曼光谱测量,已经在化学领域证明其能够识别新的NOx吸附态种类,并且能可视化多相催化过程中与NOx储存效率位置相关的信息。近期关于CO还原反应研究利用了原位增强红外和拉曼光谱技术,揭示了CO亚群的吸附过程中形成机理。早期的尝试顺序红外光谱和拉曼光谱对生物样品进行成像已显示出其在提供细胞组成全谱和增强生物标志物鉴定方面的潜力。In order to non-invasively detect the intrinsic fingerprint information of molecular vibration, infrared and Raman spectroscopy have been widely used in various fields, especially infrared spectroscopy, whose absorption cross section (about 10-18 cm2 ) is much higher than that of conventional Raman spectroscopy ( 10-28 cm2 ). However, it is difficult to determine the complete molecular structure information from a single spectral mode, because the vibration mode with Raman activity is not necessarily infrared active, and vice versa. The infrared spectrum is very sensitive to asymmetric vibrations, while the Raman spectrum is sensitive to symmetric vibrations, which leads to incomplete spectral information. Therefore, it is necessary to combine infrared spectroscopy and Raman spectroscopy for simultaneous detection to provide complementary and comprehensive vibrational spectroscopy information, so as to have a deeper understanding of complex processes. For example, by integrating infrared spectroscopy and Raman spectroscopy measurements, it has been demonstrated in the field of chemistry that it can identify new NOx adsorption states and visualize information related to the location of NOx storage efficiency in heterogeneous catalytic processes. Recent studies on CO reduction reactions have used in situ enhanced infrared and Raman spectroscopy techniques to reveal the formation mechanism of CO subpopulations during adsorption. Early attempts to sequentially image biological samples using infrared and Raman spectroscopy have shown their potential to provide a full spectrum of cellular composition and enhance biomarker identification.
然而,直接将红外和拉曼模态相结合并不可行,因为它们具有不同的物理性质(吸收与散射)和光谱窗口(中红外与可见光)之间存在显著差异。目前已经将红外和拉曼联合探测的方案。例如,IRaman将中红外光热成像与单点拉曼光谱相结合。为了克服红外和拉曼模态选择规则的冲突,引入了非线性光学过程,包括双光子激发的拉曼光谱,即超拉曼光谱。此外,还有通过傅里叶变换相干反斯托克斯拉曼散射(拉曼敏感)和脉冲内差频产生(红外敏感)过程,实现了互补振动光谱的单点光谱测量。结合相干反斯托克斯拉曼散射和三阶和频产生的三光子过程提高了红外和拉曼成像分辨率。然而,开发出具有高分辨率和定量化学成像功能的同时红外和拉曼成像平台,仍然是一项巨大的挑战。However, it is not feasible to directly combine the infrared and Raman modalities because they have different physical properties (absorption vs. scattering) and significant differences between the spectral windows (mid-infrared vs. visible light). Schemes for combined infrared and Raman detection have been proposed. For example, IRaman combines mid-infrared photothermal imaging with single-point Raman spectroscopy. To overcome the conflict of infrared and Raman modal selection rules, nonlinear optical processes have been introduced, including two-photon excited Raman spectroscopy, namely hyper-Raman spectroscopy. In addition, there are single-point spectral measurements of complementary vibrational spectra through Fourier transform coherent anti-Stokes Raman scattering (Raman sensitive) and intra-pulse difference frequency generation (infrared sensitive) processes. The three-photon process combining coherent anti-Stokes Raman scattering and third-order sum frequency generation improves the resolution of infrared and Raman imaging. However, it remains a huge challenge to develop a simultaneous infrared and Raman imaging platform with high-resolution and quantitative chemical imaging capabilities.
综上所述,目前仍然缺乏一种能够同时进行红外和拉曼模态的光谱和成像的方案,并且尚未找到有效的解决方法。这一问题需要进一步研究和开发,以满足光学成像领域对同时实现红外和拉曼模态的光谱和成像需求。In summary, there is still a lack of a solution that can simultaneously perform spectroscopy and imaging of infrared and Raman modes, and an effective solution has not yet been found. This problem requires further research and development to meet the needs of the optical imaging field for simultaneous spectroscopy and imaging of infrared and Raman modes.
发明内容Summary of the invention
基于上述所提到的技术瓶颈,本发明提供的是一种基于单光束探测的跨模态光谱与成像方法,该方法可以实现无标记、高灵敏、高光谱范围的同时红外与拉曼光谱和成像采集。Based on the technical bottlenecks mentioned above, the present invention provides a cross-modal spectroscopy and imaging method based on single-beam detection, which can achieve label-free, high-sensitivity, and high-spectral range simultaneous infrared and Raman spectroscopy and imaging acquisition.
本发明首先提供了一种基于单光束探测的跨模态光谱与成像的方法,包括以下步骤:The present invention first provides a method for cross-modal spectroscopy and imaging based on single-beam detection, comprising the following steps:
1)对一束受激辐射光和一束波长可调谐的泵浦光进行调控,使两束光在时间域和空间域上重合,再对一束波长可调谐的中红外光进行调整,使得中红外光的光路至与受激辐射光和泵浦光共线,并进一步对三束光聚焦从而使三束光的焦点在空间上重合;1) regulating a beam of stimulated emission light and a beam of wavelength-tunable pump light so that the two beams overlap in time and space, and then adjusting a beam of wavelength-tunable mid-infrared light so that the optical path of the mid-infrared light is collinear with the stimulated emission light and the pump light, and further focusing the three beams so that the focal points of the three beams overlap in space;
2)将待检测的目标样品设置在三束光聚焦的焦点上;2) Setting the target sample to be detected at the focus of the three beams;
3)调整泵浦光波长,使泵浦光与受激辐射光的能量差匹配目标分子的拉曼振动能级,实现受激拉曼散射效应,调整中红外光的波长使中红外光匹配目标分子的红外振动能级,实现目标分子周围局域的光热驰豫效应,使得三束光焦点内的目标分子同时产生受激拉曼散射效应和中红外吸收产生的中红外光热驰豫效应;从而使受激拉曼散射效应产生的受激拉曼散射信号与中红外光热驰豫效应产生的中红外光热信号被携带在泵浦光光强中;3) adjusting the wavelength of the pump light so that the energy difference between the pump light and the stimulated radiation light matches the Raman vibration energy level of the target molecule to achieve a stimulated Raman scattering effect, and adjusting the wavelength of the mid-infrared light so that the mid-infrared light matches the infrared vibration energy level of the target molecule to achieve a localized photothermal relaxation effect around the target molecule, so that the target molecules within the focus of the three beams simultaneously produce a stimulated Raman scattering effect and a mid-infrared photothermal relaxation effect produced by mid-infrared absorption; thereby causing the stimulated Raman scattering signal produced by the stimulated Raman scattering effect and the mid-infrared photothermal signal produced by the mid-infrared photothermal relaxation effect to be carried in the pump light intensity;
4)利用受激拉曼散射效应与光热驰豫效应的时间特征差异,在时间域上对受激辐射光和中红外光的光强进行调制,使得受激拉曼散射效应和光热驰豫效应的信号处在不同的频率区间;4) Using the difference in the time characteristics of the stimulated Raman scattering effect and the photothermal relaxation effect, the intensity of the stimulated radiation light and the mid-infrared light is modulated in the time domain, so that the signals of the stimulated Raman scattering effect and the photothermal relaxation effect are in different frequency ranges;
5)在不同频率区间生成的受激拉曼散射信号和中红外光热信号被泵浦光携带后,将泵浦光作为探测光输入光电探测器转换为光电压信号,采用频率域滤波技术从所述光电压信号中分离出受激拉曼散射信号与中红外光热信号;5) After the stimulated Raman scattering signal and the mid-infrared photothermal signal generated in different frequency intervals are carried by the pump light, the pump light is input into the photodetector as the detection light and converted into a photovoltage signal, and the stimulated Raman scattering signal and the mid-infrared photothermal signal are separated from the photovoltage signal by using the frequency domain filtering technology;
6)扫描目标样品的空间位置并同时采集受激拉曼散射信号与中红外光热信号,实现跨模态成像数据的采集,采集的跨模态成像数据经过光谱与成像处理设备生成成像图;6) Scan the spatial position of the target sample and simultaneously collect stimulated Raman scattering signals and mid-infrared photothermal signals to realize the collection of cross-modal imaging data. The collected cross-modal imaging data is processed by spectral and imaging processing equipment to generate an imaging image;
7)扫描泵浦光波长和中红外光的波长,对于受激辐射光、泵浦光、中红外光共同焦点内的分子进行光谱采集得到各个模态的光谱。7) Scanning the wavelength of the pump light and the wavelength of the mid-infrared light, collecting spectra of the molecules within the common focus of the stimulated radiation light, the pump light, and the mid-infrared light to obtain spectra of each mode.
作为本发明的优选方案,所述步骤1)中,受激辐射光和泵浦光为超快激光,脉冲宽度为飞秒至皮秒量级,波长范围为可见光至近红外光波段;所述泵浦光的波长短于受激辐射光,受激辐射光和泵浦光的光子能量差覆盖分子振动光谱范围。As a preferred embodiment of the present invention, in step 1), the stimulated radiation light and the pump light are ultrafast lasers with a pulse width of femtosecond to picosecond order and a wavelength range from visible light to near-infrared light; the wavelength of the pump light is shorter than that of the stimulated radiation light, and the photon energy difference between the stimulated radiation light and the pump light covers the molecular vibration spectrum range.
作为本发明的优选方案,步骤1)中的受激辐射光和泵浦光需要在时间域上调整至重合;所述的脉冲的中红外光,不与所述的受激辐射光和泵浦光形成时间域重合,以避免与受激辐射光和泵浦光产生非线性效应。As a preferred embodiment of the present invention, the stimulated radiation light and the pump light in step 1) need to be adjusted to overlap in the time domain; the pulsed mid-infrared light does not overlap with the stimulated radiation light and the pump light in the time domain to avoid nonlinear effects with the stimulated radiation light and the pump light.
本发明还提供了一种用于实现上述跨模态光谱与成像方法的跨模态光谱与成像装置,包括:The present invention also provides a cross-modal spectroscopy and imaging device for implementing the above cross-modal spectroscopy and imaging method, comprising:
光源模块,包括用于出射受激辐射光和泵浦光的第一光源、用于出射中红外光的第二光源、对受激辐射光进行时间域调制的电光调制器、对受激辐射光和泵浦光进行空间域重合调整和对中红外光共线调整的光路模块;A light source module, comprising a first light source for emitting stimulated radiation light and pump light, a second light source for emitting mid-infrared light, an electro-optic modulator for performing time domain modulation on the stimulated radiation light, and an optical path module for performing spatial domain coincidence adjustment on the stimulated radiation light and the pump light and collinear adjustment on the mid-infrared light;
显微镜模块,包括显微镜框架、用于调整光线的镜片模组、用于聚焦中红外光的和收集泵浦光的红外聚焦镜以及搭载样品的载物样品扫描台;A microscope module, including a microscope frame, a lens module for adjusting light, an infrared focusing mirror for focusing mid-infrared light and collecting pump light, and a sample scanning stage for carrying samples;
探测模块,包括用于收集信号的收集光路、用于滤出泵浦光的滤光片组、用于转换泵浦光光强为光电压信号的光电探测器以及前置放大器;A detection module, including a collection optical path for collecting signals, a filter set for filtering out pump light, a photodetector for converting the intensity of the pump light into a photovoltage signal, and a preamplifier;
光谱与成像采集模块,包括数据采集卡、滤波器件和光谱与成像处理设备;所述数据采集卡和滤波器件用于对跨模态成像数据和光谱进行采集;所述光谱与成像处理设备用于控制数据采集卡、载物样品台、第一光源、第二光源,滤波器件。The spectrum and imaging acquisition module comprises a data acquisition card, a filter device and a spectrum and imaging processing device; the data acquisition card and the filter device are used to acquire cross-modal imaging data and spectra; the spectrum and imaging processing device is used to control the data acquisition card, the sample stage, the first light source, the second light source and the filter device.
作为本发明的优选方案,所述镜片模组包括第一阔束透镜、第二阔束透镜、第三阔束透镜、第四阔束透镜、第一反射镜、第二反射镜、第三反射镜8、双色镜、第四反射镜、50-50分束镜、第五反射镜、分束镜、第六反射镜、第七反射镜、折射式物镜和红外聚焦镜。As a preferred embodiment of the present invention, the lens module includes a first wide-beam lens, a second wide-beam lens, a third wide-beam lens, a fourth wide-beam lens, a first reflector, a second reflector, a third reflector 8, a dichroic mirror, a fourth reflector, a 50-50 beam splitter, a fifth reflector, a beam splitter, a sixth reflector, a seventh reflector, a refracting objective lens and an infrared focusing lens.
与现有技术相比,本发明具有的有益效果至少包括:Compared with the prior art, the present invention has the following beneficial effects:
(一)通过利用不同物理过程的时间尺度差异特征来进行时间域调制,从根本上解决了红外光谱和拉曼光谱由于光学性质差异大与探测方案不同导致的难以集成在同一光谱采集与成像系统上的技术难题,实现了难以实现的红外和拉曼互补光谱模态的同时成像。(1) By utilizing the time scale differences of different physical processes to perform time domain modulation, the technical difficulty of integrating infrared spectroscopy and Raman spectroscopy into the same spectral acquisition and imaging system due to the large differences in optical properties and different detection schemes is fundamentally solved, and the difficult simultaneous imaging of infrared and Raman complementary spectral modes is realized.
(二)本发明不仅能够摆脱红外成像低分辨率、水吸收、拉曼光谱的弱信号等限制,而且让光热成像、受激拉曼散射成像在同一探测光上进行探测,从而大大提高了红外成像的分辨率、灵敏度,拉曼成像的灵敏度,速度。(ii) The present invention not only gets rid of the limitations of low resolution of infrared imaging, water absorption, weak signal of Raman spectrum, etc., but also allows photothermal imaging and stimulated Raman scattering imaging to be detected on the same detection light, thereby greatly improving the resolution and sensitivity of infrared imaging and the sensitivity and speed of Raman imaging.
(三)本发明利用了单一探测光束探测、单一探测器进行探测的方法,简化了系统复杂度,避免了传统多模态成像的串扰问题,实现了无串扰成像。并且提高了成像通量,实现跨光谱模态的成像能力。(III) The present invention uses a single detection beam and a single detector to simplify the system complexity, avoid the crosstalk problem of traditional multi-modal imaging, and achieve cross-talk-free imaging. It also improves the imaging flux and achieves cross-spectral imaging capabilities.
(四)本发明实现了从皮秒到微秒不同物理过程的同时成像,具有相互兼容和广泛性的特点,本方案原则上适用于任何基于时间域调制的多模态成像方法。(iv) The present invention realizes the simultaneous imaging of different physical processes from picoseconds to microseconds, and has the characteristics of mutual compatibility and universality. In principle, this scheme is applicable to any multimodal imaging method based on time domain modulation.
(五)本发明普遍适用于所有分子,并且不限于中红外的吸收。受益于光热探测的方案,本发明适用于任何基于吸收探测的成像模态;并且为皮秒超快激光激发的各种成像模式提供了更多的光谱窗口,可以兼容多种超快光学模态,例如双光子荧光、瞬态吸收、相干反斯托克斯拉曼成像、二次谐波成像、三次谐波成像等。(V) The present invention is generally applicable to all molecules and is not limited to mid-infrared absorption. Benefiting from the photothermal detection scheme, the present invention is applicable to any imaging modality based on absorption detection; and provides more spectral windows for various imaging modes of picosecond ultrafast laser excitation, and is compatible with a variety of ultrafast optical modalities, such as two-photon fluorescence, transient absorption, coherent anti-Stokes Raman imaging, second harmonic imaging, third harmonic imaging, etc.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图做简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动前提下,还可以根据这些附图获得其他附图。In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.
图1为单光束探测的跨模态光谱与成像的能级原理图;FIG1 is an energy level schematic diagram of cross-modal spectroscopy and imaging for single-beam detection;
图2为本发明实施例的实验光路部分示意图;FIG2 is a schematic diagram of a portion of an experimental optical path according to an embodiment of the present invention;
图3为本发明实施例的信号传递与系统控制图;FIG3 is a signal transmission and system control diagram of an embodiment of the present invention;
图4为本发明实施例的入射的激光脉冲,包括泵浦光,受激辐射光和中红外光脉冲在时间域的波形图;FIG4 is a waveform diagram of an incident laser pulse, including pump light, stimulated radiation light and mid-infrared light pulse in the time domain according to an embodiment of the present invention;
图5为本发明实施例探测光光强在时间域的展示图;FIG5 is a diagram showing the intensity of the detection light in the time domain according to an embodiment of the present invention;
图6为本发明实施例探测光光强在频率域的展示图;FIG6 is a diagram showing the intensity of the detection light in the frequency domain according to an embodiment of the present invention;
图7为本发明实施例在油膜上探测到的频谱图;FIG7 is a spectrum diagram detected on an oil film according to an embodiment of the present invention;
图8为本发明实施例同时测得的500nm PMMA微球上的红外和拉曼光谱数据图;FIG8 is a graph showing infrared and Raman spectra of 500 nm PMMA microspheres measured simultaneously according to an embodiment of the present invention;
图9为本发明实施例同时采集的500nm PMMA塑料微球的红外和拉曼成像图;FIG9 is an infrared and Raman image of 500 nm PMMA plastic microspheres collected simultaneously according to an embodiment of the present invention;
图10为从图9箭头指示的单一微球的红外和拉曼信号的剖面以及高斯拟合结果图;FIG10 is a cross-section of infrared and Raman signals of a single microsphere indicated by an arrow in FIG9 and a Gaussian fitting result diagram;
图11为本发明实施例采集的单一500nm PMMA塑料微球的三维红外成像图;FIG11 is a three-dimensional infrared image of a single 500nm PMMA plastic microsphere collected in an embodiment of the present invention;
图12为本发明实施例与图11同时采集的单一500nm PMMA塑料微球的三维拉曼成像图;FIG12 is a three-dimensional Raman image of a single 500 nm PMMA plastic microsphere collected simultaneously with FIG11 according to an embodiment of the present invention;
图13为从图11和图12的单一500nm PMMA塑料微球的红外和拉曼的三维重构图;FIG13 is a 3D reconstruction of the infrared and Raman images of a single 500 nm PMMA plastic microsphere of FIG11 and FIG12 ;
图14为图11和图12的单一500nm PMMA塑料微球三维成像的纵向剖面以及高斯拟合结果图;FIG14 is a longitudinal section of a single 500 nm PMMA plastic microsphere 3D imaging of FIG11 and FIG12 and a Gaussian fitting result diagram;
图15为本发明实施例中所使用的棕榈酸(PA),1,4-二苯基丁二炔(DiPhDY)和三苯基膦(TPP)的红外透射和自发拉曼光谱;FIG15 is an infrared transmission and spontaneous Raman spectra of palmitic acid (PA), 1,4-diphenylbutadiyne (DiPhDY) and triphenylphosphine (TPP) used in the examples of the present invention;
图16为本发明实施例所使用的三种化学物质在不同波数下的红外和拉曼活性;FIG16 shows the infrared and Raman activities of three chemical substances used in the embodiments of the present invention at different wave numbers;
图17为本发明实施例同时采集的三种化学物质等比例混合组成的混合物的红外和拉曼成像图;FIG17 is an infrared and Raman image of a mixture of three chemical substances mixed in equal proportions collected simultaneously in an embodiment of the present invention;
图18为由图17数据分离出的纯物质分布图及三种物质分布图的重叠图;FIG18 is a pure substance distribution diagram separated from the data of FIG17 and an overlapped diagram of the distribution diagrams of three substances;
图19示出了图17所示的成像图之间的皮尔逊相关系数的示意图;FIG19 is a schematic diagram showing the Pearson correlation coefficient between the imaging images shown in FIG17 ;
图20为本发明实施例同时采集的对照组和实验组细胞在红外1750cm-1和拉曼2850cm-1下的成像图;FIG20 is an image of cells in the control group and the experimental group collected simultaneously under infrared 1750 cm -1 and Raman 2850 cm -1 according to an embodiment of the present invention;
图21为对照组和实验组细胞在红外1750cm-1和拉曼2850cm-1下的比率成像图;FIG21 is a ratio imaging diagram of cells in the control group and the experimental group at infrared 1750 cm -1 and Raman 2850 cm -1 ;
图22为对照组和实验组细胞中脂滴的数量随相对强度变化的分布直方图;FIG22 is a distribution histogram of the number of lipid droplets in cells of the control group and the experimental group as a function of relative intensity;
图23为本发明实施例同时采集的对照组和实验组细胞在红外1080cm-1和拉曼2930cm-1下的成像图;FIG23 is an image of cells in the control group and the experimental group simultaneously collected at infrared 1080 cm -1 and Raman 2930 cm -1 in an embodiment of the present invention;
图24为对照组和实验组细胞在红外1080cm-1和拉曼2850cm-1下的比率成像图和它们的局部放大图;FIG24 is a ratio imaging diagram of cells in the control group and the experimental group at infrared 1080 cm -1 and Raman 2850 cm -1 and their local magnified diagrams;
图25为对照组和实验组细胞中的多糖蛋白颗粒的面积和相对强度的分布图;FIG25 is a distribution diagram of the area and relative intensity of polysaccharide protein particles in cells of the control group and the experimental group;
图26示出了图20和图21所示的对照组和实验组细胞的成像图之间的皮尔逊相关系数示意图;FIG26 is a schematic diagram showing the Pearson correlation coefficient between the imaging images of the control group and the experimental group cells shown in FIG20 and FIG21 ;
图27示出了本发明实施例同时采集的小鼠脑中纹状体的背向红外和前向拉曼成像图;FIG27 shows back infrared and forward Raman images of the striatum in the mouse brain collected simultaneously according to an embodiment of the present invention;
图28为从图27箭头指示的三个感兴趣位置的原位红外和拉曼光谱图;FIG28 is an in-situ infrared and Raman spectra of three locations of interest indicated by arrows in FIG27 ;
图29示出了本发明实施例采集的秀丽隐杆线虫的红外成像图;FIG29 shows an infrared image of Caenorhabditis elegans collected by an embodiment of the present invention;
图30示出了本发明实施例采集的秀丽隐杆线虫的拉曼成像图。FIG30 shows a Raman image of Caenorhabditis elegans collected according to an embodiment of the present invention.
具体实施方式Detailed ways
一般来说,本文描述的实施方式涉及利用频率复用技术实现单光束探测跨模态光谱信息获得多光谱图像。按照要求,本文公开了本发明的实施方法。然而,需要注意的是,本文涉及的实施方法仅仅是示范性的,所以本发明可以以多种不同的和替代的形式实施。In general, the embodiments described herein relate to the use of frequency multiplexing technology to achieve single beam detection of cross-modal spectral information to obtain multi-spectral images. As required, the implementation method of the present invention is disclosed herein. However, it should be noted that the implementation method involved in this article is only exemplary, so the present invention can be implemented in many different and alternative forms.
而且本文中所示的附图,为了凸显本发明的新颖的方面以及考虑到画图的简洁性,附图的部分示意图并不一定是按照实际比例,并且可能夸大或者最小化一些特征以示出具体事物的细节。同时在本文的附图中,尤其是装置图,相关元件可能已经省略以防止模糊新装置的诸多创新的方面。因此,本文中公布的方法,结构以及功能细节不应被理解为限制,而仅仅作为权利要求的基础和用于教导本领域研究人员多样性地使用本发明的代表性基础。出于引领而非限制的目的,本文公开了利用频率复用技术实现单光束探测跨模态光谱信息获得多光谱图像的方法。Moreover, in order to highlight the novel aspects of the present invention and to take into account the simplicity of the drawings, some of the schematic diagrams in the drawings shown in this article are not necessarily in actual proportion, and some features may be exaggerated or minimized to show the details of specific things. At the same time, in the drawings of this article, especially the device diagrams, related elements may have been omitted to prevent blurring the many innovative aspects of the new device. Therefore, the methods, structures, and functional details disclosed in this article should not be understood as limitations, but only as the basis for the claims and as a representative basis for teaching researchers in the field to use the present invention in a variety of ways. For the purpose of guidance rather than limitation, this article discloses a method for obtaining multi-spectral images by using frequency multiplexing technology to detect cross-modal spectral information with a single beam.
本发明的探测原理能够通过能级图进行阐述,具有特定结构的分子都有拉曼振动能级与红外振动能级,如图1所示。对特定分子引入一束泵浦光与一束受激辐射光进行照射,分子会被泵浦光激发到虚能级,当泵浦光与受激辐射光的能量差匹配到拉曼振动的能量,引入的受激辐射光会对拉曼光子产生受激辐射,将会放大拉曼信号约108倍,放大的拉曼信号称之为受激拉曼信号:在泵浦光的光强上会携带有受激拉曼损耗SRL信号,在受激辐射光的光强上会携带有受激拉曼增益SRG信号;用一束中红外光照射分子,但中红外光的能量匹配到了红外振动能级的能量,分子会直接吸收红外光能量,吸收的能量经过耗散会使得周围温度上升,从而改变局域的折射率,发生形变,此时产生显著的光热效应,会对照射在同一个空间位置的光束的传播发生光热透镜的调制,称之为中红外光热MIP信号,此时红外光谱与MIP信号强度线性相关。The detection principle of the present invention can be explained through an energy level diagram. Molecules with specific structures have Raman vibration energy levels and infrared vibration energy levels, as shown in FIG1 . A beam of pump light and a beam of stimulated radiation light are introduced to irradiate a specific molecule, and the molecule will be excited to a virtual energy level by the pump light. When the energy difference between the pump light and the stimulated radiation light matches the energy of Raman vibration, the introduced stimulated radiation light will produce stimulated radiation on the Raman photons, which will amplify the Raman signal by about 10 8 times. The amplified Raman signal is called stimulated Raman signal: the pump light intensity will carry a stimulated Raman loss SRL signal, and the stimulated Raman gain SRG signal will be carried on the intensity of the stimulated radiation light. When a beam of mid-infrared light is used to irradiate the molecule, but the energy of the mid-infrared light matches the energy of the infrared vibration energy level, the molecule will directly absorb the infrared light energy. The absorbed energy will cause the surrounding temperature to rise after dissipation, thereby changing the local refractive index and causing deformation. At this time, a significant photothermal effect is generated, which will modulate the propagation of the light beam irradiated at the same spatial position through a photothermal lens, which is called a mid-infrared photothermal MIP signal. At this time, the infrared spectrum is linearly related to the MIP signal intensity.
因此,提出一种泵浦-探测方案来同时检测这两个过程的构想是合理的。当上述提及三束光同时打在样品上时,泵浦光和受激辐射光的光强上会同时产生受激拉曼散射信号与光热信号。这样的探测方法能够将两种截然不同的模态带入到同一维度下进行探测。Therefore, it is reasonable to propose a pump-probe scheme to detect these two processes simultaneously. When the three beams mentioned above are simultaneously hit on the sample, the intensity of the pump light and the stimulated radiation light will simultaneously generate stimulated Raman scattering signals and photothermal signals. This detection method can bring two completely different modes into the same dimension for detection.
上述提到的受激拉曼过程和光热过程,如图1所示,在泵浦光、受激辐射光和中红外光为连续光和脉冲光的情况下均会发生。本发明利用到了受激拉曼过程和光热过程在时间尺度上有截然不同的特征:即受激拉曼散射过程发生在飞秒至皮秒的时间尺度范围,光热弛豫过程发生在纳秒至微秒尺度的过程,采用了脉冲光激发的探测方案,分别在受激拉曼散射过程和光热弛豫过程各自的时间窗口进行调制,以实现最小的信号串扰和最高的调制效率,即实现单光束探测的跨模态光谱成像。The stimulated Raman process and photothermal process mentioned above, as shown in Figure 1, will occur when the pump light, stimulated radiation light and mid-infrared light are continuous light and pulsed light. The present invention takes advantage of the fact that the stimulated Raman process and the photothermal process have completely different characteristics in terms of time scale: the stimulated Raman scattering process occurs in the time scale range of femtoseconds to picoseconds, and the photothermal relaxation process occurs in the process of nanoseconds to microseconds. A pulsed light excitation detection scheme is adopted, and the stimulated Raman scattering process and the photothermal relaxation process are modulated in their respective time windows to achieve minimum signal crosstalk and maximum modulation efficiency, that is, to achieve cross-modal spectral imaging of single-beam detection.
另外,泵浦光和受激辐射光都会携带有受激拉曼散射信号与中红外光热信号,均可作为探测光来实现单光束探测的跨模态光谱成像。考虑诸多因素,如常规的硅基探测器的探测量子效率与光斑衍射极限大小,本发明选择泵浦光作为探测光,以实现更优的性能,包括更高的探测灵敏度和更高的成像分辨率。In addition, both pump light and stimulated radiation light carry stimulated Raman scattering signals and mid-infrared photothermal signals, and can be used as detection light to achieve cross-modal spectral imaging of single-beam detection. Considering many factors, such as the detection quantum efficiency and spot diffraction limit size of conventional silicon-based detectors, the present invention selects pump light as the detection light to achieve better performance, including higher detection sensitivity and higher imaging resolution.
在本发明的一个具体实施例中,如图2所示,为本发明公开的一种基于单光束探测的跨模态光谱与成像装置,包括第一光源1,第一阔束透镜2,第二阔束透镜3,第三阔束透镜4,第四阔束透镜5,第一反射镜6,第二反射镜7,第三反射镜8,电光调制器9,双色镜10,第四反射镜11,50-50分束镜12,第二光源13,第五反射镜14,分束镜15,第六反射镜16,第七反射镜17,折射式物镜18,红外聚焦镜19,载物样品扫描台20,第一滤光片组21,第一光电探测器22,第二滤光片组23,第二光电探测器24。采用图2和图3所示系统实现单光束探测的跨模态光谱与成像的过程如下:In a specific embodiment of the present invention, as shown in FIG2, a cross-modal spectroscopy and imaging device based on single beam detection disclosed by the present invention includes a first light source 1, a first wide beam lens 2, a second wide beam lens 3, a third wide beam lens 4, a fourth wide beam lens 5, a first reflector 6, a second reflector 7, a third reflector 8, an electro-optical modulator 9, a dichroic mirror 10, a fourth reflector 11, a 50-50 beam splitter 12, a second light source 13, a fifth reflector 14, a beam splitter 15, a sixth reflector 16, a seventh reflector 17, a refractive objective lens 18, an infrared focusing lens 19, a sample scanning stage 20, a first filter set 21, a first photodetector 22, a second filter set 23, and a second photodetector 24. The process of implementing cross-modal spectroscopy and imaging of single beam detection using the system shown in FIG2 and FIG3 is as follows:
1)第一光源1为超快激光光源,发射出两束激光,脉冲宽度为2皮秒,基础脉冲重复频率均为80兆赫兹,其中一束为波长可调谐的泵浦光,可调谐的范围为700-990纳米,经过第一阔束透镜2,第二阔束透镜3调整为平行光,然后透过双色镜10传播。另外一束为波长固定在1031纳米的受激辐射光,分别被第三阔束透镜4,第四阔束透镜5准直并调整为平行光,然后依次经过第一反射镜6,第二反射镜7,第三反射镜8引导至电光调制器9,调制成频率为20兆赫兹,并由双色镜反射,通过调节三片反射镜的角度使受激辐射光与泵浦光合束。1) The first light source 1 is an ultrafast laser light source, emitting two laser beams with a pulse width of 2 picoseconds and a basic pulse repetition frequency of 80 MHz. One of the beams is a wavelength-tunable pump light with a tunable range of 700-990 nanometers, which is adjusted to parallel light through the first wide-beam lens 2 and the second wide-beam lens 3, and then propagates through the dichroic mirror 10. The other beam is a stimulated radiation light with a wavelength fixed at 1031 nanometers, which is collimated and adjusted to parallel light by the third wide-beam lens 4 and the fourth wide-beam lens 5, and then guided to the electro-optic modulator 9 through the first reflector 6, the second reflector 7, and the third reflector 8 in sequence, modulated to a frequency of 20 MHz, and reflected by the dichroic mirror. The stimulated radiation light and the pump light are combined by adjusting the angles of the three reflectors.
2)通过移动第二反射镜7,第三反射镜8的距离来调整受激辐射光的光程,用来调节泵浦光与受激辐射光脉冲聚焦在样品上的时间重合;移动方向由箭头指出,受激拉曼散射效应在受激辐射光和泵浦光的时间延时为0皮秒时,达到最高的信号值,通过调节受激辐射光和泵浦光脉冲的时间延时,可以实现时间分辨相干拉曼散射的探测。2) The optical path of the stimulated radiation light is adjusted by moving the distance between the second reflector 7 and the third reflector 8 to adjust the time overlap of the pump light and the stimulated radiation light pulse focused on the sample; the moving direction is indicated by the arrow, and the stimulated Raman scattering effect reaches the highest signal value when the time delay between the stimulated radiation light and the pump light is 0 picoseconds. By adjusting the time delay between the stimulated radiation light and the pump light pulse, the detection of time-resolved coherent Raman scattering can be achieved.
3)合束后的泵浦光和受激辐射光将经过反射镜11的引导,透射过50-50分束镜12,被折射式物镜18聚焦在样品上;第二光源13出射的波长可调谐的中红外光,波长可调谐的范围为5-10.5微米,通过对激光器进行电调制将重复频率调到100千赫兹,脉冲宽度为500纳秒,然后经过第五反射镜14引导,透过分束镜15;3) The combined pump light and stimulated radiation light will be guided by the reflector 11, transmitted through the 50-50 beam splitter 12, and focused on the sample by the refractive objective lens 18; the wavelength-tunable mid-infrared light emitted by the second light source 13 has a tunable wavelength range of 5-10.5 microns, and the repetition frequency is adjusted to 100 kHz by electrically modulating the laser, and the pulse width is 500 nanoseconds, and then guided by the fifth reflector 14 and transmitted through the beam splitter 15;
4)中红外光随后通过第六反射镜16,第七反射镜17引导至红外聚焦镜19并聚焦在样品上。通过调整两片反射镜的夹角与红外聚焦镜的空间位置,使得中红外光光路的光轴与泵浦光和受激辐射光的光路光轴完全重合,并且使得中红外激光聚焦的焦斑与泵浦光和受激辐射光焦斑在空间上重合;4) The mid-infrared light is then guided to the infrared focusing mirror 19 through the sixth reflector 16 and the seventh reflector 17 and focused on the sample. By adjusting the angle between the two reflectors and the spatial position of the infrared focusing mirror, the optical axis of the mid-infrared light path is completely overlapped with the optical axis of the pump light and the stimulated radiation light, and the focal spot of the mid-infrared laser is spatially overlapped with the focal spot of the pump light and the stimulated radiation light;
5)样品搭载在载物样品扫描台20上,通过调谐泵浦光的波长使得泵浦光与受激辐射光的能量差匹配对应的拉曼振动能级,调谐中红外光的波长至红外振动能级,样品产生对应的受激拉曼散射信号与中红外光热信号,信号会被携带在泵浦光与受激辐射光中;5) The sample is mounted on the sample scanning stage 20. By tuning the wavelength of the pump light so that the energy difference between the pump light and the stimulated radiation light matches the corresponding Raman vibration energy level, the wavelength of the mid-infrared light is tuned to the infrared vibration energy level. The sample generates a corresponding stimulated Raman scattering signal and a mid-infrared photothermal signal, and the signal will be carried in the pump light and the stimulated radiation light;
6)携带有受激拉曼散射信号与中红外光热信号的泵浦光作为探测光,可通过后向或者前向得到探测。所述前向为泵浦光的入射方向,后向为与泵浦光的入射方向相反的方向。后向探测的流程是;经过折射式物镜18收集后,由50-50分束镜反射,再经过第一滤光片组21滤掉受激辐射光后入射到第一光电探测器22上;前向探测的流程是:经过红外聚焦镜19进行前向收集,依次通过第七反射镜17、第六反射镜16引导,由合束镜15反射从光路中分离,再经过第二滤光片组23过滤掉受激辐射光后入射到第二光电探测器24上;当进行前向探测时,用于收集泵浦光的红外聚焦镜需要对泵浦光保持高透过率和收集效率,以减少泵浦光在探测时的损耗。6) The pump light carrying the stimulated Raman scattering signal and the mid-infrared photothermal signal is used as the detection light, which can be detected in the backward or forward direction. The forward direction is the incident direction of the pump light, and the backward direction is the direction opposite to the incident direction of the pump light. The process of backward detection is: after being collected by the refractive objective lens 18, it is reflected by the 50-50 beam splitter, and then filtered out by the first filter group 21 to be incident on the first photodetector 22; the process of forward detection is: forward collection is performed through the infrared focusing mirror 19, guided by the seventh reflector 17 and the sixth reflector 16 in turn, and separated from the optical path by reflection by the beam combiner 15, and then filtered out by the second filter group 23 to be incident on the second photodetector 24; when performing forward detection, the infrared focusing mirror used to collect the pump light needs to maintain high transmittance and collection efficiency for the pump light to reduce the loss of the pump light during detection.
7)光电探测器22、24将获得的光信号转换为电信号后,输入到前置放大器28进行进一步地电压信号放大,随后输入至滤波器件26进行数模转换后,滤波器件接收第二光源13的调制频率信号与电光调制器9的调制频率信号作为滤波的中心频率带,在100千赫兹和20兆赫兹同时进行窄带滤波,分离出信号成分,即同时探测到受激拉曼散射信号与中红外光热信号;7) After the photodetectors 22 and 24 convert the obtained optical signals into electrical signals, they are input into the preamplifier 28 for further voltage signal amplification, and then input into the filter device 26 for digital-to-analog conversion. The filter device receives the modulation frequency signal of the second light source 13 and the modulation frequency signal of the electro-optical modulator 9 as the center frequency band of filtering, and performs narrow-band filtering at 100 kHz and 20 MHz at the same time to separate the signal components, that is, the stimulated Raman scattering signal and the mid-infrared photothermal signal are detected at the same time;
8)通过载物样品台20进行空间扫描并同时采集滤波器件的输出信号,实现基于单光束探测的跨模态红外与拉曼成像数据采集,采集的数据通过光谱与成像处理设备25进行像素分割、平均后形成成像图;成像速度、视野范围、像素大小与采集时间由光谱与成像处理设备25控制;8) Performing spatial scanning through the sample stage 20 and simultaneously collecting the output signal of the filter device to realize cross-modal infrared and Raman imaging data collection based on single-beam detection, and the collected data is pixel-segmented and averaged through the spectrum and imaging processing device 25 to form an imaging image; the imaging speed, field of view, pixel size and collection time are controlled by the spectrum and imaging processing device 25;
9)通过扫描第二光源13出射的中红外光的波长和扫描第一光源1出射的泵浦光波长,对于受激辐射光、泵浦光、中红外光共同焦点内的分子进行光谱采集,以实现基于单光束探测的跨模态原位红外与拉曼光谱数据采集,跨模态原位红外与拉曼光谱采集波段和速度由光谱与成像处理设备25控制;9) By scanning the wavelength of the mid-infrared light emitted by the second light source 13 and the wavelength of the pump light emitted by the first light source 1, spectrum collection is performed on the molecules within the common focus of the stimulated radiation light, the pump light, and the mid-infrared light, so as to realize cross-modal in-situ infrared and Raman spectroscopy data collection based on single-beam detection, and the cross-modal in-situ infrared and Raman spectroscopy collection band and speed are controlled by the spectrum and imaging processing device 25;
为了更方便理解本发明的探测方案,结合图4所示展示的入射的泵浦光、受激辐射光、中红外光的脉冲序列图,其中的fP,fS,fM分别代表着泵浦光、受激辐射光和中红外光的调制频率。在本发明的一个实施例中,fP,fS,fM分别为80兆赫兹、20兆赫兹、100千赫兹。所述的脉冲宽度为2皮秒,重复频率为80兆赫兹的波长可调谐的泵浦光的传播产生光热调制。所述的重复频率为80兆赫兹的波长可调谐的泵浦光光强中编码了一组调制频率在高频20兆赫兹的受激拉曼损耗信号和一组调制频率在低频100千赫兹的光热信号。所述泵浦光光强,通过光电探测器转换为光电压信号,光电压信号经过前置放大后输入至滤波器件。为了更方便理解本实施例所产生的信号构成,结合图5、图6所示,阐述了探测光的波形图在时间域和频率域的特征,本发明选择泵浦光为探测光,以实现更优的性能。如图5所示为时间域的泵浦光波形图,其中携带有SRL信号与MIP信号。如图6所示为图5中探测光光强的频率域成分构成,携带的SRL信号呈现形式为频率为fS处的尖峰,携带的MIP信号的呈现形式为基频为fM的一系列谐波尖峰。在本实施例中红外光调制频率和受激辐射光调制频率在频谱上距离为fS-fM,为19.9兆赫兹,能避免MIP信号和SRS信号的混频。杜绝信号串扰的同时,高频调制能避免低频噪声,显著提升信噪比。图7示出了实际在橄榄油样品上测到的信号频率域构成,在fS±fM处没有观察到频率成分,这表明单光束探测的跨模态光谱与成像方法没有存在信号之间的串扰。In order to more conveniently understand the detection scheme of the present invention, the pulse sequence diagram of the incident pump light, stimulated radiation light, and mid-infrared light shown in Figure 4 is combined, where f P , f S , and f M represent the modulation frequencies of the pump light, stimulated radiation light, and mid-infrared light, respectively. In one embodiment of the present invention, f P , f S , and f M are 80 MHz, 20 MHz, and 100 kHz, respectively. The propagation of the wavelength-tunable pump light with a pulse width of 2 picoseconds and a repetition frequency of 80 MHz produces photothermal modulation. The wavelength-tunable pump light intensity with a repetition frequency of 80 MHz encodes a group of stimulated Raman loss signals with a modulation frequency of 20 MHz at a high frequency and a group of photothermal signals with a modulation frequency of 100 kHz at a low frequency. The pump light intensity is converted into a photovoltage signal by a photodetector, and the photovoltage signal is input into the filter device after preamplification. In order to more conveniently understand the signal composition generated by this embodiment, in combination with FIG. 5 and FIG. 6, the characteristics of the waveform of the detection light in the time domain and the frequency domain are described. The present invention selects pump light as the detection light to achieve better performance. As shown in FIG. 5, the pump light waveform in the time domain carries the SRL signal and the MIP signal. As shown in FIG. 6, the frequency domain component composition of the detection light intensity in FIG. 5, the SRL signal carried is presented as a peak at a frequency of f S , and the MIP signal carried is presented as a series of harmonic peaks with a fundamental frequency of f M. In this embodiment, the infrared light modulation frequency and the stimulated radiation light modulation frequency are at a frequency spectrum distance of f S -f M , which is 19.9 MHz, and can avoid mixing of MIP signals and SRS signals. While eliminating signal crosstalk, high-frequency modulation can avoid low-frequency noise and significantly improve the signal-to-noise ratio. FIG. 7 shows the frequency domain composition of the signal actually measured on the olive oil sample. No frequency component is observed at f S ±f M , which indicates that there is no crosstalk between signals in the cross-modal spectrum and imaging method of single-beam detection.
在本发明的一个具体实施例中,已试验性地实施了用单光束探测的跨模态光谱与成像方法,对500纳米的聚甲基丙烯酸甲酯PMMA塑料微球进行了跨模态光谱数据与成像数据的采集。图8示出了的红外和拉曼光谱数据。利用采集到的光谱数据,选择了红外模态为1730cm-1的C=O伸缩振动带,拉曼模态为2957cm-1的CH3非对称伸缩振动带,进行单光束探测的跨模态成像,结果如图9所示。图10示出了本发明对500纳米小球成像的剖面图,通过去卷积方法可以计算出红外和拉曼成像的分辨率。其中红外成像的分辨率为599纳米,拉曼成像的分辨率为490纳米,略优于红外成像,主要是由于拉曼成像基于非线性的受激拉曼散射方案,分辨率由泵浦光和受激辐射光共同决定,而红外成像分辨率主要是由泵浦光决定的。In a specific embodiment of the present invention, a cross-modal spectroscopy and imaging method using a single beam detection has been experimentally implemented, and cross-modal spectral data and imaging data have been collected for 500-nanometer polymethyl methacrylate PMMA plastic microspheres. FIG8 shows the infrared and Raman spectral data. Using the collected spectral data, the C=O stretching vibration band with an infrared mode of 1730 cm -1 and the CH3 asymmetric stretching vibration band with a Raman mode of 2957 cm -1 were selected to perform cross-modal imaging with a single beam detection, and the results are shown in FIG9. FIG10 shows a cross-sectional view of the imaging of a 500-nanometer microsphere by the present invention, and the resolution of infrared and Raman imaging can be calculated by the deconvolution method. The resolution of infrared imaging is 599 nanometers, and the resolution of Raman imaging is 490 nanometers, which is slightly better than that of infrared imaging, mainly because Raman imaging is based on a nonlinear stimulated Raman scattering scheme, and the resolution is jointly determined by the pump light and the stimulated radiation light, while the resolution of infrared imaging is mainly determined by the pump light.
并且对单一微球进行了单光束探测的跨膜态三维成像,如图11、图12分别为在红外1730cm-1和拉曼2957cm-1下采集的图像,其三维重构图如图13所示。图14为图13中的轴向剖面图。通过三维成像,可以看到两个模态的成像图并没有焦平面的偏移,这更加确保了本发明提供的跨膜态成像系统的可靠性,并且确保了量化分析跨模态数据的可行性。In addition, a single microsphere was subjected to three-dimensional imaging of the transmembrane state by single-beam detection, as shown in Figures 11 and 12, which are images collected at infrared 1730 cm -1 and Raman 2957 cm -1 , respectively, and its three-dimensional reconstruction is shown in Figure 13. Figure 14 is an axial cross-sectional view in Figure 13. Through three-dimensional imaging, it can be seen that the imaging images of the two modes do not have a focal plane offset, which further ensures the reliability of the transmembrane state imaging system provided by the present invention and ensures the feasibility of quantitative analysis of cross-modal data.
为了证明本发明增强的化学特异性,在本发明的一个具体实施例中,进行了包含棕榈酸PA、1,4-二苯基丁二炔DiPhDY和三苯基膦TPP三种混合物的单光束探测的跨膜态成像,其中包含如图15所示的具有相似结构和重叠光谱带图。由于本发明具有广阔并且独立的红外和拉曼光谱覆盖范围,因此可以选择具有更高特异性的化学谱带,以充分利用振动光谱的互补性。选择代表性谱带进行了成像,包括红外1477cm-1的苯半圆拉伸、拉曼1593cm-1的苯象限拉伸、红外1700cm-1的C=O拉伸和拉曼2216cm-1的C≡C拉伸。受到光谱选择定则限制,苯半圆拉伸和C=O拉伸位于拉曼光谱中是不活跃的,而红外在炔烃频带中表现为不活跃,如图16所示。因此,每种单模态成像本身不能区分该混合物。在两种模态中具有独特的振动活性模式,经过光谱解混后成功分离了这三种化合物,如图17、图18所示。此外,通过皮尔逊相关系数(PCC)评估,如图19所示,本发明在特异性和选择性方面优于单一模态测量。In order to demonstrate the enhanced chemical specificity of the present invention, in a specific embodiment of the present invention, single beam detection transmembrane state imaging of three mixtures including palmitic acid PA, 1,4-diphenylbutadiyne DiPhDY and triphenylphosphine TPP was performed, including a similar structure and overlapping spectral band diagram as shown in Figure 15. Since the present invention has a broad and independent infrared and Raman spectral coverage, chemical bands with higher specificity can be selected to fully utilize the complementarity of vibrational spectra. Representative bands were selected for imaging, including benzene semicircular stretching at 1477cm -1 in infrared, benzene quadrant stretching at 1593cm -1 in Raman, C=O stretching at 1700cm -1 in infrared, and C≡C stretching at 2216cm -1 in Raman. Limited by the spectral selection rule, benzene semicircular stretching and C=O stretching are inactive in the Raman spectrum, while infrared is inactive in the alkyne band, as shown in Figure 16. Therefore, each single modality imaging itself cannot distinguish the mixture. The three compounds were successfully separated after spectral unmixing with unique vibrational activity patterns in the two modes, as shown in Figures 17 and 18. In addition, as shown in Figure 19, the present invention is superior to single-mode measurement in terms of specificity and selectivity as evaluated by the Pearson correlation coefficient (PCC).
单细胞分析对于跟踪细胞分化过程以及研究其在疾病模型或组织工程中的作用对于各种临床应用至关重要。传统的表型表征方法常常耗时且侵入性,导致有限的时间和空间分辨率。因此,迫切需要一种新技术,可以在细胞分化的初始阶段非侵入性地监测分子动态。Single-cell analysis is essential for tracking cell differentiation processes and studying their roles in disease models or tissue engineering for various clinical applications. Traditional phenotypic characterization methods are often time-consuming and invasive, resulting in limited temporal and spatial resolution. Therefore, a new technology is urgently needed to non-invasively monitor molecular dynamics during the initial stages of cell differentiation.
在验证了本发明具有高空间分辨率和高特异性的化学成像能力后,在本发明的一个具体实施例中,对分化三天后的间充质干细胞(MSCs)作为对照组和成软骨细胞(CBs)作为实验组进行原位成像和代谢分析。为了进一步研究与脂滴合成相关的代谢变化,进行了红外1750cm-1C=O带和拉曼2850cm-1CH2对称伸缩带下的单光束探测的跨膜态成像,如图20所示、图21为两个波数,以展现相对于总脂滴中甘油三酯(TAG)水平。如图22所示,观察到脂滴数量的显著增加,并且大小分布更广,具有显著差异即p=0.007,这表明在分化诱导后脂质合成代谢发生了显著改变。考虑到脂滴在抗氧化作用中的作用,软骨细胞分化的早期阶段增加的脂滴可能表明激活了抗氧化途径,以减轻活性氧的积累。After verifying that the present invention has high spatial resolution and high specificity chemical imaging capabilities, in a specific embodiment of the present invention, in situ imaging and metabolic analysis were performed on mesenchymal stem cells (MSCs) as a control group and chondrocytes (CBs) as an experimental group after three days of differentiation. In order to further study the metabolic changes associated with lipid droplet synthesis, transmembrane state imaging with single beam detection under the infrared 1750cm -1 C=O band and Raman 2850cm -1 CH2 symmetric stretching band was performed, as shown in Figure 20 and Figure 21 are two wave numbers to show the level of triglycerides (TAG) relative to total lipid droplets. As shown in Figure 22, a significant increase in the number of lipid droplets was observed, and the size distribution was wider, with a significant difference of p=0.007, indicating that lipid anabolism was significantly changed after differentiation induction. Considering the role of lipid droplets in antioxidant effects, the increased lipid droplets in the early stages of chondrocyte differentiation may indicate the activation of antioxidant pathways to alleviate the accumulation of reactive oxygen species.
复杂大型分子的合成,如蛋白聚糖,是软骨细胞分化的重要代谢指标。尽管在各种红外光谱研究中,红外1080cm-1被认为是蛋白聚糖的特征峰,但传统红外成像在空间分辨率和与水溶液环境中细胞成像的兼容性方面存在限制,阻碍了蛋白聚糖分布的原位探测。为了有效利用红外和拉曼的灵敏度,使用红外1080cm-1描绘蛋白聚糖,并选择CH2非对称伸展带的拉曼2930cm-1代表整体蛋白质分布,如图23所示。通过对在1080cm-1/2930cm-1的跨膜态图像进行比率分析,图24可视化了细胞体内分布的大量蛋白聚糖颗粒。图25对蛋白聚糖颗粒的大小和相对强度的分布进行了量化,揭示了即使在诱导3天的分化的早期阶段,PD的合成存在显著差异,即大小分布p=0.0011,强度分布p=0.0012。差异化分析后的PCC进一步凸显了与单模态测量相比,证明本发明提供的方法提供了更高的特异性,如图26所示。总之,这些结果表明本发明提供的单光束探测的跨膜态光谱于成像方法作为一种非侵入性、快速的单细胞分析技术具有高灵敏度、高特异性和高空间分辨率,而无需任何标记或染色。The synthesis of complex large molecules, such as proteoglycans, is an important metabolic indicator of chondrocyte differentiation. Although the infrared 1080 cm -1 is considered to be the characteristic peak of proteoglycans in various infrared spectroscopy studies, the limitations of conventional infrared imaging in terms of spatial resolution and compatibility with cell imaging in aqueous environments have hindered the in situ detection of proteoglycan distribution. In order to effectively utilize the sensitivity of infrared and Raman, infrared 1080 cm -1 was used to depict proteoglycans, and Raman 2930 cm -1 of the CH2 asymmetric stretching band was selected to represent the overall protein distribution, as shown in Figure 23. By performing ratio analysis on the transmembrane state image at 1080 cm -1 /2930 cm -1 , Figure 24 visualizes a large number of proteoglycan particles distributed in the cell body. Figure 25 quantifies the distribution of the size and relative intensity of proteoglycan particles, revealing that even in the early stages of differentiation induced for 3 days, there are significant differences in the synthesis of PD, i.e., p = 0.0011 for size distribution and p = 0.0012 for intensity distribution. PCC after differential analysis further highlights the higher specificity of the method provided by the present invention compared to single-modality measurements, as shown in Figure 26. In summary, these results indicate that the single-beam probed transmembrane state spectroscopy and imaging method provided by the present invention is a non-invasive and rapid single-cell analysis technique with high sensitivity, high specificity and high spatial resolution without any labeling or staining.
在细胞水平上识别分子特征在早期疾病检测和病理机制研究中起着关键作用。虽然基于组学的技术在筛选与疾病相关的分子标记方面表现出色,但基于成像的方法提供了有价值的空间信息,从而能够更深入地了解分子机制。然而,传统的组织病理学分析需要先验知识和耗时的准备工作,例如免疫荧光染色。此外,高效地为原位组成分析标记大多数代谢物,包括脂类和胆固醇,存在相当大的挑战。本发明通过提供高分子特异性的无标记成像,解决了这些挑战,有助于在病理条件下进行代谢分析。Identifying molecular features at the cellular level plays a key role in early disease detection and the study of pathological mechanisms. While omics-based technologies excel in screening disease-associated molecular markers, imaging-based methods provide valuable spatial information, enabling a deeper understanding of molecular mechanisms. However, traditional histopathological analysis requires prior knowledge and time-consuming preparations, such as immunofluorescence staining. In addition, there are considerable challenges in efficiently labeling most metabolites, including lipids and cholesterol, for in situ compositional analysis. The present invention addresses these challenges by providing label-free imaging with high molecular specificity, facilitating metabolic analysis under pathological conditions.
在本发明的一个具体实施例中,对阿尔茨海默病(AD)模型中进行了跨膜态光谱与成像,展示了原位高信息含量分析的能力。针对大脑的纹状体,分别在红外1465cm-1、拉曼2850cm-1和红外1750cm-1、拉曼2930cm-1进行了两组单光束探测的跨膜态成像,如图27所示。成像结果显示,在纹状体内,纤维状结构均匀分布,出现明显的2930cm-1蛋白信号。值得注意的是,观察到大小从20到100微米不等的纹状小体存在,其含有高水平的脂质和蛋白质信号。借助本发明在探测方法对小颗粒具有更高探测灵敏度的能力,可视化大量脂滴,在涉及脂质C=O带的红外1750cm-1和涉及脂质CH2振动和胆固醇甲基振动的红外1465cm-1观察到了主要分布在纹状体外部,并且部分共定位在纹状体内。In a specific embodiment of the present invention, transmembrane state spectroscopy and imaging were performed in an Alzheimer's disease (AD) model, demonstrating the ability of in-situ high information content analysis. For the striatum of the brain, two sets of single-beam detection transmembrane state imaging were performed at infrared 1465cm -1 , Raman 2850cm -1 and infrared 1750cm -1 , Raman 2930cm -1 , as shown in Figure 27. The imaging results show that in the striatum, the fibrous structure is evenly distributed, and an obvious 2930cm -1 protein signal appears. It is worth noting that the presence of striatal corpuscles ranging in size from 20 to 100 microns was observed, which contained high levels of lipid and protein signals. With the ability of the present invention to have a higher detection sensitivity for small particles in the detection method, a large number of lipid droplets were visualized, and the infrared 1750cm -1 involving lipid C=O band and the infrared 1465cm -1 involving lipid CH2 vibration and cholesterol methyl vibration were observed to be mainly distributed outside the striatum, and partially co-localized in the striatum.
为了深入了解化学成分,在不同结构中同时采集了原位的红外和拉曼光谱,包括纤维结构(S1)、纹状体中共定位的液滴(S2)和纹状体外的液滴(S3),揭示了它们明显不同的成分,如图28所示。光谱s1和s2明显显示了1655cm-1(酰胺I)、1545cm-1(酰胺II)和2930cm-1(CH2不对称带)处的峰值,这些是纤维结构和纹状体中蛋白质成分的特征。值得注意的是,光谱s2中1750cm-1和2850cm-1的峰值表明与纤维结构相比,纹状体中的脂质含量较高。此外,通过多个胆固醇特征峰的验证:红外1375cm-1(CH3对称弯曲)、红外1465cm-1(CH3不对称弯曲)和拉曼2870cm-1(CH3对称伸展),证实了共定位液滴中胆固醇水平的升高。光谱s3显示了1069cm-1和1224cm-1处的显著峰值,与P-O-C和PO2 -振动有关,表明高磷脂含量。这些结果证明了互补光谱成像和原位光谱测量在识别病理条件的代谢特征方面的潜力,这有助于疾病检测和靶向治疗的发展。To gain insight into the chemical composition, in situ IR and Raman spectra were simultaneously collected in different structures, including the fiber structure (S1), the co-localized droplets in the striatum (S2), and the droplets outside the striatum (S3), revealing their distinct compositions, as shown in Figure 28. Spectra s1 and s2 clearly show peaks at 1655 cm -1 (amide I), 1545 cm -1 (amide II), and 2930 cm -1 (CH 2 asymmetric band), which are characteristic of the protein components in the fiber structure and the striatum. Notably, the peaks at 1750 cm -1 and 2850 cm -1 in spectrum s2 indicate a higher lipid content in the striatum compared to the fiber structure. In addition, the elevated cholesterol levels in the co-localized droplets were confirmed by the verification of multiple cholesterol characteristic peaks: IR 1375 cm -1 (CH 3 symmetric bending), IR 1465 cm -1 (CH 3 asymmetric bending), and Raman 2870 cm -1 (CH 3 symmetric stretching). Spectrum S3 shows significant peaks at 1069 cm -1 and 1224 cm -1 , associated with POC and PO 2 - vibrations, indicating a high phospholipid content. These results demonstrate the potential of complementary spectral imaging and in situ spectroscopic measurements to identify metabolic signatures of pathological conditions, which could aid disease detection and the development of targeted therapies.
除了单细胞分析和组织切片的代谢成像外,还展示了本发明提供的跨膜态成像方法可以成为研究微生物的多功能工具。C.elegans是一个广泛采用的与衰老和疾病相关的机制研究模型生物。重要的是,不同分子的机制研究需要至少双波数成像。在C.elegans中进行了单次的同时双波数成像:红外1655cm-1和拉曼2850cm-1,分别对应蛋白质酰胺I带和脂质CH2带,如图29和图30所示,揭示了C.elegans内部不同器官的结构和生物成分。具体地,脂质成分主要储存在腹部,可能归因于胃肠等器官。相反,富含蛋白质的区域主要位于背部,包含丰富的体壁肌肉。In addition to single-cell analysis and metabolic imaging of tissue sections, it is also demonstrated that the transmembrane state imaging method provided by the present invention can become a versatile tool for studying microorganisms. C.elegans is a widely adopted model organism for mechanism studies related to aging and disease. Importantly, mechanistic studies of different molecules require at least dual wavenumber imaging. A single simultaneous dual wavenumber imaging was performed in C.elegans: infrared 1655cm -1 and Raman 2850cm -1 , corresponding to the protein amide I band and lipid CH2 band, respectively, as shown in Figures 29 and 30, revealing the structure and biological composition of different organs inside C.elegans. Specifically, lipid components are mainly stored in the abdomen, which may be attributed to organs such as the gastrointestinal tract. In contrast, protein-rich areas are mainly located in the back and contain abundant body wall muscles.
本发明实现了单光束和探测器探测的红外和拉曼成像和光谱,解决了传统红外成像分辨率受限、拉曼成像灵敏度不足的问题,更重要的是使得同时采集红外和拉曼成像并进行更加精准的定量生化分析成为可能。展示了在各种应用场景下实现高分辨率、高灵敏度和高准确性的多个化学键的红外和拉曼同时成像。值得注意的是,由于红外和拉曼振动模式及其内在的互补性优势,使得本发明提供的光谱与成像方法具有增强的特异性和选择性,能够实现互补图像的定量分析,并适用于更广泛的场景。双模式振动成像继承了互补光谱学在结构和动态洞察力方面的丰富性,为原位化学反应监测、单细胞代谢分析、病理机制研究和生物成像等复杂生物和化学系统的光谱成像研究提供了巨大的机会和促进分子水平的理解。此外,与往往限于连续光谱范围的复杂的复用SRS技术相比,本发明的方法具有更广泛的光谱可调谐性,并且大大减少了光谱冗余信息。此外,本发明的方法的概念可以扩展到各种光谱成像模式,用于高信息维度的跨模态成像。凭借目前配备的皮秒激光器源,本发明的方法可以成为与丰富的非线性光学成像模态兼容的多功能成像和分析平台,如TA、TPEF、CARS、SHG、THG和SFG模态等,为生物医学和材料科学的光学成像和传感开辟新的可能性。The present invention realizes infrared and Raman imaging and spectroscopy detected by a single beam and detector, solves the problems of limited resolution of traditional infrared imaging and insufficient sensitivity of Raman imaging, and more importantly, makes it possible to simultaneously collect infrared and Raman images and perform more accurate quantitative biochemical analysis. It demonstrates the simultaneous infrared and Raman imaging of multiple chemical bonds with high resolution, high sensitivity and high accuracy in various application scenarios. It is worth noting that due to the complementary advantages of infrared and Raman vibrational modes and their inherent complementarity, the spectral and imaging methods provided by the present invention have enhanced specificity and selectivity, can realize quantitative analysis of complementary images, and are applicable to a wider range of scenarios. Dual-mode vibrational imaging inherits the richness of complementary spectroscopy in terms of structural and dynamic insights, and provides great opportunities and promotes molecular-level understanding for spectral imaging studies of complex biological and chemical systems such as in situ chemical reaction monitoring, single-cell metabolic analysis, pathological mechanism research and biological imaging. In addition, compared with complex multiplexed SRS techniques that are often limited to continuous spectral ranges, the method of the present invention has a wider spectral tunability and greatly reduces spectral redundant information. In addition, the concept of the method of the present invention can be extended to various spectral imaging modes for cross-modal imaging with high information dimensions. With the currently equipped picosecond laser source, the method of the present invention can become a versatile imaging and analysis platform compatible with a rich range of nonlinear optical imaging modalities, such as TA, TPEF, CARS, SHG, THG and SFG modalities, opening up new possibilities for optical imaging and sensing in biomedicine and materials science.
受限于激光器波长的范围,本实施例中红外成像的波段限制在900-2000波数,利用其他激光器,可以使得单光束探测的跨模态光谱与成像中利用光热效应的成像波段进一步扩大,可以扩大至紫外光、可见光、近红外至远红外。Limited by the range of laser wavelength, the infrared imaging band in this embodiment is limited to 900-2000 wavenumbers. By using other lasers, the imaging band using the photothermal effect in cross-modal spectrum and imaging of single-beam detection can be further expanded to ultraviolet light, visible light, near infrared to far infrared.
如本文使用的,术语“包括”应被理解为包括性和开放性,不应被理解为排他性。具体来说,当用于包括权利要求的本说明书时,术语“包括”及其变型意思是包括特定特征,步骤或组件。这些术语不被解释成排除其他特征、步骤或组件的存在。As used herein, the term "comprising" should be understood as inclusive and open, and should not be understood as exclusive. Specifically, when used in this specification including claims, the term "comprising" and its variations mean including specific features, steps or components. These terms are not to be interpreted as excluding the presence of other features, steps or components.
以上所述的具体实施方式对本发明的技术方案和有益效果进行了详细说明,应理解的是以上所述仅为本发明的最优选实施例,并不用于限制本发明,凡在本发明的原则范围内所做的任何修改、补充和等同替换等,均应包含在本发明的保护范围之内。The specific implementation methods described above have described in detail the technical solutions and beneficial effects of the present invention. It should be understood that the above is only the most preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, supplements and equivalent substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.
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