CN1156684C - Multifunctional molecular radar - Google Patents
Multifunctional molecular radar Download PDFInfo
- Publication number
- CN1156684C CN1156684C CNB011366710A CN01136671A CN1156684C CN 1156684 C CN1156684 C CN 1156684C CN B011366710 A CNB011366710 A CN B011366710A CN 01136671 A CN01136671 A CN 01136671A CN 1156684 C CN1156684 C CN 1156684C
- Authority
- CN
- China
- Prior art keywords
- fluorescence
- excitation light
- beam splitting
- splitting chip
- sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Abstract
The present invention relates to a multifunctional molecular radar which comprises an excitation light source part, a sample excition and fluorescence collecting part and a fluorescence signal detection and processing part, wherein excitation light in the visible wave band penetrates through a beam-splitting sheet and is scanned by a scanning vibration mirror group after being attenuated by an attenuation sheet and reflected by a high-flux two-colour beam-splitting sheet; then the excitation light is focused on samples by an objective lens of a microscope having high numerical aperture; the backward fluorescence emitted by specified fluorescence particles is collected by the objective lens of a microscope and reflected by the beam-splitting sheet after passing through the scanning vibration mirror group, and then the fluorescence passes through a band-pass filter to remove the scattered excitation light, and the like, and then the fluorescence without the scattered excitation light is focused on a confocal aperture by a lens and input to a signal system to be processed after being received by a single photon counter, and thereby, fluorescence images and a fluorescence correlation spectrum figure are obtained. The multifunctional molecular radar can carry out multiple different types of measurement aiming at one sample on a set of system, the experimental equipment and the instrument regulation process are simple, and the work efficiency is enhanced.
Description
Technical field
The present invention relates to the compound imaging device of a kind of biological living, is a kind of new bio surveying instrument.Integration by light path and detection system, it can realize the repertoire of the imaging of laser scanning confocal fluorescent, two-photon fluorescence imaging, single photon and two-photon fluorescence correlation spectrometer, gradient field fluorescence correlation spectrometer on same set of device, realize the imaging of specific biochemical composition in the living cells system and the kinematics of target molecule are measured.
Background technology
In the prior art, the specific fluorescent label is carried out laser scanning imaging, to carry out pair cell, the research of biological phenomena on the subcellular fraction level with laser scanning two-photon (confocal) fluorescent microscope.
Existing laser scanning confocal fluorescence microscopy basic device is as shown in Figure 1: the single photon confocal imaging adopts visible waveband laser (wavelength is generally 488nm) to carry out fluorescence excitation.Exciting light is after attenuator (11) decay, see through the double-colored beam splitting chip of low pass (5) (wavelength less than 488nm time see through), pass through X-Y scanning galvanometer group (4) again and realize beam flying, focus in solution or the living cells sample (2) by high-NA microcobjective (3) at last.The fluorescence dorsad that the interior specific fluorescent particle of focal zone (1) is sent is collected by microcobjective (3), through X-Y scanning galvanometer group (4) afterwards, reflected by the double-colored beam splitting chip of low pass (5), mating plate (6) is removed exciting light and other veiling glares of scattering after filtration again, converge on the adjustable confocal aperture in aperture on (8) by lens (7) then, be detected and be input to signal system (10) after device (9) receives and carry out signals collecting and processing.Confocal aperture is consistent with the picture size of focus point (1), utilizes spatial filtering to suppress focal area (1) fluorescence and parasitic light in addition, improves the signal to noise ratio (S/N ratio) of fluorescence measurement.The said apparatus technology maturation, the imaging research that generally is used for the living cells system, but because its excitation source that adopts is visible or inclined to one side ultraviolet, therefore bigger to the lethal effect in the living cells in long-time observation, and its wavelength that adopts is single, can't realize continuously adjustable.
The two-photon fluorescence microscopie unit is basic identical with confocal microscope, and as shown in Figure 2: the fluorescence excitation light source adopts the infrared band femto-second laser, and wave band is 680-1080nm; The double-colored beam splitting chip of high pass (12) sees through for the excitation beam of wavelength greater than 680nm, and visible waveband fluorescence is reflected.In addition, owing to only can produce two-photon fluorescence at the focus place, do not need spatial filtering, thereby do not add confocal aperture before the detector, the principal feature of this device is to have adopted the long infrared excitation light of wavelength, and is less to the lethal effect of living cells sample, applicable to long-time observational study to sample, because its wavelength is adjustable continuously, and is bigger for the fluorescent dye choice.But because this technology is newer, its practical field is still waiting further exploration.
In the prior art,,, generally adopt fluorescence correlation spectrometer for procedural informations such as the structural change of measuring biomolecule, microcosmic chemical reaction, desmoenzyme dynamics at living cells or solution system.Its principle is: by measuring the fluorescence fluctuation signal of a small amount of light emitting molecule in the microcell, and it is done association analysis, thereby obtain the physics system information that relevant fluorescence molecule concentration, rate of propagation etc. influence fluctuation.
The basic light path of single photon and two-photon fluorescence excitation correlation spectrometer is respectively Fig. 3 and Fig. 4, and its structure is similar with the two-photon fluorescence microscope to confocal fluorescence microscopy respectively.The key distinction is that the X-Y scanning galvanometer group (4) among figure one and the figure two is replaced respectively by completely reflecting mirror (13).In addition, the detector of fluorescence correlation spectrometer is a snowslide diode single photon counter (16), adopt multiscaler and auto correlation card record data in the signal system (17), data processing is also different fully with the scanning fluorescent microscope with system controlling software, comprise auto correlation data processing and least square fitting function, so that from experimental data, obtain in the coefficient of diffusion of fluorescent particles and the focus area series of parameters such as fluorescent particles number.The technology that this device adopts belongs to the single-molecule detection field, and operation easier is bigger, is applicable to the kinetic measurement of target biological molecules in the cell system.
In sum, existing scanning fluorescent microscope and fluorescence correlation spectrometer all are autonomous systems, function singleness.In the practical study, when conducting a research at a concrete living things system, the multiparameter duplex measurement is very important.The method of scanning imagery can obtain the information of cell level, and the method for fluorescence correlation spectrometer can obtain the information of molecular level.Therefore, under existence conditions, when a particular organisms object is carried out imaging research, if need simultaneously the privileged site of this object to be carried out the research of molecular dynamics aspect, need it is moved on on the fluorescence correlation spectrometer again,, will suitable difficulty also be arranged with imaging region is corresponding with the survey region of fluorescence correlation spectrometer simultaneously because the research environment in moving process can change, its operating process is also complicated, has influenced the accuracy of experimental result greatly.Simultaneously, setting up two, to overlap the required optical device number of system independently more, also has waste to a certain extent.
Summary of the invention
The objective of the invention is to design the compound imaging device of a kind of biological living, by changing light path setting and laser work pattern, realize the integrated of laser scanning two-photon (confocal) fluorescent microscope and fluorescence correlation spectrometer, overlap in the new system, one, carry out dissimilar multiple measurements at same sample, simplify experimental provision and instrument adjustment process as much as possible, reduce the experiment difficulty, effectively utilize all optical device, increase work efficiency.
The compound imaging device of biological living that the present invention designs 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 femtosecond 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 the visible excitation light attenuator, through the double-colored beam splitting chip reflection of high pass, infrared femtosecond laser beam decays through infrared femtosecond laser attenuator simultaneously, close bundle with the visible excitation light of reflection after seeing through the double-colored beam splitting chip of high pass, after two-beam leads to beam splitting chip by the multiband band, realize beam flying through X-Y scanning galvanometer group, focus on the sample by 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 diode single photon counter, be input to signal system and carry out signal Processing, obtain fluoroscopic image and fluorescence correlation spectrogram.
Use the compound imaging device of biological living of the present invention's design, realized the integrated of laser scanning two-photon (confocal) fluorescent microscope and fluorescence correlation spectrometer, on a cover system, at same sample, carried out dissimilar multiple measurements, experimental provision and instrument adjustment process are simple, have improved work efficiency.
Description of drawings
Fig. 1 is a cofocus scanning fluorescent microscope structural representation.
Fig. 2 is a two-photon scanning fluorescent microscope structural representation.
Fig. 3 is an one-photon excitation fluorescence correlation spectrometer structural representation.
Fig. 4 is a two-photon fluorescence excitation correlation spectrometer structural representation.
Fig. 5 is the compound imaging apparatus structure of the biological living synoptic diagram that the present invention designs.
Among Fig. 1～Fig. 5, the 1st, laser focusing point, the 2nd, sample, the 3rd, microcobjective, the 4th, X-Y scanning galvanometer group, the 5th, the double-colored beam splitting chip of low pass, the 6th, optical filter, the 7th, condenser lens, the 8th, confocal aperture, the 9th, detector (photomultiplier), the 10th, signal system (imaging), the 11st, visible excitation light attenuator, the 12nd, the double-colored beam splitting chip of high pass, the 13rd, optical filter, the 14th, infrared femtosecond laser attenuator, the 15th, double-colored beam splitting chip, the 16th, diode single photon counter (single photon counting module), the 17th, signal system (fluorescence correlation spectroscopy), the 18th, the logical beam splitting chip of band, the 19th, optical filter, the 20th, adjustable confocal aperture, the 21st, signal system (imaging and fluorescence correlation spectroscopy), the 22nd, the high pass beam splitting chip, I is the excitation source part, and II is sample excitation and phosphor collection part, and III is that fluorescence signal is surveyed and disposal system.
Embodiment
As shown in Figure 5, the compound imaging device of biological living that the present invention designs comprises excitation source part, sample excitation and phosphor collection part and fluorescence signal detection and processing section.Excitation source partly comprises visible excitation light attenuator 11, infrared femtosecond laser attenuator 14 and high pass beam splitting chip 22.The sample excitation partly comprises laser focusing point 1, microcobjective 3 and X-Y scanning galvanometer group 4 with phosphor collection.Fluorescence signal is surveyed with the processing section and is comprised the logical beam splitting chip 18 of band, optical filter 19, condenser lens 7, adjustable confocal aperture 20, diode single photon counter 16 and signal system 21.The visible waveband exciting light is through 11 decay of visible excitation light attenuator, through double-colored beam splitting chip 22 reflections of high pass, infrared femtosecond laser beam is through infrared femtosecond laser attenuator 14 decay simultaneously, see through the double-colored beam splitting chip of high pass 22 backs and close bundle with the visible excitation light of reflection, after two-beam leads to beam splitting chip 18 by the multiband band, realize beam flying through X-Y scanning galvanometer group 4, focus on the sample 2 by microcobjective 3 at last.The fluorescence dorsad that the specific fluorescent particle is sent in the focal zone 1 is collected by microcobjective 3, after X-Y scanning galvanometer group 4, by logical beam splitting chip 18 reflections of multiband band, remove exciting light and other veiling glares of scattering again through bandpass filter 19, converge on the adjustable confocal aperture 20 in aperture by condenser lens 7 then, after 16 receptions of diode single photon counter, be input to signal system 21 and carry out signal Processing, obtain fluoroscopic image and fluorescence correlation spectrogram.
Use the compound imaging device of biological living of the present invention, on a cover system, carried out following five kinds of different measuring processes:
1, the present invention has laser scanning confocal fluorescent imaging function, has carried out calcium imaging research in the oocyte of mouse.Visible waveband exciting light (selecting wavelength 488nm laser in the experiment) is decayed through attenuator (11), the double-colored beam splitting chip of high pass (22) (reflects the visible waveband exciting light, to the transmission of infrared band exciting light, reflection 488nm exciting light in the experiment) after the reflection, see through the logical beam splitting chip (18) of multiband band and (see through infrared and visible excitation light, reflection wavelength between infrared and visible excitation light fluorescence and wavelength less than the fluorescence of visible light), pass through X-Y scanning galvanometer group (4) again and realize beam flying, focus in solution or the living cells sample (2) by high-NA microcobjective (3) at last and (select for use in the experiment and dye the oocyte of mouse that Fluo3 is arranged).The fluorescence dorsad (Fluo3 sends the fluorescence that wavelength is 514nm in the experiment) that the interior specific fluorescent particle of focal zone (1) is sent is collected by microcobjective (3), through X-Y scanning galvanometer group (4) afterwards, by logical beam splitting chip (18) reflection of multiband band, pass through bandpass filter (19) again and remove exciting light (comprising visible excitation light and infrared excitation light) and other veiling glares of scattering, converge on the adjustable confocal aperture in aperture on (20) by lens (7) then, after being received by snowslide diode single photon counter (16).Be input to signal system (21), cooperate and with the photon counting card signal is noted down continuously, output image is carried out the intracellular Ca2+ imaging research after treatment.
2, the present invention has laser scanning two-photon fluorescence imaging function, has carried out calcium imaging research in the oocyte of mouse.The fluorescence excitation light source adopts infrared band femto-second laser (optical maser wavelength is elected 720nm as in the experiment, and fluorescent dye is selected Indo-1 for use); After infrared attenuating sheet (14) decay, see through the double-colored beam splitting chip of high pass (22) back according to exciting and imaging with the identical light path of aforementioned single photon confocal measurement.But owing to only can produce two-photon fluorescence (wavelength 490nm) at the focus place, do not need spatial filtering, the confocal aperture of avalanche diode single photon counter (20) is transferred to maximum, and fluorescence is passed through fully.Carry out the intracellular Ca2+ imaging research.
3, the present invention has single photon fluorescence correlation spectroscopy measurement function, has carried out the dynamics research of fluorescence bead in the solution.Use light path and operation and the imaging of single photon confocal fluorescent basic identical.The key distinction is: X-Y scanning galvanometer group (4) is pinned, and points to definite point in the sample, and particular location is by software control.In addition, comprise two kinds of data recording hardware of multiscaler and auto correlation card in the signal system of fluorescence correlation spectrometer (21), can utilize the computed in software auto correlation, or utilize hardware real time record auto correlation function.Signal system software can also carry out least square fitting to the auto correlation function, so that obtain in the coefficient of diffusion of fluorescent particles and the focus area series of parameters such as fluorescent particles number from experimental data.Exciting light adopts the laser of wavelength 488nm in the experiment, sends the fluorescence of wavelength 515nm after the fluorescence bead is excited, and obtains the kinetic parameter of bead at last, as: coefficient of diffusion, sample concentration etc.
4, the present invention has two-photon fluorescence correlation spectrum measurement function, has carried out the dynamics research of fluorescence bead in the solution.Use light path and operation and two-photon fluorescence imaging basic identical.The key distinction is as described in the last joint: X-Y scanning galvanometer group (4) is pinned, and points to definite point in the sample.In addition, adopt multiscaler and auto correlation card record data, least square fitting in the signal system (21).Exciting light adopts the laser of wavelength 850nm in the experiment, sends the fluorescence of wavelength 505nm after the fluorescence bead is excited, and obtains the kinetic parameter of bead at last, as: coefficient of diffusion, sample concentration etc.
5, the present invention has gradient field fluorescence correlation spectrometry function, has carried out the dynamics research of fluorescence bead in adding laser gradient field in the solution.Its light path and operating process and single photon fluorescence correlation spectrometry are basic identical, and difference is: in the experiment, femto-second laser losing lock mould uses as infrared continuous wave laser, and laser gradient field (choosing the continuous laser that wavelength is 780nm in the experiment) is provided.The exciting light of visible wavelength is as exciting light source (selecting the laser of wavelength 488nm for use) in the experiment.Two bundle laser close bundle behind beam splitting chip (22), and focus on same sensing point (1) in the sample (2) by imaging system.
Claims (1)
1, the compound imaging device of a kind of biological living is characterized in that this imaging device 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 femtosecond 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, diode single photon counter and signal system; The visible waveband exciting light is decayed through the visible excitation light attenuator, through the double-colored beam splitting chip reflection of high pass, infrared femtosecond laser beam decays through infrared femtosecond laser attenuator simultaneously, close bundle with the visible excitation light of reflection after seeing through the double-colored beam splitting chip of high pass, after two-beam leads to beam splitting chip by the multiband band, realize beam flying through X-Y scanning galvanometer group, focus on the sample by 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 diode single photon counter, be input to signal system and carry out signal Processing, obtain fluoroscopic image and fluorescence correlation spectrogram.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011366710A CN1156684C (en) | 2001-10-26 | 2001-10-26 | Multifunctional molecular radar |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011366710A CN1156684C (en) | 2001-10-26 | 2001-10-26 | Multifunctional molecular radar |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1349093A CN1349093A (en) | 2002-05-15 |
| CN1156684C true CN1156684C (en) | 2004-07-07 |
Family
ID=4673817
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB011366710A Expired - Fee Related CN1156684C (en) | 2001-10-26 | 2001-10-26 | Multifunctional molecular radar |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1156684C (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8116341B2 (en) * | 2007-05-31 | 2012-02-14 | Electro Scientific Industries, Inc. | Multiple laser wavelength and pulse width process drilling |
| CN101782518A (en) * | 2010-02-11 | 2010-07-21 | 华南师范大学 | Cell opto-acoustic microscopic imaging method and device thereof |
| DE102011051042B4 (en) * | 2011-06-14 | 2016-04-28 | Leica Microsystems Cms Gmbh | Scanning microscope and method for light microscopic imaging of an object |
| US10502830B2 (en) * | 2016-10-13 | 2019-12-10 | Waymo Llc | Limitation of noise on light detectors using an aperture |
| CN108540292A (en) * | 2018-03-30 | 2018-09-14 | 中国工程物理研究院电子工程研究所 | Verification System based on vibration mirror scanning imaging |
| CN108827478B (en) * | 2018-07-10 | 2019-11-26 | 迪瑞医疗科技股份有限公司 | A kind of photon measurement system based on the wide range of linearity of two-photon counter |
-
2001
- 2001-10-26 CN CNB011366710A patent/CN1156684C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN1349093A (en) | 2002-05-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US4407008A (en) | Method and apparatus for light-induced scanning-microscope display of specimen parameters and of their distribution | |
| US5751839A (en) | Apparatus and process for the detection and counting of rarely occurring mammalian cells | |
| US7576862B2 (en) | Measuring time dependent fluorescence | |
| US5962238A (en) | Method and apparatus for cell counting and cell classification | |
| JP3290786B2 (en) | Particle analyzer | |
| EP0834066B1 (en) | Process and device for detecting physical, chemical, biological or biochemical reactions and interactions | |
| CN1181334C (en) | Method and device for characterizing culture liquid | |
| US8921809B2 (en) | Device for microscopy having selective illumination of a plane | |
| US20050179892A1 (en) | Method and arrangement for analyzing samples | |
| JPS6332356A (en) | Device for analyzing and picture-displaying fluorescent signal | |
| WO2011035299A2 (en) | Reflective focusing and transmissive projection device | |
| CN111220590A (en) | Rapid detection instrument and detection method for pathogenic microorganism drug sensitivity | |
| CN1156684C (en) | Multifunctional molecular radar | |
| EP2524260A1 (en) | Ultra dark field microscope | |
| US20230221178A1 (en) | Apparatus and a method for fluorescence imaging | |
| EP2631631B1 (en) | Photometric analysis device and photometric analysis method using wavelength characteristic of light emitted from single illuminant particle | |
| JP2004361087A (en) | Biomolecule analyzer | |
| CN1156686C (en) | Gradient field fluorescence correlation spectrometer | |
| CN213903257U (en) | Scanning flow type cell imaging analyzer | |
| JPH11505928A (en) | Wide-angle scattered light detector | |
| US20220228968A1 (en) | Hyperspectral quantitative imaging cytometry system | |
| CN217360437U (en) | Staring type fast light sheet confocal high-dimensional imaging system | |
| Martin-Fernandez et al. | Subnanosecond polarized microfluorimetry in the time domain: An instrument for studying receptor trafficking in live cells | |
| DE60317049T2 (en) | METHOD FOR DETECTING THE IMPACT OF INTERFERENCE TO MEASUREMENT DATA | |
| CN114911044A (en) | Staring type fast light sheet confocal high-dimensional imaging system |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C06 | Publication | ||
| PB01 | Publication | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C19 | Lapse of patent right due to non-payment of the annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |