CN1953701A - Method and system for tomographic imaging using fluorescent proteins - Google Patents

Abstract

A system for optical tomography includes an apparent light source adapted to project excitation light toward a specimen having fluorescent proteins therein, wherein the excitation light enters the specimen becoming intrinsic light within the specimen, wherein the intrinsic light is adapted to excite fluorescent light from the fluorescent proteins, and wherein the intrinsic light and the fluorescent light are diffuse. A method of optical tomography includes generating the excitation light with the apparent light source, wherein the intrinsic light and the fluorescent light are diffuse.

Description

Use fluorescin to carry out the method and system of tomographic imaging

Technical field

The present invention relates generally to optical tomography, and relate more specifically to use fluorescin to extract the method and system of quantitative three-dimensional molecular and bio information from living specimen.

Background technology

Fluorescin (FPs) is the important reporter molecule that is used for different biomedical applications.In some existing application, utilize to fall to penetrating fluorescence, confocal (microscopy) or reflect (whole animal) imaging and survey engineering FPs.

Falling to penetrating fluorescence, confocal microscopy depends on to the specimen projection with from relevant (indiffusion) light of specimen reflection.Because microscopy needs relevant in essence light, so this technology is only able to image to the little degree of depth (for example less than 1mm) in the specimen.At darker imaging depth, well-known light begins diffusion, makes microscopy invalid at darker imaging depth.

Proved that the reflected fluorescent light imaging helps to survey and follows the tracks of in-vivo tumour, particularly those are attached near the body surface or the tumor in the organ that exposes of underwent operative.Yet because the image that obtains is the stack from the fluorescence signal of a plurality of degree of depth, this often causes the image that blurs, so the reflected fluorescent light imaging has intrinsic limitation.In addition, the reflected fluorescent light imaging is not a tomography, not the absolute quantification of retrieve depth information or permission fluorescence activity.This part is because nonlinear attenuation and the propagation of light in biological tissue, its with the application limitations of reflected fluorescent light imaging in the only sxemiquantitative imaging of several mm depths.

Darker image optics feature usually needs the optical excitation of application of advanced to be combined in the data that different projections are gathered with light detection device with technology and use tomographic principles in the tissue.Use the progress of diffraction light sources imaging to cause using several researchs inherent or that the external optical contrast agents of bestowing is come research organization.Especially, diffuse optical tomography (DOT) be can in dispersive medium, exist absorb and the situation of scattering under the technology of the tomographic image relevant with dispersive medium is provided.For example, DOT has been applied to the imaging of cerebral hemodynamic imaging and breast tissue.For example, submit to by Vasilis Ntziachristos and Jorge Ripoll on February 5th, 2004, title is among the International Patent Application PCT/US04/03229 of " Method and System for Free SpaceOptical Tomography of Diffuse Media ", introduced a kind of schematic DOT method and system, this application transfers assignee of the present invention.

Verified, because the low tissue in so-called " near-infrared window " absorbs, therefore have the distance that the light of the wavelength in the near infrared range can propagate by tissue and be approximately several centimetres.Near-infrared (NIR) window makes the NIR fluorescent technique developed to show the special biochemical reaction in the specimen.

The various correlation techniques that are used to handle the NIR fluorescence signal have also been developed.Especially, the development of suitable imaging system has allowed to use fluorescence molecule tomography (FMT), and it is the technology that a kind of NIR of use fluorescent probe or labelling are resolved the characterization of molecules in the dark tissue.The FMT that has showed three-dimensional imaging in the body that is applied to the enzymatic activity in the deep layer tumor.

Common hypothesis in the tradition NIR optical tomography is low absorption that the propagation in the dispersive medium has high scattering and provides as the NIR window.This hypothesis allows to derive " diffusion equation " relevant with " transport equation " by " diffusion approximation ", and this provides effective tool for the NIR photon spread modeling in the tissue.For example at " the LinearTransport Therory " of K.M.Case and P.F.Zweifel, Addison-Wesley, MA, (1967) and K.Furutsu and Y.Yamada " Diffusion Approximation for a Dissipative RandomMedium and the Applications ", introduced this transport equation among the Phys.Rev.E 50,3634 (1994).

As everyone knows, all current available fluorescins utilize the exciting light of wavelength in visible-range.And traditional fluorescin is launched visible fluorescence when being excited.The higher absorption of the visible light of propagating in the biological tissue will use the tomographic imaging as the visible light that is provided by conventional fluorescent proteins to become complicated, and this causes remarkable decay.Because high the absorption, (for example for visible light) above-mentioned traditional diffusion approximation becomes invalid.

Other of transport equation is more high-grade to be separated, and (except above-mentioned diffusion approximation) has produced and has been applied to the NIR optical tomography.The described senior deficiency that has overcome above-mentioned diffusion approximation of separating.Yet it is big and be not suitable for the tomograph system with a large amount of excitation sources that transport equation senior separated common amount of calculation, and this causes producing large data sets.

In order to provide tomography required a plurality of images, many traditional optical tomography systems use optical switch as the part of light source assembly so that use single optical element to throw in various angles or position with respect to specimen.As everyone knows, optical switch produce power loss.In addition, many optical tomography systems are used at room temperature or the CCD photographing unit under the appropriateness cooling is collected light.As everyone knows, room temperature or appropriate refrigerative CCD photographing unit have dark (heat) noise of higher level, and this has often limited the quality of final optical tomography image.

Summary of the invention

According to an aspect of the present invention, a kind of system that is used for optical tomography comprises the apparent source (apparent lightsource) that is suitable for to the specimen projection exciting light that wherein has fluorescin, wherein exciting light enters specimen, becomes the inherent light in the specimen.Inherent light is suitable for from the fluorescin fluorescence excitation.Inherent light and fluorescence spread.In certain embodiments, at least one in exciting light and the fluorescence has the wavelength in visible wavelength range.

According to a further aspect in the invention, a kind of method of optical tomography comprises uses the apparent source that is suitable for to the specimen projection exciting light that wherein has fluorescin to produce exciting light, and wherein exciting light enters specimen, becomes the inherent light in the specimen.Inherent light is suitable for from the fluorescin fluorescence excitation.Inherent light and fluorescence spread.In certain embodiments, at least one in exciting light and the fluorescence has the wavelength in visible wavelength range.

According to a further aspect in the invention, a kind of system that is used for optical tomography comprises at least one alternative parts that move with mobile apparent source optionally, thereby a plurality of light paths are pointed to specimen.

Description of drawings

From to the following specifically describes of accompanying drawing, can more fully understand above-mentioned feature of the present invention and the present invention self, wherein:

Fig. 1 illustrates a kind of block diagram that is used for the system of optical tomography, and described system provides transmission imaging;

Figure 1A illustrates a kind of block diagram that is used for the system of optical tomography, the cremasteric reflex imaging of described system;

Fig. 2 illustrates a kind of block diagram that is used for the system of optical tomography, and described system has lasing light emitter, optical fiber, optical switch, cooling CCD photographing unit and image processor;

Fig. 3 is a kind of block diagram of optical scanner, and this optical scanner has fiber coupler, position-controllable mirror and telecentric lens, and they one are used from constant operating distance (WD) and locate to produce a plurality of apparent sources with zero diopter field;

The flow chart that is used to provide according to the method for tomographic image of the present invention is provided Fig. 4;

Fig. 5 shows the series of 31 images, and 31 apparent sources of the Fig. 1 that provides corresponding to the optical scanner as Fig. 3 have shown inherent light (promptly from apparent source, enter and leave the exciting light of specimen);

Fig. 5 A shows another series of 31 images, and 31 apparent sources of the Fig. 1 that provides corresponding to the optical scanner as Fig. 3 have shown in response to the inherent light of Fig. 5 fluorescin emitted fluorescence in the specimen;

Fig. 5 B shows the another series of 31 images, and but 31 apparent sources of the Fig. 1 that provides corresponding to the optical scanner as Fig. 3 have shown and do not passed specimen passed the transillumination of homogeneous plate (being phantom);

Fig. 6 shows the fluoroscopic image of the White-light image of the dead Mus of stack, and the vial that wherein has the green fluorescent protein (GFP) of express cell is placed in the esophagus of dead Mus;

Fig. 6 A shows another fluoroscopic image of the White-light image of the dead Mus of stack, and another vial that wherein has the green fluorescent protein (GFP) of express cell is placed in the esophagus of dead Mus, and this vial has more cell than the bottle of Fig. 6;

Fig. 7 is a kind of block diagram that is used for the system of optical tomography, and this system has the planar imaging chamber;

Fig. 7 A is the block diagram that the imaging chamber of the system that is used for Fig. 7 is shown;

Fig. 8 is the block diagram that another kind is used for the system of optical tomography, and this system has Plane of rotation imaging chamber;

Fig. 8 A is the block diagram of Plane of rotation imaging chamber that can be used for the system of Fig. 8;

Fig. 9 is the block diagram that another kind is used for the system of optical tomography, and this system has the cylindrical shape imaging chamber of rotation;

Figure 10 is the block diagram that another kind is used for the system of optical tomography, and this system has the cylindrical shape imaging chamber and the alternative apparent source that moves of rotation;

Figure 10 A illustrates the cylindrical shape imaging chamber of rotation and the block diagram of a plurality of light sources;

Figure 11 is the block diagram that another kind is used for the system of optical tomography, and this system has the optical scanning head of band apparent source array; And

Figure 11 A is the block diagram of optical scanning head as shown in figure 11.

The specific embodiment

Before introducing formation method and system, explain some introductory notion and terms.When this uses, " phantom (phantom) " just refers to the tested object in imaging.Phantom has the Artifact that is similar to living tissue diffused light propagation characteristic typically, for example a slice plastics.As another example, phantom can be the vial that wherein has the cell of express fluorescent protein (being fluorescent labeling).

When this uses, term " apparent source " is used to illustrate single light source projects to a plurality of physical locations or angle, and each provides an apparent source.

When this uses, term " excites " light to be used to illustrate the light that is produced by excitation source, and (for example apparent source), before entering specimen, described light is propagated to the specimen for the treatment of imaging.In case in the arrival specimen, described light is called " inherence " light at this.This inherence light is absorbed in specimen and scattering, and also can leave specimen.

The inherent light that has left specimen has identical wavelength with the light that excitation source produces.Exciting light and inherent light can be unicolor or they can cover wideer spectrum, for example white light.

In certain embodiments, usually with light source be arranged on specimen mutually the optical detection equipment of the same side receive the inherent light that leaves specimen (for example, the catoptric imaging among Figure 1A of introduction in) below.In other embodiments, usually receive the inherent light that leaves specimen (for example, the projection imaging of Fig. 1 of introduction in) below with photo-detector that excitation source is arranged on the opposite side of specimen.In either case, when exciting light entered specimen, it became inherent light and from the internal reflection of specimen or pass specimen.

When this uses, term " emission " light is used to illustrate by biological tissue or the light that produces in biological tissue.When this uses, term " fluorescence " is used to describe by fluorescence excitation albumen to respond the radiative form that inherent light produces.

When this uses, term " image " is used to illustrate the visual representation on the basis " view data " with digital camera or computer system generation.Yet, be to be understood that when when this uses, term " image " also is used in reference to view data.

When this uses, term " diffusion " is used for having run into when explanation is propagated the light with photon of some scattering events (for example scattering events more than ten) in specimen, irrelevant with the absorption of photon in the specimen.The number of scattering events can be greater than or less than ten.

The following method and system of introducing of the present invention is applicable in diffused light propagates the visible light of propagating in the dominant biological tissue.Yet described method and system is applicable to too well in diffused light propagates any type of light of propagating in dominant any medium, for example, propagates into the NIR light of enough degree of depth in biological tissue, for example visible excitation light and NIR fluorescence (emission light).And described method and system can also be applicable to the light of propagating in the dominant medium of coherent propagation.

Although the method and system of the present invention that this place is introduced similarly is applicable to the fluorescin of emission visible fluorescence, in the visible wavelength range of 700nm, provide special benefit at about 400nm, but described method and system also goes for having the light of other wavelength, and for example about 700nm is to the interior fluorescence of near-infrared (NIR) scope of 1000nm.And described method and system is applicable to (for example in visible-range) and the fluorescin emitted fluorescence system of (for example in the NIR scope) in another wave-length coverage in a wave-length coverage of exciting light wherein equally well.Described method and system is applicable to also that wherein exciting light and fluorescin emitted fluorescence are in the NIR scope or the occasion of both in visible-range.And, can use to exceed the light of 400nm to the wave-length coverage of 1000nm.

With reference to Fig. 1, the system 10 that is used to use fluorescin to carry out optical imagery comprises that imaging source 12 and light guiding device 14 are to provide a plurality of apparent source (not shown).Apparent source provides exciting light 22a-22c in a plurality of positions of relative specimen 18.Although shown three such positions, also can have more than three or the apparent light source position below three. Exciting light 22a, 22c are radiated on the specimen 18, at light, and leave specimen 18 as inherent light 24a, 24b in becoming when entering. Inherent light 24a, 24b pass optional selectable light filter 28, pass optional image intensifier 30, and are received by photo-detector 32.Exciting light 22b also enters specimen 18 and is radiated on the fluorescin 20 in the specimen 18.Response exciting light 22b, fluorescin 20 emitting fluorescences 26, this fluorescence 26 also passes optional selectable light filter 28, passes optional image intensifier 30, and is received by photo-detector 32.

Optional white light source 40 can provide further illumination so that other light path (not shown) to be provided for specimen, this light path is from the surface reflectance of specimen, and pass optional selectable light filter 28 equally, pass optional image intensifier 30, and receive by photo-detector 32.

In certain embodiments, inherent light 24a, 24b, fluorescence 26 and receive by photo-detector simultaneously from the white light of white light source 40.In this configuration, inherent light 24a, 24b, fluorescence 26 and can separate simultaneously or in the different time different light is offered photo-detector 32 by selectable light filter 28 from the white light of white light source 40.For this reason, can with selectable filter passband in different time concentrates on the wavelength of light, fluorescence and white light.

In other embodiments, inherent light 24a, 24b, fluorescence 26 and, fluorescence 26 and from the time of reception difference of other light in the white light of white light source 40 from any one or a plurality of time of reception and inherent light 24a, 24b in the white light of white light source 40.For example in a specific embodiments, at first receive inherent light 24a, 24b, this moment, imaging source 12 extinguished.After light 24a, 24b no longer exist, receive fluorescence 26 interior.After stopping emitting fluorescence 26, open white light source 40, and receive white light.

The light that photo-detector 32 is used for receiving converts numerical data 32a (being also referred to as view data at this) to.Image processor 34 receiving digital data 32a and generation image 46.In certain embodiments, image 46 is tomographic image.

Image processor 34 can comprise (FP) processor 36 of forward problem (forward problem) with diffusion equation processor 38.For example will describe more specifically the function of forward issue processor 36 and diffusion equation processor 38 below in conjunction with Fig. 4.Compare with the light that photo-detector receives at this model (expection light) that is noted that forward problem processor 36 is propagated the light in the specimen.The difference that receives between light and the expection light (being provided by light propagation model) is relevant with the fluorescin 20 in the specimen.Diffusion equation processor 38 is provided for the modified diffusion coefficient in " diffusion equation ", is somebody's turn to do " diffusion equation " and can be used to provide " transport equation " of above-mentioned light propagation model relevant.

In certain embodiments, modified diffusion coefficient allows the light of the light (have about 400nm wavelength to 700nm) of model prediction in visible wavelength range to propagate.In other embodiments, modified diffusion coefficient allows the light of the light (have about 700nm wavelength to 1000nm) of model prediction near infrared wavelength region to propagate.In another embodiment, modified diffusion coefficient allows the model prediction wavelength to propagate at the light that 400nm arrives the light outside the 1000nm scope.

System 10 can comprise that also light direction controller 44 is to be directed to predetermined light paths with apparent source.System 10 also can comprise optional chamber positioner 42 to replace light direction controller 44 or combination with it, and it can be used for mobile imaging chamber 16 so that more apparent source to be provided, and promptly inherent light passes specimen 18 along more predetermined light paths.

Should be understood that system 10 provides a kind of transmission imaging system, wherein the light that produces of imaging source 12 passes specimen 18 and is received at the opposite side of specimen 18 basically.

Referring now to Figure 1A, show system 70, wherein the like with Fig. 1 is illustrated as having similar Reference numeral, selectable light filter 28, image intensifier 30 and photo-detector 32 usually and imaging source 12 and light guiding device 14 be positioned at the same side of specimen 18.Utilize this particular arrangement, inherent light 72a, 72b are by photo-detector 32 conduct emission light-receivings.In fact, exciting light 22a-22c enters specimen 18 and reflection, and perhaps more specifically, optical receiver 32 is got back in scattering.Fluorescence 74 is also received by photo-detector 32.System 70 generates image 76.

Should be understood that system 70 provides a kind of reflection imaging system, wherein enter specimen 18 and be received in the same side of specimen 18 basically by the light of imaging source 12 emission.In other embodiments, the angle between light guiding device 14 and the photo-detector 32 is approximately 90 degree.

Referring now to Fig. 2, device 100 comprises laser instrument 102, optical fiber 104 and optical switch 106, and it is combined and produces a plurality of apparent sources that point to the imaging chamber 112 with imaging plate 108, and specimen 110 is placed on this imaging plate 108.Imaging chamber can be full of matching fluid 114.Selectable light filter 115 and CCD photographing unit 116 receives and passes the inherent light of specimen 110 and by specimen 110 emitted fluorescence.As mentioned above, selectable light filter has the passband on the wavelength that optionally concentrates on inherent light or fluorescence, and this depends on that what generating is inherent image or fluoroscopic image.Optical switch 106 can be by computer 122 controls.Computer 122 also can be via CCD controller 118 control CCD photographing units 116.Image processor 120 is via CCD controller 118 receiving digital data 116a.Pictorial displays 124 can show final image information.

Will be appreciated that the imaging source 12 of laser instrument 102 corresponding to Fig. 1 and Figure 1A, optical switch 106 is corresponding to the light guiding device 14 of Fig. 1 and Figure 1A, imaging chamber 112 is corresponding to the imaging chamber 16 of Fig. 1 and Figure 1A, CCD photographing unit 116 is corresponding to the photo-detector 32 of Fig. 1 and Figure 1A, and image processor 120 is corresponding to the image processor 34 of Fig. 1 and Figure 1A.

The specimen 110 of Xian Shiing is the mouse that is placed in the imaging chamber 112 herein.Laser instrument 102 provides the exciting light (not shown), and this exciting light enters specimen 110 and excites fluorescin (not shown) in the specimen 110 to produce the fluorescence (not shown).CCD photographing unit 116 receives as the laser (having passed specimen 110) of inherent light via selectable light filter 115 and also receives emitted fluorescence in the specimen 110.

In a specific embodiments, laser instrument 102 is argon (Ar ⁺) laser instrument, its emission under continuous wave (CW) power of about 200mW has the laser of about 488nm wavelength.This laser can be used to excite the fluorescin in the specimen 110, for example green or yellow fluorescence protein.

In a specific embodiments, optical fiber 104 is that diameter is the multimode fibre of 100 μ m.Laser instrument 102 provides a plurality of apparent sources by optical switch 106 in the different physical locations with respect to specimen 110.In a specific embodiments, optical switch provides 31 apparent sources.Yet, in other embodiments, can provide more than 31 or the apparent source below 31.

Though shown optical switch 106, can use optical scanning head (or optical scanner) to replace optical switch 106 in another embodiment, as more specifically showing among Fig. 3.

Optical switch 106 provides apparent source in a plurality of angles or position with respect to specimen 110, allows image processor 120 to form corresponding multiple image thus, and it can be attached in the tomography method by image processor 120.

Should be understood that optical switch 106 comprises a plurality of selectable fiber path (not shown), be suitable for light is directed to corresponding a plurality of selectable fixed physicals position, thereby be provided on position and the number by the fixed apparent source of selectivity.

In a specific embodiments, living tissue (what show is mouse herein) is placed on the imaging plate 108 and with optical match liquid 114 and contacts.To further introduce matching fluid 114 below.Matching fluid is used to reduce the influence of veiling glare.Yet, in other embodiments, do not use matching fluid.

In operation, inherent light (being derived from each apparent source) and received by the CCD photographing unit and carried out tomography by image processor 120 thereafter and handle by the light of the emission of the fluorescins in the specimen 110 is as described in conjunction with Fig. 4.

In a specific embodiments, CCD photographing unit 116 is to have the cooling CCD photographing unit that reduces dark noise.For example, the CCD photographing unit 116 that provides can be Roper Scientific, and Princeton Instruments has a unitary CCD photographing unit of sub-cooled.

During operation, 118 controls of CCD controller also trigger optical switch 106, and therefore feasible each image that obtains has realized the accurate synchronization that excites and survey corresponding to the reposition (being the different light paths in the optical switch 106) of new apparent source.The each collection is made up of N image, and each apparent light source position is once gathered.Therefore, suppose the CCD photographing unit of 512 * 512 pixels, being used for every group of maximum amount of data of measuring is N * 512 * 512.Yet being used for number (being pixel) that image processor 120 carries out the detector of subsequent treatment can be less than the group fully of 512 * 512 pixels, and this depends on and each relevant visual field in the apparent source.And the number of pixels that can reduce to use is to reduce Flame Image Process required computation time.

In a specific embodiments, CCD photographing unit 116 and selectable light filter 115 are for example along near the represented direction of arrow 140,140 alternative moving so that obtain more images with respect to other angle of specimen 110 specimen 110.In another embodiment, can be provided in image from the optical scanner 117 of CCD photographing unit 116 off-lines a little with respect to other angle of specimen.

Specimen 110 can flatly be placed on the imaging plate 108 and with the coverslip (not shown) to be compressed.Then, use matching fluid 114 to be full of imaging chamber 112, in a specific embodiments, this matching fluid is by lipid Emulsion (intralipid) and prepared Chinese ink (India ink) solution composition.Matching fluid 114 provides the coupling of optical characteristics, and this often reduces refractive index and diffuse-wave mismatches in the chamber.In a specific embodiments, matching fluid 114 is made up of 1% lipid Emulsion and 2.1% prepared Chinese ink, and it corresponds respectively to μ _a=1.25cm ^-1And μ _s'=16.7 cm ^-1, μ wherein _aBe absorptance and μ _s' be the scattering coefficient that reduces.To further introduce the scattering coefficient that reduces below.

Referring now to Fig. 3, the optical scanner 117 that optical scanner 150 can be used to replace the optical switch 106 of Fig. 2 and also can be used as Fig. 2.In a specific embodiments, optical scanner 150 can comprise that centering on roughly orthogonal two galvanometers that rotate on pivot controls mirrors (at this for the sake of clarity, show a galvanometer control mirror 156) and a scanning lens 158, scanning lens 158 is used to scan and laser beam 156 is focused on the input window of imaging chamber 162 so that a plurality of apparent sources to be provided, each apparent source is represented by light beam 160a and 160b at this at different physical locations.In a specific embodiments, scanning lens 158 is telecentric lenses.In a specific embodiments, the beam diameter of plane 162 on the scene (being the imaging chamber 112 of Fig. 2) is approximately 300 μ m.Can use the single light source (not shown), it is received by fiber coupler 152.

Compare with the light loss consumption of traditional optical switch (for example optical switch 106 of Fig. 2), optical scanner 150 provides the consumption of light loss still less.Therefore, optical scanner 150 provides low-loss apparent light source system.In addition, utilize this optical scanner 150, infinite basically variation can be carried out in the quantity of scan area, beam shape and apparent source and the position of apparent source, and this is different from the optical switch 106 of Fig. 2, and wherein the light path of fixed qty is in fixed position.And, can obtain higher luminous power and wideer optical wavelength range (visible light is to near-infrared (NIR) light).

Optical scanner 150 has the multiple advantage that surpasses optical switch, includes but not limited to low energy consumption, consistent reliability that responds and improve and vigorousness on a plurality of apparent sources.And the quantity of scan area and apparent source and spatial configuration can be by software controls, so it can change according to the characteristic that is scanned specimen.In addition, can obtain more high-power illumination and wideer wave-length coverage (for example from 400nm to 1000nm).

Referring now to Fig. 4, the method 200 of optical tomography is in step 202 beginning, produces exciting light herein and it is launched to specimen.In certain embodiments, for example causing producing among the embodiment of tomographic image, from providing exciting light corresponding to a plurality of apparent sources with respect to the diverse location of specimen.

In certain embodiments, a plurality of light sources at a plurality of apparent sources place provide exciting light usually simultaneously.In other embodiments, a plurality of apparent sources sequentially provide exciting light.

At square frame 204, receive inherent light.As mentioned above, inherent light is corresponding to the exciting light that enters and leave specimen.Can use photo-detector, for example the photo-detector 32 of Fig. 1 receives this inherence light.In certain embodiments, the inherent light of reception is corresponding to transillumination, and wherein photo-detector and apparent source are basically in the relative both sides of specimen.In other embodiments, the inherent light of reception is corresponding to reflected light, and wherein photo-detector and apparent source are basically in the same side of specimen.

At square frame 206, receive fluorescence.Fluorescin in the specimen responds inherent light and emitting fluorescence.Can use photo-detector, for example the photo-detector 32 of Fig. 1 receives this fluorescence.In certain embodiments, for example by being suitable for that fluorescence and the isolating light filter of inherent light (for example 28 of Fig. 1) received fluorescence and inherent light simultaneously.In other embodiments, after extinguishing, exciting light also receives fluorescence by light filter.

At square frame 208, convert the inherent light that receives to first image information, for example the numerical data 32a of Fig. 1.At square frame 210, convert the inherent light that receives to second image information, it also can be numerical data 32a.

At square frame 212, produce model and propagate with the light in the prediction specimen.Described model can be based on diffusion equation, and this diffusion equation has the modified diffusion coefficient of following will more specifically description the (for example in the equation below (4)) and has as the correction wave number in the following equation (6).

Can be relevant at the optical model that square frame 212 produces with the propagation in the homogenous medium (medium that does not promptly have optical heterostructure).In other embodiments, also can utilize more advanced model to resolve and utilize then information about the background optical heterogeneity.

By following argumentation, it is evident that light in the tissue is propagated can come modeling by the modified diffusion equation that use has a modified diffusion coefficient, wherein said modified diffusion coefficient is suitable for predicting the characteristic that the light of dispersive medium is propagated, wherein said medium has higher absorption, in the situation as the propagation of the visible light in biological tissue.Have modified diffusion coefficient as described below, modified diffusion equation for example can be predicted the propagation of visible light in biological tissue, and it will spread and have higher absorption for visible light as everyone knows.Yet, modified diffusion equation also be suitable for the calculating to a nicety propagation of the light in dispersive medium, propagated, the near infrared light of for example in biological tissue, propagating with other wavelength.

General diffusion equation can be derived from radiation transport equation.The inherent light field that the lasers excites of propagating in medium (inherent light) produces and by the position

The fluorescin at place and the fluorescence field that produces in medium is all calculated separately is used to calculate normalization Born field (diffusion approximation) then.As following more specifically as described in, modified diffusion equation can be used for forward problem so that the image of the fluorescin in the medium to be provided.

Because in the position So the apparent source at place is as in the photo-detector position The Born field that light in the medium of being surveyed is propagated

Have modified diffusion coefficient and revise propagation wave-numbers, the both illustrates high the absorption, and described Born field is provided by following formula:

$U_c({\stackrel{\→}{r}}_s,{\stackrel{\→}{r}}_d)=S_o\frac{U_{\mathrm{fl}}({\stackrel{\→}{r}}_s,{\stackrel{\→}{r}}_d)-{\mathrm{\Θ}}_f{U}_{\mathrm{inc}}({\stackrel{\→}{r}}_s,{\stackrel{\→}{r}}_d)}{U_{\mathrm{inc}}({\stackrel{\→}{r}}_s,{\stackrel{\→}{r}}_d)}=---\left(1\right)$

$\frac{1}{U_o({\stackrel{\→}{r}}_s-\stackrel{\→}{r},k^{{\mathrm{\λ}}_1})}\∫d^{3}r\·U_o({\stackrel{\→}{r}}_s-\stackrel{\→}{r},k^{{\mathrm{\λ}}_1})n\left(\stackrel{\→}{r}\right)\frac{\mathrm{\υ}}{D^{{\mathrm{\λ}}_2}}G({\stackrel{\→}{r}}_d-\stackrel{\→}{r},k^{{\mathrm{\λ}}_2})$

Here

With

Be respectively to excite (λ ₁) (laser instrument) and emission (λ ₂) measured value of (fluorescence) wavelength. $U_{\mathrm{bl}}({\stackrel{\→}{r}}_s,{\stackrel{\→}{r}}_d)={\mathrm{\Θ}}_f{U}_{\mathrm{inc}}({\stackrel{\→}{r}}_s,{\stackrel{\→}{r}}_d)$ Be diafiltration signal (bleedthrough signal), Θ _fBe for example relevant logical light attenuating filter factor of band with the selectable light filter 28 of Fig. 1, S _oBe that explanation is exciting (λ ₁) and emission (λ ₂) gain term of instrument gain poor (for example photo-detector gain inequality) of wavelength,

Be the product of fluorescin absorptance and fluorescence quantum yield, k ^{λ 1}, k ^{λ 2}Be respectively at λ ₁And λ ₂Correction photon ripple propagation wave-numbers, its explanation is high to be absorbed, υ is the speed of light in medium, D ^{λ 2}Be at λ ₂Modified diffusion coefficient when having high the absorption, $U_o(\stackrel{\→}{r_s}-\stackrel{\→}{r},k^{{\mathrm{\λ}}_1})$ Be to be described in λ ₁, the position in medium

The item of the photon field of setting up, and $G(\stackrel{\→}{r_d}-\stackrel{\→}{r,}{k}^{{\mathrm{\λ}}_2})$ The Green's function that is diffusion approximation is separated, and diffusion approximation is described the ballistic phonon ripple from the position

Fluorescin propagate into photo-detector.Function $G(\stackrel{\→}{r_d}-\stackrel{\→}{r},k^{{\mathrm{\λ}}_2})$ Provide by following formula:

$G({\stackrel{\→}{r}}_d-\stackrel{\→}{r},k^{{\mathrm{\λ}}_2})=\frac{\exp \left(i{k}^{{\mathrm{\λ}}_2}({\stackrel{\→}{r}}_d-\stackrel{\→}{r})\right)}{({\stackrel{\→}{r}}_d-\stackrel{\→}{r})}---\left(2\right)$

Top equation (1) is substantially by U _IncNormalization.Use the normalized advantage in the equation (1) to be to have eliminated the position relative influence, and, even, also can calculate this existing under the situation of fluorescin.This means that not needing to carry out background before bestowing fluorescin measures, this is important for research in the body.

Expression is by diffusion coefficient is write as with a kind of useful mode that medium absorbs the absorption dependency of irrelevant diffused light propagation:

$D_{\mathrm{\α}}=\frac{1}{3({\mathrm{\μ}}_s^{\text{'}}+\mathrm{\α}{\mathrm{\μ}}_a)}---\left(3\right)$

Here α is absorption, scattering and the anisotropic constant that depends on medium usually.Coefficient μ _s' be the scattering coefficient that reduces, and μ _aIt is absorptance.The scattering coefficient μ that reduces _s' can be write as ${\mathrm{\μ}}_s^{\text{'}}=(1-g){\mathrm{\μ}}_s,$ μ wherein _sIt is scattering coefficient.Be used to illustrate the high modified diffusion coefficient D that absorbs _αExpression formula can obtain by derivation from radiation transport equation, obtain:

$D_{\mathrm{\α}}=\frac{1}{3({\mathrm{\μ}}_s^{\text{'}}+{\mathrm{\μ}}_a)}(1-\frac{4}{5}\frac{{\mathrm{\μ}}_a}{{\mathrm{\μ}}_s^{\text{'}}(1+g)+{\mathrm{\μ}}_a}{)}^{-1}---\left(4\right)$

Wherein g is an anisotropy factor.Here, according to the scattering coefficient μ that reduces _s' express D _α, scattering coefficient μ _s' be the correlative in the scattering experiment of anisotropic medium.A main difference between the most frequently used expression formula of equation (4) and standard diffusion coefficient D is: in the most frequently used expression formula, the value of α a priori is fixed as α=0 or α=1.Yet the more generally expression formula of α can illustrate the background absorption of various degree by selecting anisotropy factor g suitably, and this depends on the spectral region of consideration.By analyzing or testing and to find desired value.The equation that obtains α from equation (4) and equation (3) is:

$\mathrm{\α}=1-\frac{4}{5}\frac{{\mathrm{\μ}}_s^{\text{'}}+{\mathrm{\μ}}_a}{{\mathrm{\μ}}_s^{\text{'}}(1+g)+{\mathrm{\μ}}_a}---\left(5\right)$

The scope of the representative value of α is 0.2 to 0.6.For the visible light of in biological tissue, propagating, suppose anisotropy factor g～0.8 (this is typical for biological tissue), α is approximately 0.50 to 0.55.The dependency of α and g is little, and the variation (g 0.5 to 0.9 between) of g value in actual biological value causes that the variation of α value is little.

Should be understood that if use traditional diffusion coefficient (being α=0 or α=1), then for having the high medium that absorbs, diffusion approximation produces coarse result.The reason (for example in biological tissue, propagating) of diffusion approximation failure under the high situation about absorbing of the existence that Here it is considers for a long time for visible light.Yet when using the modified diffusion coefficient of equation (4), diffusion approximation keeps accurately.For this reason, must be defined as revising wave number:

$k^{\mathrm{\λ}}=\sqrt{-\frac{{\mathrm{\μ}}_{\mathrm{\α}}^{\mathrm{\λ}}}{D_{\mathrm{\α}}^{\mathrm{\λ}}}+\frac{\mathrm{i\ω}}{v{D}_{\mathrm{\α}}^{\mathrm{\λ}}}}---\left(6\right)$

Wherein ω is modulating frequency (the continuous wave exciting light is represented in ω=0).The ripple propagation wave-numbers of the modified diffusion coefficient of equation 4 and equation 6 is combined, can derive the Green's function of diffusion approximation and separate, it is suitable for having imaging under the high situation about absorbing, and the visible light that for example is adapted in the biological tissue is propagated.

Above-mentioned model (equation 1 and explanatory equation subsequently) can utilize the attribute of apparent source, for example their position and intensity.

At square frame 214, with for example combination in so-called " forward model " of first image information, second image information and light propagation model.Have inner fluorescin and the inhomogeneous place in inside thus in specimen, " image problem " of the following form of combination results of square frame 214 or " forward model ": measured value=(theoretical prediction value) * (unknown distribution), here usually according to equation (1)-(6), measured value is provided by photo-detector 32 (Fig. 1) at square frame 204-210, and the theoretical prediction value is provided by image processor 34 (Fig. 1) at square frame 212.Unknown distribution is corresponding to the fluorescin emitted fluorescence.Image processor 34 can parse unknown distribution so that determine the physical location and the characteristic of the fluorescin 20 (Fig. 1) in the specimen 18 (Fig. 1).

At square frame 216, produce the figure of fluorescin concentration in the specimen.In certain embodiments, this figure is a tomographic image.In order to produce this figure, above-mentioned forward model quilt " inverting " is to parse above-mentioned unknown distribution.

In producing fluorescent protein map, the volume of being paid close attention to can be divided into a plurality of axially (level) layers (for example 21 layers), and every layer comprises a plurality of (for example 651) voxel (voxel).The size of selecting voxel based on the size and the sectional quantity in the visual field.

The volume of being paid close attention to can be divided into a plurality of voxels on three-dimensional.These can be regarded as level, vertical or transverse layers, be similar to and abutting one another the square that piles up in three dimensions.Each voxel has the fluorescin and the unknown decay of unknown number.If fluorescence in each voxel and decay are known, then can predict the image of measurement.Yet fluorescence in each voxel and decay are not known.Therefore can parse (inverting) above-mentioned forward problem to find fluorescent protein map.

In certain embodiments, for the intelligibility that makes image strengthens, it is desirable to White-light image with specimen and be added on the fluorescin concentration map.For this reason, can utilize white light source that specimen is illuminated, and receive white light, and can generate White-light image at square frame 222 at square frame 220 at square frame 218.

White-light image that generates at square frame 222 and the fluorescent protein map that produces at square frame 216 can be superposeed.For this reason, in a specific embodiments, with White-light image with the fluorescent protein map registration or aim at.

For White-light image is aimed at fluorescent protein map, for example can make the image of apparent source to allow to determine the apparent source coordinate by phantom.This program has improved the common registration that can be superimposed upon the White-light image on the tomographic image, has reduced relative site error.

Referring now to Fig. 5,31 images 300 of inherent light are corresponding to 31 apparent sources that light are directed to specimen and the inherent light that receives therefrom as transillumination.Apparent source one next or simultaneously or with combination in any light is directed to specimen.

Can use the logical interference light filter of band on the light wavelength (for example scheme l 28) that excites that concentrates on from light source (for example 12 of Fig. 1) to generate image 300.

Referring now to Fig. 5 A, 31 images 310 of fluorescence are corresponding to 31 apparent sources that light are directed to specimen and the emitting fluorescence that receives therefrom.This apparent source one next or simultaneously or with combination in any light is directed to specimen.Can when apparent source just is directed to specimen with light or after apparent source is directed to specimen with light, receive fluorescence.For example, the inherent light image of Fig. 5 and the fluoroscopic image of Fig. 5 A are combined at the square frame 214 of Fig. 4.

Can use to concentrate on and generate image 310 from the logical interference light filter of the band on the radiative wavelength of fluorescin (for example 20 of Fig. 1) (for example 28 of Fig. 1).

Measured value (or image) 300,310 is used to produce the field that equation (1) is described

Time of exposure can change between single image so that the dynamic range maximization.

Referring now to Fig. 5 B, 31 images 320 of apparent source are corresponding to the light guiding being passed through 31 apparent sources of phantom and the light that receives therefrom.Apparent source one next or simultaneously or with combination in any light is directed to phantom.The image of apparent source for example is used for randomly with White-light image and fluorescin concentration map registration, as shown in the square frame 224 of Fig. 4.

Can use the logical interference light filter of band on the light wavelength (for example 28 of Fig. 1) that excites that concentrates on from white light source (for example 40 of Fig. 1) to generate image 320.

Referring now to Fig. 6 and 6A, shown the example of the image that method and system of the present invention provides.Use two varying numbers (10 ⁵And lO ⁶) the green fluorescent protein (GFP) of expressing tumor cell.Fluorescin is placed in the thin glass tubing, and after kill animals, is inserted in the esophagus of animal.Animal is placed in imaging chamber's (for example 16 of Fig. 1), and imaging chamber is full of above-mentioned matching fluid then.Use 31 apparent source arrays of transmission animal chest peripheral region to carry out imaging.

At first with reference to Fig. 6, image 350 comprises 10 ⁵Figure 35 2 of individual fluorescin, White-light image 354 stacks of mouse thereon.

Then with reference to Fig. 6 A, image 370 comprises 10 ⁶Figure 37 2 of individual fluorescin, White-light image 374 stacks of mouse thereon.

Referring now to Fig. 7, the system 400 that is used for optical tomography 400 comprises the lasing light emitter 402 that is connected to optical scanner 412, and is for example described in conjunction with Fig. 3, and this optical scanner provides a plurality of apparent sources.System 400 also comprises laser intensity controller 406 (LIC).This LIC406 provides strength control for lasing light emitter 402.

System 400 further comprises image intensifier 420 and CCD photographing unit.Selectable light filter 418 optionally frequency band is concentrated on apparent source emission excite on the light wavelength or specimen 416 in the fluorescin wavelength of light emitted on.Computer 424 can be controlled LIC 406 and optical scanner 412.Computer also can be exercised the function of image processor, for example exercises the function of the image processor 34 of Fig. 1, thereby provide image on pictorial displays 426.

In operation, optical scanner 412 provides a plurality of apparent sources in a plurality of positions with respect to specimen 416.The CCD photographing unit is collected as transillumination and is passed the inherent light of specimen 416 and the fluorescin institute emitted fluorescence in the specimen 416.The light that the CCD photographing unit will receive converts numerical data to, and as described in conjunction with Fig. 4, this numerical data of Computer Processing is to provide tomographic image on pictorial displays 426.

In this specific embodiments, the required a plurality of images of tomographic imaging are relevant with the position of a plurality of apparent sources that optical scanner 412 provides, and imaging chamber keeps motionless substantially, but can move to influence picture quality along axle 417.

Referring now to Fig. 7 A, the imaging chamber 414 of Fig. 7 comprises cylinder 450, and is for example described in conjunction with Fig. 2, and it can be full of matching fluid.Imaging plate 454 and coverslip 456 are around specimen 416.

Referring now to Fig. 8, wherein the like with Fig. 7 is illustrated as having similar Reference numeral, and the system 500 that is used for optical tomography comprises the imaging chamber 504 of fixed preparation 510.Imaging chamber 504 is suitable for rotating and being suitable for doing translational motion along axle 508 like that according to arrow 506 is represented.System 500 can comprise that rotation platform 502 is with fixing imaging chamber 504.

As described in conjunction with Fig. 7, the required a plurality of images of tomographic imaging are relevant with the position of a plurality of apparent sources that optical scanner 412 provides.In addition, imaging chamber 504 for example can do rotation and translational motion so that more apparent light source position and/or angle to be provided under the control of computer 424, thereby more images of specimen 510 are provided.

Referring now to Fig. 8 A, wherein the like with Fig. 7 and 8 is illustrated as having similar Reference numeral, the imaging chamber 504 of Fig. 8 can be with imaging chamber 550 (for example, shown in Fig. 7 A) replace, but be suitable for rotating like that and doing translational motion like that according to arrow 554 is represented according to arrow 552 is represented.

Imaging chamber 550 also can do translational motion along arrow 556 represented axles, and it has considered the influence to the CCD photographing unit 442 resultant images of Fig. 8.

Referring now to Fig. 9, the system 600 that another kind is used for optical tomography comprises wherein places specimen 604 and is roughly columnar imaging chamber 602, in system 600, the like of Fig. 7 is illustrated as having similar Reference numeral.Imaging chamber 602 can be according to arrow 606 represented such rotations.This rotation can be by computer control, the rotation platform controller 612 of 608 controls that for example use a computer.Though the LIC 406 of displayed map 7 not, it can be included in the system 600.

As described in conjunction with Fig. 7, the required a plurality of images of tomographic imaging are relevant with the position of a plurality of apparent sources that optical scanner 412 provides.In addition, imaging chamber 602 for example can rotate under the control of computer, and more apparent light source position and/or angle promptly are provided, thereby more images of specimen 604 are provided.

The advantage of cylindrical shape imaging chamber 602 is rotation and imaging algorithm is simple and fast, and can not cause picture quality generation defective.

Referring now to Figure 10, the system 700 that another kind is used for optical tomography comprises lasing light emitter 710, and it provides light to optical fiber 712.Optical fiber 712 for example can optionally move along at least two axles 718,720 by structure 716.System 700 can comprise also and be roughly columnar imaging chamber 722 that specimen 724 is placed in the imaging chamber 722.Imaging chamber 722 can be according to arrow 723 represented such rotations.This rotation can be by computer control, the rotation platform controller 744 of 740 controls that for example use a computer.In addition, the motion of structure 716 (it provides the motion of optical fiber 712 at least two axles 718,720) can be by computer control, the XY platform controller 746 of 740 controls that for example use a computer.Though the LIC 406 of displayed map 7 not, it can be included in the system 700.

Identical with said system, pass the inherent light of specimen 724 and in the specimen 724 emitted fluorescence pass selectable light filter 734 and receive by image intensifier 736 and CCD photographing unit 738.The CCD photographing unit provides numerical data 738a to computer 740, and this computer 740 provides the processing shown in the square frame 208-216 of Fig. 4 at least, and the fluorescent protein map in the specimen 724 is provided on pictorial displays 742.

Another optical fiber 714 can provide secondary light source 726 in a side opposite with optical fiber 712 of specimen 724.Secondary light source 726 can be the single white light source of White-light image that is used to provide the square frame 218 to 224 of Fig. 4.In other embodiments, being very similar to for example mode shown in Figure 1A, secondary light source 726 can be optionally moves other a plurality of apparent sources with the catoptric imaging that is provided for specimen 724 along at least two axles 730,732.

The same high-quality 3D rendering that the system 600 that uses the system 700 of Figure 10 can obtain Fig. 9 is provided.Yet the advantage of system 700 is that it can work and can realize any geometry under reflective-mode and transmission mode, for example the cylindrical shape geometry of free space (promptly do not contact and do not have matching fluid).

Referring now to Figure 10 A, wherein the like with Figure 10 is illustrated as having similar Reference numeral, can provide the 3rd light source 760 to obtain even more apparent source.In certain embodiments, the 3rd light source 760 optionally moves along at least two axles 762,764, for example not only uses secondary light source 726 that side lighting is provided but also use the 3rd light source 760 that front illumination is provided.

Side lighting and front illumination can be used to improve low-level optical signal, especially the signal collection efficiency outside the signal of being hidden by big absorber in the transmission mode that uses first light source 719.The measured value that produces from first, second and the 3rd light source 719,726,760 can be handled combine and be used for solution and be similar to above in conjunction with the described forward problem of Fig. 4 with tomography respectively.

Referring now to Figure 11, another imaging system 800 comprises lasing light emitter 802 and the single optical fiber 804 that is used to produce exciting light.Optical fiber 804 is connected to beam splitter 806, and it is not only assigned to exciting light a plurality of optical fiber 808a-808N but also assign to optical fiber 810, and each optical fiber carries exciting light.Optical fiber 808a-808N is connected to probe 812, and it can do translational motion along arrow 814,816 at least two represented axles.In a specific embodiments, optical fiber 808a-808N provide apparent source, and this apparent source is the 822 projection exciting lights of the specimen in being arranged on imaging chamber 820 (or imaging chamber) simultaneously.

Identical with said system, leave the inherent light of specimen 822 and pass selectable light filter 828 by the fluorescin emitted fluorescence in the specimen 822, pass image intensifier 830 and enter CCD photographing unit 832.CCD photographing unit 832 converts this light to numerical data 832a, and this numerical data 832a is received by computer 834.Computer 834 is for example handled to digital data by the described method 200 of Fig. 4, and can show the pictorial display of tomographic image on pictorial displays 836.

White light source 826 can produce white light, and it is from specimen 822 reflection, thereby provides by selectable light filter 828, by image intensifier 830 and enter the white light of CCD photographing unit.As described in conjunction with Fig. 4, the White-light image of specimen 822 can with the fluorescent protein map stack in the specimen 822, make tomographic image easy to understand more.

Computer 834 can also be via the position of XY platform controller 838 gated sweeps 812, according to arrow 814,816 represented like that around the axle movable scan heads so that more apparent source to be provided, make the better tomography figure that produces fluorescin.

In a specific embodiments, can scan probe 812 along arrow 814,816 at least two represented axles, and can throw light on to all optical fiber 808a-808N that are connected to probe 812 simultaneously.The advantage of this embodiment includes but not limited to tomographic imaging faster, and particularly under the situation that has the low amplitude optical signal, it causes long time of exposure.

Can make crosstalk minimization between the optical fiber 808a-808N by selecting distance between each optical fiber 808a-808N suitably, make that the path of propagating photon is not overlapping.In other embodiments, particularly for those short embodiment of time of exposure, among the lighting fiber 808a-808N is to eliminate from the noise of crosstalking between the optical fiber 808a-808N at every turn.In this embodiment, beam splitter 806 can be replaced by optical switch (for example 106 of Fig. 2).In alternative, can scan single optical fiber along arrow 814,816 represented axles.

Referring now to Figure 11 A, wherein the like with Figure 11 is illustrated as having similar Reference numeral, and the probe 812 of Figure 11 has a plurality of perforate 850a-850N, and wherein each perforate is corresponding to separately apparent source, thereby forms N apparent source.When probe 812 is mobile along axle (for example by the represented axle of arrow 814,816), form more apparent source, for example N apparent source in addition.

Method and system of the present invention can use any fluorescin, includes but not limited to DsRed and HcRed fluorescin.These special fluorescins are provided at the HONGGUANG of visible spectrum or the fluorescence of near infrared region.Because compare with other wavelength of visible light, the red light district of the visible spectrum of light has the more high efficiency deep penetration degree of depth in biological tissue, and can provide than long wavelength's the higher resolution of NIR system more, have higher-quality fluorescent protein map so these special fluorescins can cause producing.

When method and system of the present invention was used for the tumor cell of express fluorescent protein (being similar to GFP), method and system of the present invention can be used to study tumor propagation and monitoring tumor shifts formation.

Method and system of the present invention can be used for GFP expressing tumor cell and YFP expresses gene transmission and the gene therapy of virocyte with research particular patient targeted therapy.

Method and system of the present invention also can utilize the imaging pattern that uses algorithm with under the situation that does not need matching fluid to the random geometry imaging.Be used for the embodiment that light modeling of propagating and the algorithm that solves forward problem go for all said system.

Should be understood that when using visible light, compare with the traditional tomography method that uses near-infrared (NIR) light, system and method for the present invention provides higher spatial resolution.

By the exciting light that the above embodiment of the present invention provided and the emitting fluorescence of generation can be continuous wave (CW) light, intensity modulated (IM) or time resolution (TR) light or both combinations.System and method of the present invention can provide about the information as the system dynamic characteristic of time function, and final image can with the common registrations of the image that formation method obtained such as another kind such as NMR (Nuclear Magnetic Resonance)-imaging (MRI), computed tomography imaging (CT), ultrasonic or bioluminescence imaging.

This said system and method are used in conjunction with suitable normalized modified diffusion approximation, its allow have the higher absorption coefficient (for example＞0.3cm ^-1) medium in (being that it spreads) and 400nm at least in the visible wavelength range of 700nm to body in fluorescin carry out the three-dimension disclocation photographic imagery.Modified diffusion approximation does not need to use more complicated transport equation.Therefore, this correction is separated and is obtained computational efficiency.

In some above-mentioned embodiment, use non-contact tissue illumination and/or noncontact light-receiving, wherein above-mentioned apparent source and/or photo-detector and the specimen that just is being scanned separate.In other embodiments, apparent source and/or photo-detector are placed to specimen contact substantially.

By using various fluorescin, can be with said method and system applies in multiple biology and molecular process.For example, in various embodiments, fluorescin can be used for monitoring tumor growth, neoplasm metastasis formation, gene expression and therapeutic effect.In addition, this method and system can be used to provide noinvasive, whole body molecular imaging to produce the information relevant with the activity of subcellsular level with noinvasive ground.

Method and system of the present invention can provide the understanding to specific molecular and biological anomalies, this abnormal formation the basis of numerous disease, for example cancer, tumor propagation and neoplasm metastasis form.Form contrast because the height of hematochrome absorbs the fluorescence background that makes blood vessel and tumor cell, so this method and system can be used for to blood vessel imaging also.In addition, this method and system can be used for the effect at the new targeted therapy of molecular level assessment.Conversely, this can exert an influence to the selection of drug development, drug test, appropriate therapies and given patient's therapy variation again.In addition, this method and system allows the origin of study of disease in the intact microenvironment of living systems.And further, this method and system helps to test new gene transmission policy.Compare with using the current possibility traditional background science technology with effort consuming time, this formation method and system allow to gather quickly three-dimensional information.

Method and system of the present invention is being used for promoting the control of various diseases (comprising cancer, neurodegenerative disease, inflammation, infectious disease and other disease) and various biologies, immunity and the gene therapy of elimination to have extensive use.In addition, this method and system is widely used in not having scar disease detection and treatment in combination is provided with.

All reference as cite herein cited herein is incorporated into herein as a reference.

By the agency of the preferred embodiments of the present invention, now it is evident that other embodiment that can use the notion that comprises them for those of ordinary skill in the art.Therefore be to be understood that these embodiment should not be limited to the disclosed embodiments, but should be only by the spirit and scope restriction of appended claim.

Claims (43)

1, a kind of system that is used for optical tomography comprises:

Be suitable for apparent source to the specimen projection exciting light that wherein has fluorescin, wherein said exciting light enters specimen, become the inherent light in the described specimen, wherein said inherent light is suitable for from described fluorescin fluorescence excitation, and wherein said inherent light and described fluorescence spread.

2, system according to claim 1, at least one in wherein said exciting light and the described fluorescence has the wavelength in visible wavelength range.

3, system according to claim 1, wherein said fluorescence has the wavelength in visible wavelength range.

4, system according to claim 1, wherein said fluorescence has the wavelength in the red light portion of visible wavelength range.

5, system according to claim 1, wherein said fluorescence has the wavelength in near-infrared (NIR) district.

6, system according to claim 1 also comprises:

Photo-detector, be suitable for receiving the described inherent light that leaves described specimen and be suitable for receiving the described fluorescence that leaves described specimen, the described inherent light that also is suitable for receiving converts first image information to, and the described fluorescence that is suitable for receiving converts second image information to; With

Image processor, be connected to described photo-detector and be suitable for producing light propagation model, wherein said model is suitable for predicting the light propagation in dispersive medium, wherein said image processor also is suitable for making up described first image information, described second image information and described light propagation model, and is suitable for providing the image of described fluorescin.

7, system according to claim 6, wherein said image processor comprises the diffusion equation processor of using the diffusion equation with modified diffusion coefficient, this modified diffusion coefficient is relevant with in described inherent light and the described fluorescence at least one.

8, system according to claim 6, wherein said photo-detector move selectively with on respect to a plurality of light paths of specimen, receive described at light and fluorescence.

9, system according to claim 6 also comprises optical scanner, will offer described photo-detector at light and fluorescence in described on respect to a plurality of light paths of specimen.

10, system according to claim 1, wherein said apparent source comprises the light guiding device, to move described apparent source selectively and then guide described exciting light to described specimen on a plurality of light paths.

11, system according to claim 10, wherein said smooth guiding device comprises optical switch, to move described apparent source selectively and then to provide described a plurality of light path to described specimen.

12, system according to claim 10, wherein said smooth guiding device comprises movably reflecting mirror, to move described apparent source selectively and then to provide described a plurality of light path to described specimen.

13, system according to claim 10, wherein said smooth guiding device is suitable for moving described apparent source with translation mode selectively along at least one apparent source translation shaft.

14, system according to claim 1, wherein said specimen can move selectively to provide described exciting light on a plurality of light paths with respect to described specimen.

15, system according to claim 14, wherein specimen can rotate selectively around the specimen rotating shaft.

16, system according to claim 14, wherein specimen can be done translational motion selectively along at least one specimen translation shaft.

17, system according to claim 14, wherein specimen can rotate selectively around the specimen rotating shaft, and described specimen also can optionally be done translational motion along at least one specimen translation shaft.

18, system according to claim 1, wherein said apparent source comprises the light guiding device, moving described apparent source selectively and then guide described exciting light to described specimen on a plurality of light paths, and described specimen can move selectively to provide described exciting light on a plurality of light paths with respect to described specimen.

19, system according to claim 1, wherein said inherent light passes described specimen as transillumination.

20, system according to claim 1, wherein said inherent light reflects from described specimen as reflected light.

21, a kind of method of optical tomography comprises:

Use is suitable for producing exciting light to the apparent source of the specimen projection exciting light that wherein has fluorescin, wherein said exciting light enters described specimen, become the inherent light in the described specimen, wherein said inherent light is suitable for from described fluorescin fluorescence excitation, and wherein said inherent light and described fluorescence spread.

22, method according to claim 21, at least one in wherein said exciting light and the described fluorescence has the wavelength in visible wavelength range.

23, method according to claim 21, wherein said fluorescence has the wavelength in visible wavelength range.

24, method according to claim 21, wherein said fluorescence has the wavelength in the red light portion of visible wavelength range.

25, system according to claim 21, wherein said fluorescence has the wavelength in near-infrared (NIR) district.

26, method according to claim 21 also comprises:

The described inherent light of described specimen is left in reception;

The described fluorescence of described specimen is left in reception;

Convert the described inherent light that receives to first image information;

Convert the described fluorescence that receives to second image information;

Generation is suitable for predicting the model that the light in the dispersive medium is propagated; With

Make up described first image information, described second image information and described model so that the image of described fluorescin to be provided.

27, method according to claim 26, wherein receive described inherent light and receive described fluorescence and comprise that use can receive described inherent light and receive described fluorescence by selective mobile photo-detector, this photo-detector be suitable on respect to a plurality of light paths of specimen, receiving described at light and fluorescence.

28, method according to claim 26 wherein produces described model according to the separating of diffusion equation with modified diffusion coefficient, and this modified diffusion coefficient is relevant with in described inherent light and the described fluorescence at least one.

29, method according to claim 21 also includes and optionally moves described apparent source to guide described exciting light to described specimen on a plurality of light paths.

30, method according to claim 29, wherein said apparent source comprises optical switch, to move described apparent source selectively.

31, method according to claim 29, wherein said apparent source comprise that the alternative reflecting mirror that moves is to move described apparent source selectively.

32, method according to claim 29 wherein saidly optionally moves described apparent source and comprises along at least one apparent source translation shaft and move described apparent source with translation mode selectively.

33, method according to claim 21 also includes and optionally moves described specimen to provide described exciting light on a plurality of light paths with respect to specimen.

34, method according to claim 33, wherein said mobile selectively described specimen comprises around the specimen rotating shaft rotates mobile described specimen selectively.

35, method according to claim 33 wherein saidly moves described specimen selectively and comprises along at least one specimen translation shaft and move described specimen with translation mode selectively.

36, method according to claim 33, wherein said optionally mobile described specimen comprises:

Rotate mobile described specimen selectively around the specimen rotating shaft; With

Move described specimen with translation mode selectively along at least one specimen translation shaft.

37, method according to claim 21 also comprises:

Move described apparent source selectively on a plurality of light paths, to guide described exciting light to described specimen; With

Move described specimen selectively on other a plurality of light paths, to provide described exciting light with respect to described specimen.

38, method according to claim 21, wherein said inherent light passes described specimen as transillumination.

39, method according to claim 21, wherein said inherent light reflects from described specimen as reflected light.

40, a kind of system that is used for optical tomography comprises:

At least one can selective mobile parts, guide to specimen with mobile apparent source selectively and then with a plurality of light paths.

41, according to the described system of claim 40, wherein said at least one alternative reflecting mirror that moves that can selective mobile parts comprise.

42, according to the described system of claim 40, wherein said can selective mobile parts comprising can selective mobile structure.

43,, also comprise the optical fiber that is connected to described can be selective mobile structure according to the described system of claim 42.

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