CN1349093A - Multifunctional molecular radar - Google Patents

Abstract

The invention relates to a multifunctional molecular radar, which includes an excitation light source part, a sample excitation and fluorescence collection part, and a fluorescence signal detection and processing part. Scanning is carried out through the scanning galvanometer group, and finally the sample is focused on the sample through a high numerical aperture microscopic objective lens. The band-pass filter removes the scattered excitation light, etc., and then it is converged on the confocal aperture by the lens. After being received by the single photon counter, it is input to the signal system for processing, and the fluorescence image and the fluorescence correlation spectrum are obtained. By using the multifunctional molecular radar of the present invention, various measurements of different types can be carried out on the same sample on a set of systems, and the adjustment process of the experimental device and the instrument is simple, and the working efficiency is improved.

Description

multifunctional molecular radar

technical field

The invention relates to a multifunctional molecular radar, which is a novel biological measuring instrument. Through the integration of optical path and detection system, it can realize all the functions of laser scanning confocal fluorescence imaging, two-photon fluorescence imaging, single-photon and two-photon fluorescence correlation spectrometer, gradient field fluorescence correlation spectrometer on the same device, and realize the Imaging of specific biochemical components and measurement of the kinematics of target molecules in living cell systems.

Background technique

In the prior art, a laser-scanning two-photon (confocal) fluorescence microscope is used to perform laser-scanning imaging of specific fluorescent markers to carry out research on life phenomena at the cell and subcellular levels.

The basic device of the existing laser scanning confocal fluorescence microscope is shown in Figure 1: single-photon confocal imaging uses visible-band laser (wavelength is generally 488nm) for fluorescence excitation. After the excitation light is attenuated by the attenuator (11), it passes through the low-pass two-color beam splitter (5) (passes through when the wavelength is less than 488nm), then passes through the X-Y scanning galvanometer group (4) to realize beam scanning, and finally passes through the high numerical aperture The microscope objective lens (3) is focused on the solution or living cell sample (2). The back fluorescence emitted by specific fluorescent particles in the focusing area (1) is collected by the microscope objective lens (3), after passing through the X-Y scanning galvanometer group (4), it is reflected by the low-pass two-color beam splitter (5), and then filtered The light sheet (6) removes the scattered excitation light and other stray light, and then the lens (7) converges on the confocal aperture (8) with adjustable aperture, and is received by the detector (9) and then input to the signal system ( 10) Carry out signal acquisition and processing. The image size of the confocal pinhole and the focus point (1) is consistent, and the fluorescence and stray light outside the focus area (1) are suppressed by spatial filtering, and the signal-to-noise ratio of the fluorescence measurement is improved. The above devices have mature technology and are generally used for imaging research of living cell systems. However, because the excitation light source used is visible or partial ultraviolet, it has a greater killing effect on living cells during long-term observation, and the wavelength it uses is single. , cannot achieve continuous adjustment.

The two-photon fluorescence microscope device is basically the same as the confocal microscope, as shown in Figure 2: the fluorescence excitation light source adopts an infrared band femtosecond laser, and the wave band is 680-1080nm; , visible wavelength fluorescence is reflected. In addition, since two-photon fluorescence can only be generated at the focal point, no spatial filtering is required, so there is no confocal aperture in front of the detector. The killing effect is small, and it can be applied to the observation and research of samples for a long time. Because of its continuously adjustable wavelength, there is a large choice for fluorescent dyes. However, due to the relatively new technology, its practical field remains to be further explored.

In the prior art, for living cells or solution systems, in order to measure process information such as structural changes of biomolecules, microscopic chemical reactions, and intracellular enzyme kinetics, fluorescence correlation spectrometers are generally used. The principle is: by measuring the fluorescence fluctuation signals of a small number of luminescent molecules in the micro-area and performing correlation analysis on them, the physical mechanism information about the concentration of fluorescent molecules, diffusion speed, etc. that affect the fluctuations can be obtained.

The basic optical paths of single-photon and two-photon excitation fluorescence correlation spectrometers are shown in Figure 3 and Figure 4, respectively, and their structures are similar to those of confocal fluorescence microscopes and two-photon fluorescence microscopes. The main difference is that the X-Y scanning galvanometer group (4) in Fig. 1 and Fig. 2 is respectively replaced by a total reflection mirror (13). In addition, the detector of the fluorescence correlation spectrometer is an avalanche diode single photon counter (16). The signal system (17) uses a multi-channel scaler and an auto-correlation card to record data. The data processing and system control software are also related to the scanning fluorescence Microscopes are completely different, including autocorrelation data processing and least squares fitting functions, in order to obtain a series of parameters such as the diffusion coefficient of fluorescent particles and the number of fluorescent particles in the focal area from the experimental data. The technology adopted by the device belongs to the field of single-molecule detection, which is difficult to operate and is suitable for the dynamic measurement of target biomolecules in cell systems.

To sum up, the existing scanning fluorescence microscope and fluorescence correlation spectrometer are both independent systems with a single function. In actual research, when conducting research on a specific biological system, multi-parameter composite measurement is very necessary. The method of scanning imaging can obtain information at the cell level, and the method of fluorescence correlation spectrometer can obtain information at the molecular level. Therefore, under the current conditions, when performing imaging research on a specific biological object, if it is necessary to carry out molecular dynamics research on a specific part of the object at the same time, it needs to be moved to the fluorescence correlation spectrometer. The research environment will change during the process. At the same time, it is quite difficult to match the imaging area with the research area of the fluorescence correlation spectrometer. The operation process is also complicated, which greatly affects the accuracy of the experimental results. At the same time, the number of optical devices required to establish two sets of independent systems is large, and there is also a certain degree of waste.

Contents of the invention

The purpose of the present invention is to design a multifunctional molecular radar, by changing the optical path setting and laser working mode, realize the integration of laser scanning two-photon (confocal) fluorescence microscope and fluorescence correlation spectrometer, on a set of new system, for The same sample is used to perform multiple measurements of different types, simplify the experimental device and instrument adjustment process as much as possible, reduce the difficulty of the experiment, effectively use all optical devices, and improve work efficiency.

The multifunctional molecular radar designed by the invention includes an excitation light source part, a sample excitation and fluorescence collection part, and a fluorescence signal detection and processing part. The excitation light source part includes a visible excitation light attenuator, an infrared laser attenuator and a high-pass beam splitter; the sample excitation and fluorescence collection part includes a laser focus point, a microscope objective lens and an X-Y scanning galvanometer group; the fluorescence signal detection and processing part includes a band pass Beamsplitters, filters, focusing lenses, adjustable confocal apertures, detectors and signal systems. The excitation light in the visible band is attenuated by the attenuator, reflected by the high-pass two-color beam splitter, passed through the multi-band band-pass beam splitter, and then passed through the X-Y scanning galvanometer group to realize beam scanning, and finally focused on the sample through a high numerical aperture microscope objective. The back fluorescence emitted by specific fluorescent particles in the focusing area is collected by the microscope objective lens, after passing through the X-Y scanning galvanometer group, it is reflected by the multi-band band-pass beam splitter, and then passes through the band-pass filter to remove the scattered excitation light and other The stray light is then converged by the lens on the confocal aperture with adjustable aperture, received by the avalanche diode single photon counter, and then input to the signal system for signal processing to obtain the fluorescence image and fluorescence correlation spectrum.

Using the multifunctional molecular radar designed by the present invention, the integration of laser scanning two-photon (confocal) fluorescence microscope and fluorescence correlation spectrometer is realized, and various measurements of different types are carried out for the same sample on one set of systems , the experimental device and the adjustment process of the instrument are simple, and the work efficiency is improved.

Description of drawings

Figure 1 is a schematic diagram of the structure of a confocal scanning fluorescence microscope.

Fig. 2 is a schematic diagram of the structure of a two-photon scanning fluorescence microscope.

Fig. 3 is a schematic diagram of the structure of a single-photon excitation fluorescence correlation spectrometer.

Fig. 4 is a schematic diagram of the structure of a two-photon excitation fluorescence correlation spectrometer.

Fig. 5 is a schematic structural diagram of the multifunctional molecular radar designed by the present invention.

In Figures 1 to 5, 1 is the laser focusing point, 2 is the sample, 3 is the microscope objective lens, 4 is the X-Y scanning galvanometer group, 5 is the low-pass two-color beam splitter, 6 is the filter, and 7 is the focusing lens , 8 is the confocal pinhole, 9 is the detector (photomultiplier tube), 10 is the signal system (imaging), 11 is the visible excitation light attenuator, 12 is the high-pass two-color beam splitter, 13 is the filter, 14 is Infrared laser attenuator, 15 is a two-color beam splitter, 16 is a detector (single photon counting module), 17 is a signal system (fluorescence correlation spectrum), 18 is a bandpass beam splitter, 19 is a filter, 20 is an optional Adjust the confocal aperture, 21 is the signal system (imaging and fluorescence correlation spectrum), 22 is the high-pass beam splitter, I is the excitation light source part, II is the sample excitation and fluorescence collection part, III is the fluorescence signal detection and processing system.

Detailed ways

As shown in Figure 5, the multifunctional molecular radar designed by the present invention includes an excitation light source part I, which is composed of a visible excitation light attenuation sheet 11, an infrared laser attenuation sheet 14 and a high-pass beam splitter; sample excitation and fluorescence collection part II , this part is composed of laser focusing point 1, sample 2, microscope objective lens 3 and X-Y scanning galvanometer group 4; fluorescence signal detection and processing part III is composed of bandpass beam splitter 18, filter 19, focusing lens 7, Confocal aperture 20, detector (single photon counting module) 16 and signal system (imaging and fluorescence correlation spectrum) 21. See the band excitation light is attenuated by the attenuation sheet 11, after the reflection of the high-pass two-color beam splitter 22 (reflecting the visible-band excitation light and transmitting the infrared-band excitation light) (infrared ultrafast laser is attenuated by the attenuation sheet 14, and passes through the high-pass two-color beam splitter After the sheet 22) is reflected, it passes through the multiband bandpass beam splitter (18) (through infrared and visible excitation light, the reflection wavelength is between the fluorescence between the infrared and visible excitation light and the fluorescence with a wavelength smaller than the visible light), and then passes through The X-Y scanning galvanometer group 4 realizes beam scanning, and finally focuses on the solution or living cell sample 2 through the high numerical aperture microscopic objective lens 3 . The back-facing fluorescence emitted by specific fluorescent particles in the focusing area 1 is collected by the microscope objective lens 3, and after passing through the X-Y scanning galvanometer group 4, it is reflected by the multi-band band-pass beam splitter 18, and then passes through the band-pass filter 1 to remove scattering The excitation light (including visible excitation light and infrared excitation light) and other stray light are then converged by the lens 7 on the confocal aperture 20 with adjustable aperture, and received by the avalanche diode single photon counter 16. The signals are input to the signal system 21 for signal processing to obtain fluorescence images and fluorescence correlation spectra.

Using the multifunctional molecular radar of the present invention, carried out following five different measurement processes on a set of systems:

1. The present invention has the function of laser scanning confocal fluorescence imaging, and the research on calcium imaging in mouse oocytes has been carried out. Visible band excitation light (laser with a wavelength of 488nm in the experiment) is attenuated by the attenuation sheet (11), and the high-pass two-color beam splitter (22) (reflects the visible band excitation light, transmits the infrared band excitation light, and reflects the 488nm excitation light in the experiment) reflection Afterwards, through the multi-band bandpass beam splitter (18) (passing infrared and visible excitation light, reflecting fluorescence with a wavelength between infrared and visible excitation light and fluorescence with a wavelength smaller than visible light), and then passing through the X-Y scanning galvanometer Group (4) realizes beam scanning, and finally focuses on the solution or living cell sample (2) through a high numerical aperture microscopic objective lens (3) (mouse oocytes stained with Fluo3 are used in the experiment). The back fluorescence emitted by specific fluorescent particles in the focusing area (1) (in the experiment, Fluo3 emits fluorescence with a wavelength of 514nm) is collected by the microscope objective lens (3), and after passing through the X-Y scanning galvanometer group (4), it is captured by the multi-band band Reflected by the beam splitter (18), then the scattered excitation light (including visible excitation light and infrared excitation light) and other stray light are removed by the bandpass filter (19), and then converged by the lens (7) on the adjustable-aperture On the confocal aperture (20), after being received by the avalanche diode single photon counter (16). Input to the signal system (21), cooperate with the photon counting card to continuously record the signal, output the image after processing, and carry out intracellular calcium imaging research.

2. The present invention has the function of laser scanning two-photon fluorescence imaging, and the research on calcium imaging in mouse oocytes has been carried out. The fluorescent excitation light source adopts an infrared band femtosecond laser (in the experiment, the laser wavelength is selected as 720nm, and the fluorescent dye is selected as Indo-1); Single-photon confocal measurements use the exact same optical path for excitation and imaging. However, since two-photon fluorescence (wavelength 490nm) can only be generated at the focal point, no spatial filtering is required, and the confocal aperture (20) of the detector is adjusted to the maximum to allow the fluorescence to completely pass through. Conduct intracellular calcium imaging studies.

3. The present invention has the measurement function of single-photon fluorescence correlation spectrum, and the kinetic research of fluorescent beads in solution has been carried out. The optical path and operation are basically the same as single-photon confocal fluorescence imaging. The main difference is that the X-Y scanning galvanometer group (4) is locked and pointed to a certain point in the sample, and the specific position is controlled by the software. In addition, the signal system (21) of the fluorescence correlation spectrometer includes two kinds of data recording hardware, a multi-channel scaler and an autocorrelation card, and software can be used to calculate the autocorrelation function, or the hardware can be used to record the autocorrelation function in real time. The signal system software can also perform least squares fitting on the self-correlation function, so as to obtain a series of parameters such as the diffusion coefficient of fluorescent particles and the number of fluorescent particles in the focal area from the experimental data. In the experiment, a laser with a wavelength of 488nm was used as the excitation light, and the fluorescent beads emitted fluorescence with a wavelength of 515nm after being excited, and finally the kinetic parameters of the beads, such as diffusion coefficient, sample concentration, etc. were obtained.

4. The present invention has the function of two-photon fluorescence correlation spectrum measurement, and the kinetic research of fluorescent beads in solution has been carried out. The optical path and operation are basically the same as for two-photon fluorescence imaging. The main difference is as described in the previous section: the X-Y scanning head unit (4) is locked and pointed at a defined point in the sample. In addition, in the signal system (21), a multi-channel scaler and an autocorrelation card are used to record data, and the least square method is used for fitting. In the experiment, a laser with a wavelength of 850nm was used as the excitation light, and the fluorescent beads emitted fluorescence with a wavelength of 505nm after being excited, and finally the dynamic parameters of the beads, such as: diffusion coefficient, sample concentration, etc. were obtained.

5. The present invention has the measurement function of gradient field fluorescence correlation spectrum, and has carried out dynamic research of fluorescent beads in solution in an external laser gradient field. Its optical path and operation process are basically the same as single-photon fluorescence correlation spectroscopy measurement, the difference is that in the experiment, the femtosecond laser lost mode locking and was used as an infrared continuous laser to provide a laser gradient field (a continuous laser with a wavelength of 780nm was selected in the experiment). The excitation light of visible wavelength is used as the excitation light source (laser with a wavelength of 488nm is used in the experiment). The two laser beams combine after passing through the beam splitter (22), and are focused on the same detection point (1) in the sample (2) by the imaging system.

Claims (1)

1, a kind of multifunctional molecular radar is characterized in that this molecular radar comprises excitation source part, sample excitation and phosphor collection part and fluorescence signal detection and processing section; Described excitation source partly comprises visible excitation light attenuator, infrared laser attenuator and high pass beam splitting chip; Described sample excitation partly comprises laser focusing point, microcobjective and X-Y scanning galvanometer group with phosphor collection: described fluorescence signal is surveyed with the processing section and is comprised the logical beam splitting chip of band, optical filter, condenser lens, adjustable confocal aperture, detector and signal system; The visible waveband exciting light is decayed through attenuator, after the double-colored beam splitting chip reflection of high pass, see through the logical beam splitting chip of multiband band and realize beam flying through X-Y scanning galvanometer group again, focus on the sample by the high-NA microcobjective at last, the fluorescence dorsad that the specific fluorescent particle is sent in the focal zone is collected by microcobjective, after X-Y scanning galvanometer group, by the logical beam splitting chip reflection of multiband band, remove exciting light and other veiling glares of scattering again through bandpass filter, converge on the adjustable confocal aperture in aperture by lens then, after the reception of snowslide diode single photon counter, be input to signal system and carry out signal Processing, obtain fluoroscopic image and fluorescence correlation spectrogram.

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