CN213030631U

CN213030631U - A standard template for fluorescence calibration

Info

Publication number: CN213030631U
Application number: CN202021590238.1U
Authority: CN
Inventors: 邵鹏飞; 陈伟; 刘鹏; 马小鹏
Original assignee: University of Science and Technology of China USTC
Current assignee: University of Science and Technology of China USTC
Priority date: 2020-08-04
Filing date: 2020-08-04
Publication date: 2021-04-23
Anticipated expiration: 2030-08-04

Abstract

The utility model provides a standard template for fluorescence calibration, comprising: a base, on which M*N filter paper sheets are arranged in an array, where M is the number of rows and N is the number of columns; the base serves as a standard template The carrier of the filter paper is black frosted cardboard, which is used to fix the filter paper; the filter paper is used as a fluorescent powder carrier, and the fluorescent powder is attached to it; the substrate and the filter paper are fixed by double-sided adhesive bonding; Each of the filter paper sheets is obtained by infiltrating fluorescent powder solutions of different concentrations and then drying in the shade; the M*N filter paper sheets include L reference paper sheets that have not been infiltrated with fluorescent powder solutions. The utility model can basically eliminate the problem of excitation light reflection in most fluorescent imaging devices; adding a paper piece of the same shape that is not contaminated with any fluorescent agent as a reference point can eliminate most of the background fluorescent noise and improve the accuracy of calibration; the template can An objective lateral comparison of the detection sensitivity of different fluorescence imaging instruments can also be used in the instrument development process.

Description

Standard template for fluorescence calibration

Technical Field

The utility model belongs to fluorescence detection and biological medical treatment are field alternately, relate to a calibration equipment of fluorescence imaging device and tissue autofluorescence, can be used to the demarcation of imaging instrument based on near-infrared or as the imitative body of autofluorescence tissue.

Background

Fluorescence imaging devices are used more and more widely in the medical field, and in particular some non-invasive fluorescence devices, such as near infrared fluorescence systems used for imaging lymphatic vessels and blood vessels, have the unique advantage of causing less harm to patients. Some newer fluorescent devices, such as FLUOBEAM series imaging devices of FLUOPICS, PDE II devices of Hamamatsu, etc., can be used for parathyroid gland confirmation, improving the situation of simply relying on the experience of surgeons in the traditional way, and some clinical experiments also prove that the new devices have effective auxiliary lifting effects for detecting and confirming parathyroid glands.

However, despite the availability of numerous fluorescence imaging systems on the market, there is still a lack of standards in the laboratory or clinically to assess the fluorescence imaging capabilities of these instruments. And due to different parameters such as exposure time, imaging device sensitivity, image processing algorithm and the like, numerical values obtained by different instruments cannot be directly compared, so that the transverse comparison of the instrument detection capabilities of different manufacturers is difficult. In addition, the common fluorescer solution sample cannot be stored for a long time under the conventional environment, and is not suitable for being used as a standard sample for calibrating the instrument. Therefore, a relatively objective, standard, and stable device for evaluating the detection sensitivity of different fluorescence imaging devices for a long time is in need.

In addition, for the developers of new fluorescence detection devices, the autofluorescence of some tissues has no standard control system, and the fluorescence detection capability of the instrument cannot be separated from the fluorescence characteristics of biological tissues, so that the fluorescence detection capability of the instrument is difficult to evaluate.

SUMMERY OF THE UTILITY MODEL

In order to solve the technical problem, the utility model provides a standard template for fluorescence is markd provides feasible scheme for the solution of these problems, the utility model discloses specifically can be applied to fluorescence imaging equipment, for example based on parathyroid autofluorescence's check out test set, fluorescence navigation endoscope etc. are to the horizontal comparison of fluorescence detection's sensitivity. And can also be used as a substitute for autofluorescent tissue for testing the instrument's ability to detect tissue fluorescence.

The technical scheme of the utility model as follows: a standard template for fluorescence calibration, comprising:

the device comprises a substrate, wherein M × N filter paper sheets are arranged on the substrate in an array mode, M is the number of rows, and N is the number of columns;

the substrate is used as a carrier of a standard template, and a black frosted paperboard is used for fixing the filter paper sheet;

the filter paper sheet is used as a fluorescent powder carrier, and fluorescent powder is attached to the filter paper sheet; the substrate and the filter paper sheet are fixed by adopting a double-sided adhesive tape bonding mode;

each filter paper sheet is obtained by drying in the shade after being soaked with fluorescent powder solutions with different concentrations;

the M x N filter paper sheets comprise L reference paper sheets which are not soaked by the fluorescent powder solution.

Furthermore, M × N filter paper sheets are arranged in the array, where M is 2 and N is 5, that is, 2 rows and 5 columns of filter paper sheets include 2 reference paper sheets that are not soaked with the fluorescent powder solution.

Furthermore, M x N-L filter paper sheets in the M x N filter paper sheets are soaked in fluorescent reagent solutions with different concentration gradients, and are taken out and dried in the shade after a preset time, so that the filter paper sheets with the fluorescent powder are formed.

Furthermore, the filter paper sheets are consistent in shape and are round paper sheets, and the black frosted paper board of the substrate is rectangular.

Further, the adopted fluorescent powder is indocyanine green ICG powder.

Has the advantages that:

the utility model designs a standard fluorescence template can be used to the contrast to different fluorescence imaging instrument, also can be used as the imitative body of autofluorescence tissue for the detectability of inspection instrument to fluorescence. This design has the following advantages:

firstly, black abrasive paper is used as a substrate in the manufacturing process, and a dry paper sheet soaked with fluorescent agent is used as a point to be detected, so that the problem of exciting light reflection in most fluorescent imaging equipment can be basically eliminated.

Secondly, the same-shaped paper sheet which is not stained with any fluorescent agent is added as a reference point, so that most background fluorescent noise can be eliminated, and the calibration accuracy is improved.

Thirdly, the calibration template manufactured by the method can be used for objectively and transversely comparing the detection sensitivity of different fluorescence imaging instruments, and can also be used as a substitute of an autofluorescence tissue in the instrument development process to evaluate the detection performance of the instrument.

Fourth, the template is easy to store for a long time under dry and cool conditions.

Drawings

FIG. 1: the utility model discloses a manufacturing process of a standard template;

FIG. 2: the utility model discloses a schematic diagram of a standard template;

FIG. 3: data shown is compared to a graph.

Detailed Description

The technical solution in the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, rather than all embodiments, and all other embodiments obtained by a person of ordinary skill in the art without any inventive work belong to the protection scope of the present invention based on the embodiments of the present invention.

According to an embodiment of the present invention, a standard template for fluorescence calibration is provided, including:

According to an embodiment of the present invention, the array is arranged with M × N filter paper sheets, M is 2, N is 5, i.e. 2 rows and 5 columns of filter paper sheets, including 2 reference paper sheets that are not soaked with the fluorescent powder solution.

According to one embodiment of the present invention, M × N-L of the M × N filter paper sheets are soaked with fluorescent reagent solutions having different concentration gradients and are taken out and dried in the shade after a predetermined time, thereby forming a filter paper sheet with fluorescent powder.

According to one embodiment of the invention, the filter paper sheet is uniform in shape and is a circular paper sheet, and the black frosted paper sheet of the substrate is rectangular in shape.

According to one embodiment of the invention, the fluorescent powder used is indocyanine green ICG powder.

According to an embodiment of the present invention, the method for manufacturing the standard template for fluorescence calibration includes the following steps:

step one, manufacturing standard template paper sheets with different concentration gradient fluorescent reagents and manufacturing reference template paper sheets without fluorescent reagents;

collecting data used for instrument calibration, carrying out integral imaging or point-by-point data collection on the standard template by using an instrument to be calibrated, and respectively measuring fluorescence data at a plurality of template paper sheets;

and step three, obtaining fluorescence data of each position by using different instruments according to the step two, and obtaining instrument calibration results.

Further, the first step specifically includes:

step 1, preparing a fluorescent reagent and pure water, and preparing solutions with different concentrations according to the detection range of an instrument to be calibrated;

2, cutting paper sheets with consistent shapes by adopting filter paper, soaking the paper sheets into prepared solutions with different concentrations, taking out the paper sheets after a preset time, and drying the paper sheets in the shade;

and 3, sequentially fixing the paper sheets dried in the shade on a black frosted paper board according to the concentration gradient of the soaking solution, and simultaneously fixing a plurality of paper sheets which are not stained with the fluorescent agent for eliminating background noise.

Fig. 1 shows a manufacturing flow of the standard template, and the specific manufacturing steps. The first step specifically comprises:

1-1 several phosphor powders, such as ICG indocyanine green, a piece of black frosted cardboard, several pure water and a clean container were prepared.

1-2 weighing e mg of fluorescent powder by using a precision balance, and putting the fluorescent powder into a clean container. F ml of pure water is taken by a standard pipette or a precise measuring cylinder and is stirred uniformly, and the concentration of the fluorescent powder in the container is

0.5 fml of the solution was removed and injected into container No. 1 for storage.

1-3 to the remaining solution, 0.5 fml of pure water was further added and stirred uniformly, and the concentration of the fluorescent powder in the vessel became

0.5 fml of the solution was continuously withdrawn and injected into container No. 2 for storage.

1-4, repeating the steps 1-2 for a plurality of times, so that the concentration of the fluorescent powder solution is reduced in sequence, and the repetition times are properly selected according to the fluorescence detection range of the instrument to be calibrated. When repeating n-1 times, n different solutions can be prepared and sequentially contained in containers No. 1 to n, the concentrations of which are:

wherein (i is more than or equal to 1 and less than or equal to n)

1-5, preparing a piece of filter paper, and cutting n + m pieces of paper with the same shape, wherein m is more than or equal to 1. Putting n paper sheets into the solution from No. 1 to No. n in sequence. And the n containers are placed in a refrigerator at 4-8 ℃ and kept still for more than 1 hour.

1-6 taking out n paper sheets in the container, and placing the paper sheets in a shade place for airing. And (3) sequentially bonding and fixing the n dried paper sheets and the remaining m paper sheets which are not soaked in the solution on the black frosted fingerboard. And placing the manufactured standard template in a shade place for storage.

According to an embodiment of the present invention, as shown in fig. 2, a schematic diagram of a standard template is shown, and the standard template includes the following components:

1: a substrate, typically black frosted cardboard;

2: and the fluorescent powder carrier is typically a filter paper sheet.

The substrate 1 is used as a carrier of a standard template, has the function of fixing the fluorescent powder carrier 2, and can be fixed by adopting a double-sided adhesive tape bonding mode. The fluorescent powder carrier 2 is manufactured by the first step, a plurality of fluorescent powder carriers 2 can be fixed on the black frosted paper board 1, and the concentration of the fluorescent powder solution soaked by each fluorescent powder carrier 2 is different.

And (5) obtaining the calibration value of each position according to the second step by using different instruments, and drawing a data comparison graph shown in the figure 3. The detection sensitivity of different instruments is obtained according to the data diagram, for example, according to the graph shown in fig. 3, the horizontal axis is the position points in the calibration template, such as position No. 1, position No. 2, and the like, without units, and the concentration of the fluorescent powder at each position is different. The ordinate is the Ci value for each point measured by the different instruments, which is the intensity of the fluorescence signal normalized to a certain position, for example position n, also without units. In the data of fig. 3, the larger the last calibration value of each curve is, i.e., the higher the calibration value is, the higher the detection sensitivity of the corresponding instrument is, and the instrument three is superior to the instrument two and both are superior to the instrument one.

The first embodiment is as follows: ICG indocyanine green template manufacturing

An ICG indocyanine green calibration template is manufactured and can be used as an autofluorescence mimic of parathyroid gland. The manufacturing process comprises the following steps: 2.56mg of ICG powder was prepared, 40ml of pure water was added thereto, and the mixture was stirred to obtain a solution having a concentration of 64ng/ml, and 20ml of the solution was taken and poured into a No. 1 reagent bottle. Adding 20ml of pure water into the remaining 20ml of solution, stirring uniformly to obtain 32ng/ml of solution, taking 20ml of solution, injecting the solution into a No. 2 bottle, and repeating the operation for 6 times to finally obtain 8 bottles of ICG solution, wherein the concentration of the ICG solution is as follows: bottle No. 1: 64ng/ml, vial No. 2: 32ng/ml, vial No. 3: 16ng/ml, vial No. 4: 8ng/ml, vial No. 5: 4ng/ml, vial No. 6: 2ng/ml, flask 7: 1ng/ml, No. 8 bottle: 0.5 ng/ml.

Preparing 10 circular filter paper sheets with the diameter of 10mm as a fluorescent powder carrier 2, taking 8 of the circular filter paper sheets, respectively putting the circular filter paper sheets into No. 1 to No. 8 reagent bottles for soaking for more than one hour, and leaving 2 circular filter paper sheets to be unprocessed for standby application. Then 8 filter paper sheets are taken out and put in a dark place for drying in the shade. A black frosted cardboard sheet of 100mm by 30mm in size was prepared as a substrate 1, and 10 filter paper sheets were fixed to the substrate 1 by double-sided adhesive tape as shown in fig. 2. Thus, the ICG template is manufactured.

The template and the parathyroid gland tissue to be detected are imaged simultaneously by a small animal fluorescence imager, a filter paper sheet with the autofluorescence of the parathyroid gland equivalent to the fluorescence intensity in the fluorescent powder carrier 2 can be found, and the filter paper sheet can be selected to replace the parathyroid gland for instrument development or evaluation.

Although illustrative embodiments of the invention have been described above to facilitate the understanding of the invention by those skilled in the art, it should be understood that the invention is not limited to the scope of the embodiments, and that various changes will become apparent to those skilled in the art once they are within the spirit and scope of the invention as defined and defined in the appended claims.

Claims

1. a standard template for fluorescence calibration, characterized in that, comprising:

a substrate, on which M*N filter paper sheets are arranged in an array, where M is the number of rows and N is the number of columns;

The base is used as the carrier of the standard template, and black matte cardboard is used to fix the filter paper;

The filter paper is used as a fluorescent powder carrier, and the fluorescent powder is attached to it; the substrate and the filter paper are fixed by double-sided adhesive bonding;

Each of the filter paper sheets is obtained by drying in the shade after being infiltrated with fluorescent powder solutions of different concentrations;

The M*N filter paper pieces include L reference paper pieces that have not been soaked with the fluorescent powder solution.

2 . A standard template for fluorescence calibration according to claim 1 , wherein M*N filter paper sheets are arranged in the array manner, where M is 2, and N is 5, that is, 2 rows and 5 columns. 3 . Filter paper discs, including 2 reference paper discs not soaked in fluorescent powder solution.

3. a kind of standard template for fluorescence calibration according to claim 1, is characterized in that, in described M*N filter paper sheet, M*N-L filter paper sheet is soaked through the fluorescent reagent solution with different concentration gradients and After a predetermined time, it was taken out and dried in the shade to form a filter paper sheet with fluorescent powder.

4. a kind of standard template for fluorescence calibration according to claim 1, is characterized in that,

The filter paper sheets have the same shape and are circular paper sheets, and the black matte cardboard shape of the base is rectangular.

5. a kind of standard template for fluorescence calibration according to claim 1, is characterized in that,

The fluorescent powder used is indocyanine green ICG powder.