CN103405215A - Method for measuring multiple parameters of retina of living mouse - Google Patents
Method for measuring multiple parameters of retina of living mouse Download PDFInfo
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- CN103405215A CN103405215A CN2013103578998A CN201310357899A CN103405215A CN 103405215 A CN103405215 A CN 103405215A CN 2013103578998 A CN2013103578998 A CN 2013103578998A CN 201310357899 A CN201310357899 A CN 201310357899A CN 103405215 A CN103405215 A CN 103405215A
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Abstract
The invention discloses a method for measuring multiple parameters of retina of a living mouse. The method comprises the following steps: fixing a mouse, which is successfully anesthetized, on a three-dimensional experiment table; assembling a plano-concave lens, a front lens and an optical coherence tomography (OCT) instrument; adjusting the three-dimensional experiment table to ensure that the eyeballs of the mouse are positioned right ahead the front lens, and that a clear retina scanning eye fundus image appears on a display screen of the OCT instrument; selecting a high-definition one-line scanning mode to obtain a cross-sectional image of the total retina of the mouse, and measuring the thickness of each layer on the retina of the mouse; selecting a retinal volume mode, aligning the center of a cross scanning line with the optic disk of the mouse to measure the average thickness and volume of nine subareas of the retina of the mouse, and reconstructing the form of the retina of the mouse in a three-dimensional mode; and finally, selecting an optic disk scanning mode to acquire continuous thickness data of a nerve fiber layer of the retina. The method has the characteristics of high simplicity and feasibility, high repeatability, accurate result and zero external environment interference and is suitable for popularization and application.
Description
Technical field
The present invention relates to Small Rodents living animal retina field of measuring technique, specifically a kind of method of carrying out intravital mouse retina measuring multiple parameters with optical coherence tomography.
Background technology
Mice, as a kind of and the similar laboratory animal of human gene's type height, is widely used in medical research, so same in the ophthalmology basic research.Therefore, a kind of Mouse Retina measuring technique of mature and reliable has great significance to the Research of Animal Model for Study of ophthalmic diseases.The retina of mice is measured mainly and is observed by after the execution mice, extracing the capable Histological section of eyeball at present.Yet eyeball of mouse is too little, the integrity that guarantees tissue in slicing processes is a difficulty job that needs the protracted experience accumulation; Secondly, the retinopathy of mice is very trickle, the situation of omitting while often section occurring; Finally, for the research of longitudinal 2 observation in time needs to sacrifice a large amount of mices, not only wasted funds but also be unfavorable for the requirement of animal protection.Therefore mice live body retina measuring method has the Practical significance of height.
Optical coherence tomography (Optical Coherence Tomography, OCT), it is a kind of diagnostic techniques that is applied in recent years clinical ophthalmology, have the advantages such as not damaged, cell grade high-resolution, fast imaging and precise quantitative measurement, use OCT can solve well the problem of mice live body retina research.
Summary of the invention
The objective of the invention is the problem existed for prior art, provide a kind of simple, measurement parameter is many, measurement parameter is wide and measure the conveniently method of intravital mouse retina measuring multiple parameters.
The objective of the invention is to solve by the following technical programs:
A kind of method of intravital mouse retina measuring multiple parameters is characterized in that the step of the method is as follows:
(a) mice after anaesthetizing successfully is fixed on the three-dimensional experiment platform;
(b) after the loose large mice pupil of mydriatic, form with coverslip and concavees lens the planoconcave lens that is positioned at the eyeball of mouse front surface;
(c) on the camera lens of OCT instrument, supplementary lens is installed, is adjusted the three dimensional taest platform and make eyeball of mouse be positioned at the dead ahead of supplementary lens, and by far away and near until scanning retina eye fundus image comparatively clearly on the display screen of OCT instrument, occurs;
(d) select high definition one-line scanning pattern, by scanning line center aligned mice optic disc place, obtain the panretinal faultage image of mice and record the thickness of each layer on Mouse Retina;
(e) then select retina volume pattern, mice optic disc place is aimed in the center of cross scanning line, record and take the optic disc center and be average thickness and the volume of the Mouse Retina in the center of circle, and the form of three-dimensional reconstruction Mouse Retina;
(f) finally select the optic disc scan pattern, obtain and take the continuous thickness data of optic disc center as the retinal nerve fibre layer of certain area in the center of circle.
During high definition one-line scanning pattern in described step (d) on Mouse Retina the thickness of each layer by the survey tool on the OCT instrument, record.
During high definition one-line scanning pattern in described step (d), the scale conversion equation that the OCT instrument is imaged on Mouse Retina horizontal plane direction is: actual value=0.224*OCT measured value+72.61, and wherein the unit of actual value, OCT measured value and constant 72.61 is μ m; There is not amplification in the measurement of OCT instrument on retina vertical meridian direction.
Retina volume pattern in described step (e) is selected macula retinae volume scan pattern.
The retina scanning area that retina volume pattern in described step (e) obtains is determined according to the scale conversion equation that sweep diameter and the OCT instrument of OCT instrument is imaged on Mouse Retina horizontal plane direction.
Retina scanning area in described step (e) is for take the optic disc center in the center of circle, diameter are respectively the concentric circular of central area, 3 ㎜ and 6 ㎜ (the corresponding diameter on Mouse Retina as 0.297,0.745 and 1.417mm) of 1 ㎜, up and down each four quadrants Mouse Retina average thickness and volume of totally 9 sub regions.
The layer of fibers of optic nerve of retina area that optic disc scan pattern in described step (f) obtains is determined according to the scale conversion equation that sweep diameter and the OCT instrument of OCT instrument is imaged on Mouse Retina horizontal plane direction.
The present invention has the following advantages compared to existing technology:
The present invention adopts the optical instruments such as clinical OCT instrument to measure the intravital mouse retina, and resolution is high, and the distinguishable Mouse Retina tomography of measuring is more than ten layers, substantially corresponding with Histological section viewed layer of structure; Measurement effect can replace and even surmount tradition section measuring method, is conducive to the progression of longitudinal 2 observation Mouse Retina pathological changes, has reduced simultaneously the quantity of required laboratory animal.
The present invention is easy to location and quantitative check, this mice live body retina measuring method can obtain scan laser eye fundus image and OCT tomoscan image simultaneously, synchronous optical fundus and OCT image make research worker can obtain the faultage image of Mouse Retina any part, only need to by the position of mouse drag scanning line, the angle of definition laser scanning line, get final product simply.
The present invention can to Mouse Retina fast, measuring multiple parameters in batches, as retina volume pattern, pass through single pass (approximately 1 second), can obtain mice centered by optic disc, diameter is respectively in the concentric circular of central area, 3 ㎜ and 6 ㎜ of 1 ㎜, each four quadrants average thickness of totally 9 sub regions up and down, obtain amphiblestroid form and carry out three-dimensional reconstruction (or automatic measurement altogether) at 512 * 128 at 200 * 200; The optic disc scan pattern can also go out the indexs such as Mouse Retina nerve fibre layer average thickness by automatic measurement & calculation simultaneously, and these fast, high flux is measured is all that traditional Histological section measures incomparable.
Method of the present invention is simple, favorable repeatability, and result is accurate, is not subjected to external environmental interference, can obtain Mouse Retina faultage image clearly and measure for mice live body retina, effectively reduces the quantity of experiment mice, saves reasearch funds; Retinal structure form to viviperception ophthalmic diseases animal model plays an important role, suitable promoting the use of.
The accompanying drawing explanation
The apparatus structure schematic diagram that accompanying drawing 1 adopts for inspection method of the present invention;
Measurement result gained image when accompanying drawing 2 is high definition one-line scanning pattern of the present invention on retina horizontal plane direction;
Accompanying drawing 3 is the Histological section gained image corresponding with accompanying drawing 2;
The loose point of OCT measured value when accompanying drawing 4 is high definition one-line scanning pattern of the present invention on retina horizontal plane direction and paving sheet measured value and matched curve figure;
Accompanying drawing 5 is the Mouse Retina faultage image of high definition one-line scanning pattern gained of the present invention;
The OCT measured value that when accompanying drawing 6 was high definition one-line scanning pattern of the present invention, thickness measure recorded and the loose point of section measured value and matched curve figure;
Accompanying drawing 7 is Mouse Retina nine sub regions average thicknesss and general area thickness and the volumetric values that retina volume pattern of the present invention is measured gained, wherein Fig. 7 A means mice optical fundus OCT scintigram, Fig. 7 B means the retina average thickness values of nine sub regions centered by the mice optic disc, and Fig. 7 C means subarea, center thickness, the retina volume in scanning area and the average thickness (ILM-RPE distance) of Mouse Retina;
Wherein: the 1:OCT instrument; 2: supplementary lens; 3: planoconcave lens; 4: coverslip; 5: concavees lens; 6: the three dimensional taest platform; 7: eyeball of mouse.
GCL: ganglion cell layer of retina; IPL: inner plexiform layer of retina; INL: inner nuclear layer of retina; OPL: outer plexiform layer of retina; ONL: outer nuclear layer; IS-OS: retinal light injury photoreceptor is inside and outside to be connected; RPE: layer of retina,pigment epithelium; ILM-RPE: inner limiting membrane-layer of retina,pigment epithelium; RNFL: retinal nerve fibre layer.
The specific embodiment
The present invention is further illustrated below in conjunction with accompanying drawing and embodiment.
As shown in Figure 1: a kind of method of intravital mouse retina measuring multiple parameters, the step of the method is as follows: (a) adopt anesthetis that the mixed solution of ketamine and xylazine makes according to 0.2-0.5ml/20g body weight intraperitoneal injection of anesthesia mice, mice is fixed on three-dimensional experiment platform 6 after anaesthetizing successfully; (b) after the loose large mice pupil of the mydriatic that then adopts the Tropicamide and Phenylephrine after normal saline dilution to make, concavees lens 5 are covered to eyeball of mouse 7 surfaces, get coverslip 4 and be placed on concavees lens 5 and make coverslip 4 and concavees lens 5 form the planoconcave lens 3 that is positioned at eyeball of mouse 7 front surfaces; (c) supplementary lens 2 is installed on the camera lens of OCT instrument 1, is adjusted three dimensional taest platform 6 and make eyeball of mouse 7 be positioned at the dead ahead of supplementary lens 2, and by far away and near until scanning retina eye fundus image comparatively clearly on the display screen of OCT instrument 1, occurs; (d) select high definition one-line scanning pattern, by scanning line center aligned mice optic disc place, obtain the panretinal faultage image of mice, and by the survey tool on OCT instrument 1, record the thickness of each layer on Mouse Retina; (e) then select retina volume pattern, mice optic disc place is aimed in the center of cross scanning line, record average thickness and the volume of Mouse Retina 9 sub regions, and the form of three-dimensional reconstruction Mouse Retina; (f) finally select the optic disc scan pattern, can obtain the continuous thickness data of retinal nerve fibre layer.In above-mentioned steps, during high definition one-line scanning pattern, the measured value of OCT instrument 1 and optical microscope measuring value height correlation, there is not amplification in the measurement that OCT instrument 1 is imaged on Mouse Retina vertical meridian direction; And OCT instrument 1 is imaged in the measurement of Mouse Retina horizontal plane direction and has significant the amplification, there is fixing conversion in this amplification in measuring system of the present invention, the scale conversion equation that OCT instrument 1 is imaged on Mouse Retina horizontal plane direction is: actual value=0.224*OCT apparatus measuring value+72.61, wherein the unit of actual value, OCT apparatus measuring value and constant 72.61 is μ m.The continuous thickness data of the Mouse Retina nerve fibre layer obtained when the average thickness in 9 zones of Mouse Retina that obtain during retina volume pattern in addition and volume and optic disc scan pattern, the actual size of optic disc is determined according to the scale conversion equation that sweep diameter and the OCT instrument 1 of OCT instrument 1 is imaged on Mouse Retina horizontal plane direction; Retina scanning area during retina volume pattern refers to and take the optic disc center in the center of circle, diameter are respectively the concentric circular of central area, 3 ㎜ and 6 ㎜ of 1 ㎜ in addition, up and down each four quadrants Mouse Retina average thickness and volume of totally 9 sub regions; With respect to diameter on optic disc, be respectively the concentric circular of central area, 3 ㎜ and 6 ㎜ of 1 ㎜, the corresponding diameter on Mouse Retina is respectively 0.297,0.745 and 1.417mm.
For after the optical imagery instruments such as checking interpolation supplementary lens, OCT instrument 1 imaging is erect image or inverted image in addition, and before checking, we,, to mice row Fundus laser, form a laser spot above its optic disc temporo; Use subsequently 1 pair of Mouse Retina imaging of OCT instrument, viewed laser spot position still, above the optic disc temporo, draws thus: by add after supplementary lens 2 optical instruments such as grade become image to remain erect image.
Embodiment mono-
Select C57BL mouse, before checking, notice checking whether the mice refracting media is transparent; Adopt simultaneously clinical modal OCT instrument 1:Zeiss Cirrus HD-OCT 4000 or 400.Get anesthetis that the mixed solution of ketamine and xylazine makes according to 0.2ml/20g body weight intraperitoneal injection of anesthesia mice, solidifying to mice row Fundus laser light after anaesthetizing successfully, at the Mouse Retina random site, form a laser spot (diameter 50 μ m, puncture the Bruch film).Mice is fixed on three-dimensional experiment platform 6 subsequently; Then after the loose large mice pupil of the mydriatic that adopts the Tropicamide and Phenylephrine after normal saline dilution to make, medical hyaluronic acid viscoelastic agent (as concavees lens 5) is covered to eyeball of mouse 7 surfaces, get coverslip 4 and be placed on the hyaluronic acid viscoelastic agent and form the planoconcave lens 3 that is positioned at eyeball of mouse 7 front surfaces; Before the one diopter Volk Superfield NC supplementary lens 2 that is 90D is fixedly installed on to the camera lens of OCT instrument 1, adjusts three dimensional taest platform 6 and make eyeball of mouse 7 be positioned at the dead ahead 5cm left and right of supplementary lens 2, and by far away and near until eye fundus image occurs; Adjust the position of three dimensional taest platform 6 until scanning retina eye fundus image comparatively clearly on the display screen of OCT instrument 1, occurs.Select the high definition one-line scanning pattern of OCT instrument 1 to the Mouse Retina imaging, adjust the scanning line angle and be located on the line of optic disc and laser spot, record optic disc under the imaging of OCT instrument between laser spot apart from (as shown in Figure 2).Put to death mice, get the capable whole retina paving sheet of its eyeball, under optical microscope, measure optic disc to the distance (as shown in Figure 3) between laser spot, totally 8 mices, every each eyeball of mice has a laser spot.
Adopt the SPSS19.0 statistical software, OCT instrument 1 data measured and retina paving sheet the data obtained are carried out to correlation and regression analysis, the loose point of OCT measured value during high definition one-line scanning pattern and section measured value and matched curve figure as shown in Figure 4, square R of regression analysis neutral line correlation coefficient
2=0.887.Finally obtain the scale conversion equation on OCT instrument 1 imaging Mouse Retina horizontal plane direction (x axle, y axle): the actual value=0.224*OCT value+72.61(wherein unit of actual value, OCT apparatus measuring value and constant 72.61 is μ m).Paving sheet measurement actual value and the regression analysis of OCT measured value are as shown in the table:
In said process, due to the physiological characteristics of mice, the eye birth prevalence is high, and it is muddy that refracting media easily is subjected to medicine, pressure, hydropenia and environment etc. to affect generation very soon, so before experiment, need to check the eye situation, avoid selecting the poor animal of refractive media in order to avoid affect result; During experiment, need careful operation, cover in case after anaesthetizing successfully, namely adopt the viscoelastic agent eye dripping to add with coverslip 4, namely can form a planoconcave lens 3 and participate in optical imagery, can reduce again the evaporation of mice eye moisture, prevent its refractive media muddiness.
Embodiment bis-
Select C57BL mouse, before checking, notice checking whether the mice refracting media is transparent; Adopt simultaneously clinical modal OCT instrument 1:Zeiss Cirrus HD-OCT 4000 or 400.Get anesthetis that the mixed solution of ketamine and xylazine makes according to 0.2ml/20g body weight intraperitoneal injection of anesthesia mice, mice is fixed on three-dimensional experiment platform 6 after anaesthetizing successfully; Then after the loose large mice pupil of the mydriatic that adopts the Tropicamide and Phenylephrine after normal saline dilution to make, medical hyaluronic acid viscoelastic agent (as concavees lens 5) is covered to eyeball of mouse 7 surfaces, get coverslip 4 and be placed on the hyaluronic acid viscoelastic agent and form the planoconcave lens 3 that is positioned at eyeball of mouse 7 front surfaces; Before the one diopter Volk Superfield NC supplementary lens 2 that is 90D is fixedly installed on to the camera lens of OCT instrument 1, adjusts three dimensional taest platform 6 and make eyeball of mouse 7 be positioned at the dead ahead 5cm left and right of supplementary lens 2, and by far away and near until eye fundus image occurs; Adjust the position of three dimensional taest platform 6 until scanning retina eye fundus image comparatively clearly on the display screen of OCT instrument 1, occurs.Select high definition one-line scanning pattern, by scanning line center aligned mice optic disc place, according to the difference of required look-out station, adjust the angle of scanning line, in order to obtain the panretinal faultage image of mice (as shown in Figure 5).With the subsidiary survey tool of OCT instrument, measure the holostrome thickness of Mouse Retina different parts and the thickness of this position inner nuclear layer, outer nuclear layer.
Put to death the mice of OCT instrument data measured, get the capable Histological section of its eyeball.Above in Histological section, choosing respectively, below, endocanthion, outer canthus four direction be apart from position measurement retina holostrome thickness and the inside and outside stratum nucleare thickness of optic nerve center 200 μ m, 300 μ m, 400 μ m and 500 μ m, according to the scale conversion equation of trying to achieve in embodiment mono-, on the OCT instrument, measure retina holostrome thickness and inside and outside stratum nucleare thickness with the corresponding position of Histological section.The loose point of the OCT measured value that during high definition one-line scanning pattern, thickness measure records and section measured value and matched curve figure are as shown in Figure 6, OCT data measured and section data measured are compared, adopt the SPSS19.0 statistical software to carry out correlation and regression analysis, analysis result is as shown in the table:
By statistical analysis, can obtain: the ratio of section value and OCT value is approximately equal to 1.Due to eyeball of mouse, need in the section statining process through the step such as fixing of dewatering, shrinkage to a certain degree can occur in its retina after dehydration, and certain measurement error in addition, so the measured value of cutting into slices is more smaller than OCT apparatus measuring value.Operation principle by the OCT instrument can be known, what the OCT instrument was measured is the luminous reflectance signal of laser on the retina different levels, according to the time difference of luminous reflectance signal, carry out detect thickness, the imaging of OCT image on retina z axle can not zoom in or out because additional optical components causes in theory, and this is also consistent with our measurement result.Can think: the OCT instrument is imaged on Mouse Retina z axle and does not have the amplification phenomenon, and the measurement result of optical microscope gained is basically identical when the imaging of OCT instrument and Histological section.
In said process, due to the physiological characteristics of mice, the eye birth prevalence is high, and it is muddy that refracting media easily is subjected to medicine, pressure, hydropenia and environment etc. to affect generation very soon, so before experiment, need to check the eye situation, avoid selecting the poor animal of refractive media in order to avoid affect result; During experiment, need careful operation, cover in case after anaesthetizing successfully, namely adopt the viscoelastic agent eye dripping to add with coverslip 4, namely can form a planoconcave lens 3 and participate in optical imagery, can reduce again the evaporation of mice eye moisture, prevent its refractive media muddiness.
Embodiment tri-
Select C57BL mouse, before checking, notice checking whether the mice refracting media is transparent; Adopt simultaneously clinical modal OCT instrument 1:Zeiss Cirrus HD-OCT 4000 or 400.Get anesthetis that the mixed solution of ketamine and xylazine makes according to 0.2ml/20g body weight intraperitoneal injection of anesthesia mice, mice is fixed on three-dimensional experiment platform 6 after anaesthetizing successfully; Then after the loose large mice pupil of the mydriatic that adopts the Tropicamide and Phenylephrine after normal saline dilution to make, medical hyaluronic acid viscoelastic agent (as concavees lens 5) is covered to eyeball of mouse 7 surfaces, get coverslip 4 and be placed on the hyaluronic acid viscoelastic agent and form the planoconcave lens 3 that is positioned at eyeball of mouse 7 front surfaces; Before the one diopter Volk Superfield NC supplementary lens 2 that is 90D is fixedly installed on to the camera lens of OCT instrument 1, adjusts three dimensional taest platform 6 and make eyeball of mouse 7 be positioned at the dead ahead 5cm left and right of supplementary lens 2, and by far away and near until eye fundus image occurs; Adjust the position of three dimensional taest platform 6 until scanning retina eye fundus image comparatively clearly on the display screen of OCT instrument 1, occurs.Select retina volume pattern, mice optic disc centre is aimed in the center of cross scanning line, by this pattern, draw and refer to take that the optic disc center is in the center of circle, diameter are respectively the concentric circular of central area, 3 ㎜ and 6 ㎜ of 1 ㎜, each four quadrants Mouse Retina average thickness and volume of totally 9 sub regions up and down, with respect to diameter on optic disc, be respectively the concentric circular of central area, 3 ㎜ and 6 ㎜ of 1 ㎜, the corresponding diameter on Mouse Retina is respectively 0.297,0.745 and 1.417mm; And can the panretinal form of three-dimensional reconstruction mice (as shown in Figure 7).In the explanation of OCT instrument, the sweep diameter of this scan pattern is 6mm, amplifies to some extent after additional optical lens, and in substitution example 1, the scale conversion equation of gained is as can be known, and the sweep diameter on actual Mouse Retina is 1.417mm.
Embodiment tetra-
Select C57BL mouse, before checking, notice checking whether the mice refracting media is transparent; Adopt simultaneously clinical modal OCT instrument 1:Zeiss Cirrus HD-OCT 4000 or 400.Get anesthetis that the mixed solution of ketamine and xylazine makes according to 0.2ml/20g body weight intraperitoneal injection of anesthesia mice, mice is fixed on three-dimensional experiment platform 6 after anaesthetizing successfully; Then after the loose large mice pupil of the mydriatic that adopts the Tropicamide and Phenylephrine after normal saline dilution to make, medical hyaluronic acid viscoelastic agent (as concavees lens 5) is covered to eyeball of mouse 7 surfaces, get coverslip 4 and be placed on the hyaluronic acid viscoelastic agent and form the planoconcave lens 3 that is positioned at eyeball of mouse 7 front surfaces; Before the one diopter Volk Superfield NC supplementary lens 2 that is 90D is fixedly installed on to the camera lens of OCT instrument 1, adjusts three dimensional taest platform 6 and make eyeball of mouse 7 be positioned at the dead ahead 5cm left and right of supplementary lens 2, and by far away and near until eye fundus image occurs; Adjust the position of three dimensional taest platform 6 until scanning retina eye fundus image comparatively clearly on the display screen of OCT instrument 1, occurs.Select optic disc volume scan pattern, the analytical tool that OCT instrument 1 carries can obtain take retinal nerve fibre layer (the Retinal Nerve Fiber Layer of mice optic disc center on the 0.848mm diameter line in the center of circle, RNFL) thickness scintigram, and show RNFL varied in thickness (as shown in Figure 8) with 12 hour zones.The circle diameter that in 1 explanation of OCT instrument, this scan pattern RNFL measures is 3.46mm, amplifies to some extent after additional optical lens, and in substitution example 1, the scale conversion equation of gained is as can be known, and it is 0.848mm that actual Mouse Retina RNFL measures diameter.
By the measurement of above embodiment to Mouse Retina, OCT instrument 1 is imaged on upper existence of Mouse Retina horizontal plane direction (x axle, y axle) and amplifies phenomenon, and does not have the amplification phenomenon at the z axle.This mice live body retina measuring method can replace the application of Histological section in Mouse Retina is measured.In addition, some process of measurement instruments that OCT instrument 1 carries all can be used for the somatometry of mice, such as the average thickness that can record Mouse Retina under retina volume pattern, mice RNFL thickness etc., these data are all that conventional organization section can't accurately obtain, and can run away with result by this mice live body retina measuring method.Should be noted, OCT instrument 1 optical instrument such as grade exists and amplifies phenomenon Mouse Retina plane (x axle, y axle), need obtain correspondence position by conversion when measuring.
Method of the present invention is simple, favorable repeatability, and result is accurate, is not subjected to external environmental interference, can obtain Mouse Retina faultage image clearly and measure for mice live body retina, effectively reduces the quantity of experiment mice, saves reasearch funds; Retinal structure form to viviperception ophthalmic diseases animal model plays an important role, suitable promoting the use of.
Above embodiment only, for explanation technological thought of the present invention, can not limit protection scope of the present invention with this, every technological thought proposed according to the present invention, and any change of doing on the technical scheme basis, within all falling into protection domain of the present invention; The technology that the present invention does not relate to all can be realized by prior art.
Claims (7)
1. the method for an intravital mouse retina measuring multiple parameters is characterized in that the step of the method is as follows:
(a) mice after anaesthetizing successfully is fixed on the three-dimensional experiment platform;
(b) after the loose large mice pupil of mydriatic, form with coverslip and concavees lens the planoconcave lens that is positioned at the eyeball of mouse front surface;
(c) on the camera lens of OCT instrument, supplementary lens is installed, is adjusted the three dimensional taest platform and make eyeball of mouse be positioned at the dead ahead of supplementary lens, and by far away and near until scanning retina eye fundus image comparatively clearly on the display screen of OCT instrument, occurs;
(d) select high definition one-line scanning pattern, by scanning line center aligned mice optic disc place, obtain the panretinal faultage image of mice and record the thickness of each layer on Mouse Retina;
(e) then select retina volume pattern, mice optic disc place is aimed in the center of cross scanning line, record and take the optic disc center and be average thickness and the volume of the Mouse Retina in the center of circle, and the form of three-dimensional reconstruction Mouse Retina;
(f) finally select the optic disc scan pattern, obtain and take the continuous thickness data of optic disc center as the retinal nerve fibre layer of certain area in the center of circle.
2. the method for intravital mouse retina measuring multiple parameters according to claim 1, while it is characterized in that the high definition one-line scanning pattern in described step (d) on Mouse Retina the thickness of each layer by the survey tool on the OCT instrument, record.
3. the method for intravital mouse retina measuring multiple parameters according to claim 1, while it is characterized in that the high definition one-line scanning pattern in described step (d), the scale conversion equation that the OCT instrument is imaged on Mouse Retina horizontal plane direction is: actual value=0.224*OCT measured value+72.61, wherein the unit of actual value, OCT measured value and constant 72.61 is μ m.
4. the method for intravital mouse retina measuring multiple parameters according to claim 1, is characterized in that the retina volume pattern in described step (e) is selected macula retinae volume scan pattern.
5. the method for intravital mouse retina measuring multiple parameters according to claim 3, is characterized in that retina scanning area that the retina volume pattern in described step (e) obtains determines according to the scale conversion equation that sweep diameter and the OCT instrument of OCT instrument is imaged on Mouse Retina horizontal plane direction.
6. the method for intravital mouse retina measuring multiple parameters according to claim 1 or 5, it is characterized in that retina scanning area in described step (e) is for take the optic disc center in the center of circle, diameter are respectively the concentric circular of central area, 3 ㎜ and 6 ㎜ of 1 ㎜, up and down each four quadrants Mouse Retina average thickness and volume of totally 9 sub regions.
7. the method for intravital mouse retina measuring multiple parameters according to claim 3, is characterized in that layer of fibers of optic nerve of retina area that the optic disc scan pattern in described step (f) obtains determines according to the scale conversion equation that sweep diameter and the OCT instrument of OCT instrument is imaged on Mouse Retina horizontal plane direction.
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