CN110161671A - Microscopic imaging device is imaged in dark field, light field, phase contrast, fluorescence multi-modal synchronization - Google Patents
Microscopic imaging device is imaged in dark field, light field, phase contrast, fluorescence multi-modal synchronization Download PDFInfo
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- CN110161671A CN110161671A CN201910361630.4A CN201910361630A CN110161671A CN 110161671 A CN110161671 A CN 110161671A CN 201910361630 A CN201910361630 A CN 201910361630A CN 110161671 A CN110161671 A CN 110161671A
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/06—Means for illuminating specimens
- G02B21/08—Condensers
- G02B21/10—Condensers affording dark-field illumination
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/06—Means for illuminating specimens
- G02B21/08—Condensers
- G02B21/12—Condensers affording bright-field illumination
- G02B21/125—Condensers affording bright-field illumination affording both dark- and bright-field illumination
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/06—Means for illuminating specimens
- G02B21/08—Condensers
- G02B21/14—Condensers affording illumination for phase-contrast observation
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/18—Arrangements with more than one light path, e.g. for comparing two specimens
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
- G02B21/361—Optical details, e.g. image relay to the camera or image sensor
Abstract
The invention discloses a kind of dark field, light field, phase contrast, fluorescence multi-modal synchronizations, and microscopic imaging device is imaged, with sample stage, for placing sample at the sample stage object focal point, beam emissions unit made of sample stage side setting is arranged as several light sources with different wavelengths, beam treatment unit is set in sequence in the sample stage other side, for amplifying light beam to guarantee that hot spot is completely radiated at the light beam amplifying unit, filtered light beam unit and beam reception unit of filtered light beam unit.The present invention is detected suitable for biological observation, and easy to use, at low cost, has good imaging effect.
Description
Technical field
The present invention relates to imaging device, more particularly, to dark field, light field, phase contrast, the imaging of fluorescence multi-modal synchronization it is micro- at
As device.
Background technique
Optical microscopy is tool generally existing in different subjects, provides the detailed visualizations of material and biological sample.
In the past few decades, microscopical be constantly progressive has had been incorporated into many new imaging modes.However, light field, dark field and
Phase contrast microscope still represents most common and widely used non-staining imaging method.Light field (BF) microscope is by reflecting
The intensity modulated of the light of sample was shot through to provide image.Although it is the most simple and most common microscope form, it is not
It is suitble to observation translucent sample, such as unlabeled cells and thin tissue sample, because these samples will not be shown under visible light
Strong decaying.The high-contrast image of thin object can be generated in dark field (DF) microscope, sensitive to sample edge.DF microscope
It is irradiated using the oblique light beyond the trappable maximum angle of optical imaging system, to minimize the background not scattered, simultaneously
Collect the scattering light from sample.Phasecontrast microscope, such as Zernike and differential interference compare (DIC) microscope, by that will shine
The optical phase put-off for penetrating the light of sample is rendered as intensity distribution to provide image.In paper " Quantitative phase
imaging using a partitioned detection aperture”(OPTICS LETTERS/Vol.37,No.19/
October 1,2012) in, describe a kind of non-interfering technology to carry out quantitative phase contrast imaging, but still can only realize it is single at
As mode.
Although light field, dark field and phase contrast image provide the complementary information of sample, same in conventional microscope
When to acquire these images be infeasible because every kind of mode requires different optical arrangements and dedicated optical element.In addition,
Waste of the conversion along with time and additional optical elements between imaging pattern, inadequate energy-saving facile.In paper " Real-
time brightfield,darkfield,and phase contrast imaging in a light-emitting
diode array microscope”(Journal of Biomedical Optics 19(10),106002(October
) and Microscopy refocusing and dark-field imaging by using a simple LED 2014)
In array (October 15,2011/Vol.36, No.20/OPTICS LETTERS), propose a kind of using LED array
The method for realizing multi-mode illumination imaging, but can not all solve the problems, such as synchronous imaging, need continually toggle lights obtain it is pre-
Phase effect.
In addition, the existing fluorescence detection microscope functions in market are single, the fluorescence detection of predetermined substance can be only realized.
Summary of the invention
The purpose of the present invention is to solve the above-mentioned problems, and it is same to provide a kind of dark field, light field, phase contrast, fluorescence multi-mode
Step imaging microscopic imaging device, it is intended to be suitable for biological observation and detect, and it is easy to use, it is at low cost, while have well at
As effect.
To achieve the goals above, the present invention adopts the following technical scheme: a kind of dark field, light field, phase contrast, fluorescence multi-mode
Synchronous imaging microscopic imaging device has sample stage, is used to place sample, the sample stage one at the sample stage object focal point
Beam emissions unit made of side setting is arranged as several light sources with different wavelengths, light beam is set in sequence in the sample stage other side
Processing unit, for amplifying light beam to guarantee that hot spot is completely radiated at the light beam amplifying unit of filtered light beam unit, light beam mistake
Filter unit and beam reception unit.
Further, the beam emissions unit has 405nm laser, 488nm laser, 532nm laser, 638nm
Laser；The 405nm laser is with the incident angles less than 48.6 ° to sample stage；The 488nm laser and 405nm
Laser is symmetrical about optical axis；The 532nm laser is with the incident angles greater than 48.6 ° to sample stage；The 638nm swashs
Light device is symmetrical about optical axis with 532nm laser.
Further, the beam treatment unit by enlargement ratio is 20 times, the object lens that numerical aperture NA is 0.75 form.
Further, the light beam amplifying unit amplification factor is 1.67, it is by the first lens that focal length is 30mm, focal length
The second lens and diaphragm of 50mm form, and first lens are arranged in side, and second lens are arranged in the other side, institute
Aperture arrangement is stated at the image space focal plane of the first lens.
Further, the filtered light beam unit has two groups of lens array A sequentially arranging, one group of filtering chip arrays and
One group of lens array B；Every group of lens array A is made of four sub-lens A, with the distribution of sphere of movements for the elephants shape, for light beam to be divided into 4 beams
Light beam, accordingly directive filters four windows of chip arrays；The filtering chip arrays are made of four sub- filter plates, with sphere of movements for the elephants
Shape distribution, for by the light received correspond to different wave length filtering, make each window only penetrate corresponding wavelength light simultaneously directive it is saturating
Lens array B；The lens array B is made of four sub-lens B, with the distribution of sphere of movements for the elephants shape, for focusing four bundles light line respectively
In the imaging being made of imaging sensor light beam receiving unit.
Compared with prior art, the invention has the following beneficial effects:
(1) for the present invention because of the light source different containing four sets of wavelength, and two-by-two in groups, every group of incident angle is different, can shape
At dark field, light field, phase contrast synchronous imaging, realize to the observation of different articles and the simultaneous observation of various modes.
(2) present invention uses two kinds of fluorescence bands light sources simultaneously, it can be achieved that detection to specific fluorescent substance.
(3) lens array according to the present invention and filtering chip arrays, are divided into four beams for light, project four to filter plate
Channel, by filter action, it can be achieved that multi-section display, finally forms the picture in four regions on the image sensor.
(4) present invention is able to solve conventional microscope in the market and has a single function, and can not synchronize and realize asking for multi-modal imaging
Topic, it is easy to operate, it is easy to accomplish, the vacancy of multifunctional microscope in the market can be filled up, and have easy to operate, at low cost
Advantage.
Detailed description of the invention
Fig. 1 is dark field, light field, phase contrast, fluorescence multi-modal synchronization imaging microscopic imaging device structure chart.
Fig. 2 is that sub-lens A arranges schematic diagram in lens array A.
Fig. 3 is that four sub- filter plates arrange schematic diagram in filtering chip arrays.
Specific embodiment
Referring to Fig. 1 to Fig. 3, microscopic imaging device is imaged in a kind of dark field, light field, phase contrast, fluorescence multi-modal synchronization, has sample
Sample platform 2, for placing sample at 2 object focal point of sample stage, the sample stage side is arranged by several different wavelengths of light
Beam emissions unit 1 made of the arrangement of source, beam treatment unit 3 is set in sequence, for amplifying light beam in 2 other side of sample stage
To guarantee that hot spot is completely radiated at the light beam amplifying unit 4, filtered light beam unit 5 and beam reception of filtered light beam unit 5
Unit 6.
Sample stage 2 be can upper and lower displacement platform, to guarantee that light beam convergent point is radiated on sample.
In the present embodiment, the beam emissions unit is used to swash with the light of different angle transmitting different wave length with 405nm
Light device 11,488nm laser 12,532nm laser 13,638nm laser 14；The 405nm laser 11 is less than 48.6 °
Incident angles to sample stage, in use, need to adjust first makes its collimation, and guarantee that light injects object lens；The 488nm swashs
Light device 12 and 405nm laser 11 is symmetrical about optical axis, and application method is same as above, this two groups of laser are used to realize light field and phase contrast
Imaging；The 532nm laser 13 arrives sample stage with the incident angles greater than 48.6 °, and when use need to adjust first keeps its quasi-
Directly, and guarantee that light via sample, is not injected into object lens but；The 638nm laser 14 is with 532nm laser 13 about optical axis
Symmetrically, application method is same as above.
There are two types of operating modes for beam emissions unit.Mode one: opening 405nm laser 11,488nm laser 12,
630nm laser, for the detection to fluorescent material EGFP (eGFP), the excitation wavelength of EGFP is in 480nm
Left and right, launch wavelength is 530 or so.Mode two: open 405nm laser, 488nm laser, 530nm laser, for pair
The detection of fluorescent material PPIX (protoporphyrin), the excitation wavelength of PPIX is in 405nm or so, and launch wavelength is in 638nm or so.
It should be noted that being the light source of four sets of different wave lengths in beam emissions unit 1, subject to every set light source emergent light is equal
Direct light, wavelength are respectively λ1、λ2、λ3、λ4.(respectively correspond 630nm laser 13,488nm laser 12,405nm laser 11,
530nm laser 14) wherein, λ1、λ2、λ3The light source of wavelength is one group, λ2、λ3、λ4The light source of wavelength is one group, in use, only
Open wherein one group of light source, i.e. λ1Or λ4The light source of wavelength is closed.As unlatching λ1、λ2、λ3When the light source of wavelength, λ1The light source of wavelength
For dark-field imaging, λ2、λ3The light source of wavelength is used for light field, phase contrast, fluorescence imaging, wherein λ2The light source of wavelength swashs as fluorescence
Light emitting source, the wavelength of fluorescence emission are λ4；As unlatching λ2、λ3、λ4When the light source of wavelength, λ4The light source of wavelength for dark field at
Picture, λ2、λ3The light source of wavelength is used for light field, phase contrast, fluorescence imaging, wherein λ3The light source of wavelength is as fluorescence excitation light source, fluorescence
The wavelength for emitting light is λ1.When light source disposes, it need to ensure λ2、λ3When the light irradiating sample of wavelength light source, with objective lens optical axis in symmetrical
Angle ± α, 0 < α < θNA,θNACorresponding relationship with numerical aperture of objective is θNA=sin-1NA, wherein θ NA is numerical aperture angle,
NA is the numerical aperture of object lens；Also, it need to ensure λ1、λ4It is in certain angle with objective lens optical axis when the light irradiating sample of wavelength light source
Spend β, β > θNA。
In the present embodiment, the beam treatment unit 3 by enlargement ratio is 20 times, the objective lens that numerical aperture NA is 0.75
At.
In the present embodiment, 4 amplification factor of light beam amplifying unit be 1.67, by focal length be 30mm the first lens 41,
The second lens 42 and diaphragm composition, first lens 41 that focal length is 50mm are arranged in side, 42 cloth of the second lens
It sets in the other side, the diaphragm 43 is arranged at the image space focal plane of the first lens, i.e. the object space focal plane of the second lens.Light beam
Amplifying unit 4, which will receive, emits light (in figure 1. light is light field or phase contrast optical path, 2. light is dark field optical path), and behind
It focuses on a bit.
In the present embodiment, the filtered light beam unit 5 has the two groups of lens array A51 sequentially arranged, one group of filter plate
Array 52 and one group of lens array B53；Every group of lens array A51 is made of four sub-lens A, with the distribution of sphere of movements for the elephants shape, is used
In light beam is divided into 4 light beams, accordingly directive filters four windows of chip arrays 52；The filtering chip arrays 52 are by four sons
Filter plate composition is filtered for the light received correspond to different wave length with the distribution of sphere of movements for the elephants shape, penetrates each window only pair
Answer the light and directive lens array B53 of wavelength；The lens array B53 is made of four sub-lens B, is distributed with sphere of movements for the elephants shape,
For four bundles light line to be focused on the imaging being made of imaging sensor light beam receiving unit 6 respectively.
With in the filter plate of sphere of movements for the elephants shape distribution, the sub-lens unit in each optical filter and lens array corresponds shape
At four windows, the wavelength that four windows are acted on is λ1、λ2、λ3、λ4, make finally to focus on imaging sensor (beam reception list
The image in four regions of member 6) is respectively λ1、λ2、λ3、λ4Image.
Beam reception unit 6 for receiving the picture eventually formed, four regions respectively correspond 532nm, 408nm, 488nm,
The image of 630nm wavelength.Operating mode for the moment, region one be EGFP fluorescence imaging, region two, three be light field and phase contrast imaging,
Region four is dark-field imaging；When operating mode two, region one is dark-field imaging, and region two, three is light field and phase contrast imaging, region
Four be PPIX fluorescence imaging.
It is emphasized that imaging sensor is long or wide size need to be greater than the size for filtering chip arrays each edge, image
The target surface of sensor is divided into four regions, and with the distribution of sphere of movements for the elephants shape, region one is for receiving λ1The optical signal of wavelength, region two are used
In reception λ2The optical signal of wavelength, region three is for receiving λ3The optical signal of wavelength, region four is for receiving λ4The light of wavelength is believed
Number.Work as λ1、λ2、λ3When the light source of wavelength is opened, region one is used for light field and phase contrast for dark-field imaging, region two and region three
Imaging, region four are used for λ2The λ inspired by fluorescent samples4Corresponding fluorescence imaging；Work as λ2、λ3、λ4The light source of wavelength is opened
When, region one is used for λ3The λ inspired by fluorescent samples1Corresponding fluorescence imaging, region two and region three are used for light field and phase
Lining imaging, region four are used for dark-field imaging.X-Y coordinate is established to imaging sensor receiving surface, X indicates horizontal axis coordinate, and Y indicates vertical
Axial coordinate, the light distribution in region two and region three are Iλ2(x, y) and Iλ3(x, y), the light distribution of light field imaging are IBF(x,
Y), the light distribution of phase contrast imaging is IDPC(x, y), wherein IBF(x, y) and IDPC(x, y) is respectively by formula IBF(x, y)=Iλ2
(x, y)+Iλ3(x, y),It obtains.Work as λ1The light source of wavelength be used for dark-field imaging when, dark field at
The light distribution of picture is IDF(x, y)=Iλ1(x, y), the light distribution of fluorescence imaging are IFL=Iλ4(x,y)；Work as λ4The light of wavelength
When source is used for dark-field imaging, the light distribution of dark-field imaging is IDF(x, y)=Iλ4(x, y), the light distribution of fluorescence imaging are IFL
=Iλ1(x,y)。
The foregoing is merely the preferred embodiment of the present invention, protection scope of the present invention is not limited in above-mentioned embodiment party
Formula, all technical solutions for belonging to the principle of the invention all belong to the scope of protection of the present invention.For those skilled in the art and
Speech, several improvement carried out without departing from the principles of the present invention, these improvement also should be regarded as protection model of the invention
It encloses.
Claims (5)
1. microscopic imaging device is imaged in a kind of dark field, light field, phase contrast, fluorescence multi-modal synchronization, which is characterized in that have sample stage
(2), for placing sample at sample stage (2) object focal point, the sample stage side is arranged by several different wavelengths of light
Beam emissions unit (1) made of the arrangement of source, beam treatment unit (3) is set in sequence, for putting in sample stage (2) other side
Big light beam is to guarantee that hot spot is completely radiated at the light beam amplifying unit (4) of filtered light beam unit (5), filtered light beam unit (5)
And beam reception unit (6).
2. microscopic imaging device, feature is imaged in dark field according to claim 1, light field, phase contrast, fluorescence multi-modal synchronization
Be: the beam emissions unit have 405nm laser (11), 488nm laser (12), 532nm laser (13),
638nm laser (14)；The 405nm laser (11) is with the incident angles less than 48.6 ° to sample stage；The 488nm
Laser (12) and 405nm laser (11) are symmetrical about optical axis；The 532nm laser (13) is with the incidence greater than 48.6 °
It is incident on sample stage in angle；The 638nm laser (14) and 532nm laser (13) are symmetrical about optical axis.
3. microscopic imaging device, feature is imaged in dark field according to claim 1, light field, phase contrast, fluorescence multi-modal synchronization
Be: the beam treatment unit (3) by enlargement ratio is 20 times, the object lens that numerical aperture NA is 0.75 form.
4. microscopic imaging device, feature is imaged in dark field according to claim 1, light field, phase contrast, fluorescence multi-modal synchronization
Be: light beam amplifying unit (4) amplification factor is 1.67, the first lens (41), the focal length 50mm for being 30mm by focal length
The second lens (42) and diaphragm composition, first lens (41) are arranged in side, and second lens (42) are arranged in
The other side, the diaphragm (43) are arranged at the image space focal plane of the first lens.
5. microscopic imaging device, feature is imaged in dark field according to claim 1, light field, phase contrast, fluorescence multi-modal synchronization
Be: the filtered light beam unit (5) has two groups of lens array A (51) sequentially arranging, one group of filtering chip arrays (52) with
And one group of lens array B (53)；Every group of lens array A (51) is made of four sub-lens A, and with the distribution of sphere of movements for the elephants shape, being used for will
Light beam is divided into 4 light beams, accordingly four windows of directive filtering chip arrays (52)；The filtering chip arrays (52) are by four sons
Filter plate composition is filtered for the light received correspond to different wave length with the distribution of sphere of movements for the elephants shape, penetrates each window only pair
Answer the light and directive lens array B (53) of wavelength；The lens array B (53) is made of four sub-lens B, with sphere of movements for the elephants shape point
Cloth, for four bundles light line to be focused on the imaging being made of imaging sensor with light beam receiving unit (6) respectively.
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