U.S. patent application number 15/305416 was filed with the patent office on 2017-02-16 for medium for resin particles containing fluorescent dye.
The applicant listed for this patent is KONICA MINOLTA, INC.. Invention is credited to Fuminori OKADA, Masaru TAKAHASHI.
Application Number | 20170045452 15/305416 |
Document ID | / |
Family ID | 54332376 |
Filed Date | 2017-02-16 |
United States Patent
Application |
20170045452 |
Kind Code |
A1 |
TAKAHASHI; Masaru ; et
al. |
February 16, 2017 |
Medium For Resin Particles Containing Fluorescent Dye
Abstract
An object of the present invention is to provide a medium which
is capable of inhibiting precipitation and/or aggregation of
fluorescent dye-containing resin particles and enables to use the
fluorescent dye-containing resin particles for staining after a
long-term storage without having to perform complicated operations.
The present invention provides a medium for storing fluorescent
dye-containing resin particles, wherein, in a particle-containing
liquid obtained by adding fluorescent dye-containing resin
particles to the medium, the rate of change in the backscatter
intensity (transmitted light) at the center of the height of the
particle-containing liquid left to stand for 24 hours after the
addition is not less than -1% based on the particle-containing
liquid immediately after the addition.
Inventors: |
TAKAHASHI; Masaru;
(KoKubunji-shi, JP) ; OKADA; Fuminori; (Taito-Ku,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KONICA MINOLTA, INC. |
Chiyoda-ku |
|
JP |
|
|
Family ID: |
54332376 |
Appl. No.: |
15/305416 |
Filed: |
April 15, 2015 |
PCT Filed: |
April 15, 2015 |
PCT NO: |
PCT/JP2015/061567 |
371 Date: |
October 20, 2016 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C09K 2211/1416 20130101;
G01N 21/64 20130101; G01N 2021/6439 20130101; C09K 11/025 20130101;
C09K 2211/1433 20130101; C08K 5/3437 20130101; G01N 33/545
20130101; G01N 33/582 20130101; C09K 11/06 20130101; G01N 21/59
20130101; G01N 21/6428 20130101 |
International
Class: |
G01N 21/64 20060101
G01N021/64; C09K 11/06 20060101 C09K011/06; C09K 11/02 20060101
C09K011/02; G01N 33/545 20060101 G01N033/545; C08K 5/3437 20060101
C08K005/3437 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 23, 2014 |
JP |
2014-089287 |
Claims
1. A medium for storing fluorescent dye-containing resin particles,
wherein, in a particle-containing liquid obtained by adding
fluorescent dye-containing resin particles to said medium, the rate
of change in the backscatter intensity (transmitted light) at the
center of the height of said particle-containing liquid left to
stand for 24 hours after said addition is not less than -1% based
on said particle-containing liquid immediately after said
addition.
2. The medium according to claim 1, comprising a buffer, a protein
and a surfactant.
3. The medium according to claim 2, wherein said surfactant is a
nonionic surfactant.
4. The medium according to claim 1, wherein said fluorescent
dye-containing resin particles have a particle size of 40 nm to 200
nm.
5. The medium according to claim 1, wherein the wavelength of a
light irradiated at said center of said height is longer than the
particle size of said fluorescent dye-containing resin
particles.
6. The medium according to claim 1, wherein said fluorescent
dye-containing resin particles are used for pathological
staining.
7. The medium according to claim 1, wherein said fluorescent
dye-containing resin particles further comprise a reactive
functional group.
8. The medium according to claim 1, wherein a resin constituting
said fluorescent dye-containing resin particles is a thermosetting
resin.
9. The medium according to claim 2, wherein said fluorescent
dye-containing resin particles have a particle size of 40 nm to 200
nm.
10. The medium according to claim 2, wherein the wavelength of a
light irradiated at said center of said height is longer than the
particle size of said fluorescent dye-containing resin
particles.
11. The medium according to claim 2, wherein said fluorescent
dye-containing resin particles are used for pathological
staining.
12. The medium according to claim 2, wherein said fluorescent
dye-containing resin particles further comprise a reactive
functional group.
13. The medium according to claim 2, wherein a resin constituting
said fluorescent dye-containing resin particles is a thermosetting
resin.
Description
TECHNICAL FIELD
[0001] The present invention relates to a medium used for storing
fluorescent dye-containing resin particles.
BACKGROUND ART
[0002] In recent years, fluorescent dye-containing resin particles
have begun to be used as fluorescent labels in the field of
biology. Fluorescent dye-containing resin particles are particles
having a structure in which a fluorescent dye is encapsulated by an
appropriate resin particle. As fluorescent dye-containing resin
particles, those in the form of a complex with a functional group
or molecule that is capable of binding to a biological substance
such as an antibody may also be used in applications such as
immunostaining.
[0003] When such fluorescent dye-containing resin particles are
used, they are not always used immediately after the production and
may be stored for a certain period until use. In that case, the
fluorescent dye-containing resin particles are often stored in a
state of being diluted in a medium so that the functions as a
fluorescent label can be maintained.
[0004] As a medium for storing fluorescent dye-containing resin
particles, an appropriate buffer containing a small amount of a
blocking agent or a surfactant-containing liquid is used in many
cases so that aggregation and the like of the fluorescent
dye-containing resin particles can be inhibited as much as
possible. For example, Patent Document 1 describes the use of 1%
BSA/PBS buffer as a medium for storing fluorescent dye-containing
resin particles.
[0005] However, even in those cases where such a conventional
medium is used, when fluorescent dye-containing resin particles
stored for a long period are directly used for various staining
processes such as immunostaining, coarse aggregates are generated
in the resulting stained cellular tissue image, which may interfere
with correctly counting the number of bright spots. In order to
avoid such a situation, conventionally, those fluorescent
dye-containing resin particles that have been stored in a state of
being diluted with a medium over a long time are required to be
subjected to pretreatments such as solvent substitution, which is
performed by repeating appropriate times the operations of
centrifugation, supernatant removal, dilution with a staining
solvent and redispersion by ultrasonication, and subsequent
filtering treatment, prior to being used for staining; therefore,
there is a problem of having to perform complicated operations.
CITATION LIST
Patent Document
[0006] Patent Document 1: WO2012/029342
SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
[0007] When fluorescent dye-containing resin particles are stored
for a long period using a conventional medium, coarse aggregates
are often generated upon staining cellular tissues with the stored
fluorescent dye-containing resin particles. Such coarse aggregates
can usually be observed as aggregates of a size equivalent to a 2.5
to 5-.mu.m square or larger and may reach a size equivalent to a
10-.mu.m square or larger in some cases. It is believed that the
fluorescent dye-containing resin particles undergo precipitation
and/or aggregation after a long-term storage thereof.
[0008] Therefore, an object of the present invention is to provide
a medium which is capable of inhibiting precipitation and/or
aggregation, particularly aggregation, of fluorescent
dye-containing resin particles and enables the fluorescent
dye-containing resin particles to be used for staining after a
long-term storage without having to perform complicated
operations.
Technical Solution
[0009] In order to realize at least one of the above-described
objects, the present invention provides the following medium:
[0010] a medium for storing fluorescent dye-containing resin
particles, wherein, in a particle-containing liquid obtained by
adding fluorescent dye-containing resin particles to the medium,
the rate of change in the backscatter intensity (transmitted light)
at the center of the height of the particle-containing liquid left
to stand for 24 hours after the addition is not less than -1% based
on the particle-containing liquid immediately after the
addition.
Advantageous Effects of Invention
[0011] By storing fluorescent dye-containing resin particles in the
medium of the present invention, cellular tissues can be stained
using the fluorescent dye-containing resin particles even after a
long-term storage with only a simple operation such as pipetting
(stirring), without requiring pretreatments such as solvent
substitution, which is performed by repeating appropriate times the
operations of centrifugation, supernatant removal, dilution with a
staining solvent and redispersion by ultrasonication, and
subsequent filtering treatment, before the use for staining as in
conventional technologies.
BRIEF DESCRIPTION OF DRAWINGS
[0012] FIG. 1 shows the staining result obtained in Example 5 using
streptavidin-modified fluorescent dye-containing resin particles
immediately after the synthesis thereof.
[0013] FIG. 2 shows the staining result obtained in Example 5 using
streptavidin-modified fluorescent dye-containing resin particles
after one month of storage in the medium of the present
invention.
[0014] FIG. 3 shows the staining result obtained in Example 11
using streptavidin-modified fluorescent dye-containing resin
particles immediately after the synthesis thereof.
[0015] FIG. 4 shows the staining result obtained in Example 11
using streptavidin-modified fluorescent dye-containing resin
particles after one month of storage in the medium of the present
invention.
[0016] FIG. 5 shows the staining result obtained in Comparative
Example 7 using streptavidin-modified fluorescent dye-containing
resin particles immediately after the synthesis thereof.
[0017] FIG. 6A shows the staining result obtained in Comparative
Example 7 using streptavidin-modified fluorescent dye-containing
resin particles after one month of storage in a medium.
[0018] FIG. 6B is a sketch illustrating the positions of coarse
aggregates in the staining result of Comparative Example 7 shown in
FIG. 6A.
[0019] FIG. 7 is a chart showing the relationship of the average
particle size of the resulting fluorescent dye-containing resin
particles with respect to the amount of the added resin material in
the production of the fluorescent dye-containing resin particles
used in Examples and Comparative Examples.
MODE FOR CARRYING OUT THE INVENTION
[0020] The medium according to the present invention will now be
described concretely.
[Medium]
[0021] The medium according to the present invention is:
[0022] a medium for storing fluorescent dye-containing resin
particles, wherein, in a particle-containing liquid obtained by
adding fluorescent dye-containing resin particles to the medium,
the rate of change in the backscatter intensity (transmitted light)
at the center of the height of the particle-containing liquid left
to stand for 24 hours after the addition is not less than -1% based
on the particle-containing liquid immediately after the
addition.
[0023] That is, when a particle-containing liquid is prepared by
adding fluorescent dye-containing resin particles to the medium of
the present invention, with the backscatter intensity (transmitted
light) measured at the center of the height of the
particle-containing liquid immediately after the addition being
defined as "I.sub.0" and the backscatter intensity (transmitted
light) measured at the center of the height of the
particle-containing liquid that is left to stand for 24 hours after
the addition being defined as "I.sub.24", the rate of change in the
backscatter intensity (transmitted light) at the center of the
height of this particle-containing liquid, which is D (%)
determined by the following formula:
D=(I.sub.24-I.sub.0)/I.sub.0.times.100
satisfies the relationship of D.gtoreq.-1. This rate of change, D,
represents the degree of aggregation of fluorescent dye-containing
resin particles stored in the medium of the present invention and
serves as an index for evaluating the performance of the medium in
storing fluorescent dye-containing resin particles.
[0024] In other words, from a different perspective, whether or not
a medium for storing fluorescent dye-containing resin particles
corresponds to the medium of the present invention, that is,
whether or not a medium for storing fluorescent dye-containing
resin particles satisfies the relationship of D.gtoreq.-1, can be
verified by an evaluation method comprising the following steps (1)
to (4):
[0025] (1) the step of obtaining a particle-containing liquid by
adding fluorescent dye-containing resin particles to the medium of
interest;
[0026] (2) the step of measuring the backscatter intensity
(transmitted light) I.sub.0 at the center of the height of the
particle-containing liquid immediately after the addition;
[0027] (3) the step of again measuring the backscatter intensity
(transmitted light) I.sub.24 at the center of the height of the
particle-containing liquid after leaving the particle-containing
liquid to stand for 24 hours; and
[0028] (4) the step of determining whether or not the following
requirement is satisfied based on the thus measured I.sub.0 and
I.sub.24.
(I.sub.24-I.sub.0)/I.sub.0.times.100.gtoreq.-1
[0029] In the present invention, the "backscatter intensity
(transmitted light)", based on which the rate of change (D) is
determined, refers to the intensity of a transmitted light or
back-scattered light that is generated when a light emitted from a
light source travels straight while transmitting or being
repeatedly scattered through a sample.
[0030] In the present invention, the reason why the backscatter
intensity (transmitted light) is measured at the center of the
height of a particle-containing liquid in as follows.
[0031] In cases where fluorescent dye-containing resin particles to
be used for pathological staining are stored in a medium,
aggregation of the particles during a long-term storage causes
coarse aggregates to be generated when the pathological staining is
performed and this interferes with making a correct determination.
On the other hand, even if precipitation of the fluorescent
dye-containing resin particles occurred during a long-term storage
in the medium, by re-dispersing the precipitated particles, the
pathological staining can be performed without generation of coarse
aggregates. In view of this, for the evaluation of the performance
of a medium in storing fluorescent dye-containing resin particles,
it is required to be able to observe particle aggregation
separately from particle precipitation.
[0032] When a particle-containing liquid is left to stand and the
particles dispersed therein aggregate, since a change occurs in the
amount of the back-scattered light throughout the
particle-containing liquid regardless of its height position, the
backscatter intensity (transmitted light) at the center of the
height is reduced accordingly.
[0033] Meanwhile, when the dispersed particles simply precipitate,
although the backscatter intensity (transmitted light) at the upper
and lower parts of the particle-containing liquid changes with time
as the precipitation of the particles proceeds, the backscatter
intensity (transmitted light) at the center of the height hardly
changes.
[0034] Therefore, by setting the position of measuring the
backscatter intensity (transmitted light) at the center of the
height of a particle-containing liquid, the degree of aggregation
of fluorescent dye-containing resin particles in a medium can be
properly evaluated, so that the performance of the medium in
storing the fluorescent dye-containing resin particles can be
appropriately evaluated. In the most ideal mode of the medium of
the present invention, no aggregation of fluorescent dye-containing
resin particles occurs in the medium even after a long-term storage
and, in this case, D=0.
[0035] In the present invention, the value of D is prescribed to be
not less than -1 because, from the present inventors' experiences,
it was considered appropriate to make judgment based on this value
when evaluating the performance of a medium in inhibiting the
aggregation of fluorescent dye-containing resin particles. This has
also be confirmed from its relationship with the evaluation results
of immunostaining and morphological staining that were obtained in
the below-described Examples and Comparative Examples using
fluorescent dye-containing resin particles after one month of
storage in a medium. It is noted here that the present inventors
presume that the rate of change in the backscatter intensity
(transmitted light) at the center of the height of a
particle-containing liquid left to stand for one month also has a
certain correlation with the value of D.
[0036] In the medium of the present invention, the value of D may
be larger than 0 (D>0) depending on the measurement conditions;
however, such a value of D presents no problem.
[0037] In the present invention, the wavelength of the light to be
irradiated at the center of the height for the measurement of the
backscatter intensity (transmitted light) is not necessarily
particularly restricted as long as the backscatter intensity
(transmitted light) of the particle-containing liquid of interest
can be appropriately measured in relation to fluorescent
dye-containing resin particles. However, for an appropriate
measurement, it is desired that the wavelength of the irradiated
light be longer than the particle size of the fluorescent
dye-containing resin particles. Here, a light having a wavelength
of about 880 nm is preferably used since it does not require a
special measuring instrument.
[0038] Further, in the present invention, the measuring instrument
used for the evaluation is also not particularly restricted as long
as it is capable of appropriately measuring the backscatter
intensity (transmitted light) at the center of the height of the
particle-containing liquid of interest, and examples of a preferred
measuring instrument include TURBISCAN (trademark) manufactured by
Formulaction SA. According to this measuring instrument, it is also
possible to measure the backscatter intensity (transmitted light)
while changing the height position. This is, however, not to forbid
use of other spectrophotometer in the measurement of the
backscatter intensity (transmitted light).
(Constituents)
[0039] In the medium of the present invention, as described above,
the rate of change (D (%)) satisfies the specific range prescribed
in the present invention. The concrete constitution of the medium
of the present invention that satisfies such a rate of change (D
(%)) varies depending on the type, the condition of surface
modification and the like of the fluorescent dye-containing resin
particles to be stored and is thus not uniformly and strictly
specified here; however, the medium of the present invention
typically comprises a buffer, a protein and a surfactant.
[0040] Protein
[0041] The protein that can constitute the medium of the present
invention is not particularly restricted as long as it does not
impair the functions of fluorescent dye-containing resin particles
and is capable of inhibiting aggregation of fluorescent
dye-containing resin particles. However, when the medium of the
present invention is used for storing fluorescent dye-containing
resin particles to be used for pathological staining, it is desired
that the protein be capable of inhibiting non-specific adsorption
to the cellular tissue to be stained. Accordingly, examples of a
preferred protein include those proteins that are generally used as
a blocking agent, such as BSA and casein.
[0042] The content of the protein in the medium of the present
invention can be adjusted as appropriate within a range where
aggregation of fluorescent dye-containing resin particles can be
inhibited; however, it is desired to be, for example, 10% by weight
or less (e.g., in a range of 1 to 10% by weight) with respect to
the whole medium.
[0043] Surfactant
[0044] The surfactant that can constitute the medium of the present
invention is not particularly restricted as long as it does not
impair the functions of fluorescent dye-containing resin particles
and is capable of inhibiting aggregation of fluorescent
dye-containing resin particles. However, when the medium of the
present invention is used for storing fluorescent dye-containing
resin particles to be used for pathological staining, there are
cases where the fluorescent dye-containing resin particles are
directly used for pathological staining in a state of being diluted
with the medium of the present invention. In the cellular tissues,
parts where the cell nucleus is located are negatively charged
because of the phosphate residues constituting the nucleic acid,
whereas those parts other than the cell nucleus tend to be
positively charged. Therefore, in order to minimize the
non-specific adsorption to the cellular tissues, it is desired to
use a nonionic surfactant as the surfactant. Particularly,
polyoxyethylene sorbitan fatty acid esters such as Tween
(registered trademark)-based surfactants can be preferably used
and, thereamong, Tween (registered trademark) 20 can be
particularly preferably used.
[0045] The content of the surfactant in the medium of the present
invention can be adjusted as appropriate within a range where
aggregation of fluorescent dye-containing resin particles can be
inhibited; however, it is desired to be, for example, in a range of
0.1% by weight or less with respect to the whole medium.
[0046] Buffer
[0047] The buffer that can constitute the medium of the present
invention is not particularly restricted as long as it does not
impair the functions of fluorescent dye-containing resin particles,
and a variety of conventionally known buffers can be used.
[0048] In a preferred mode of the present invention, the medium of
the present invention is used for storing fluorescent
dye-containing resin particles to be used for pathological
staining. In this case, as the fluorescent dye-containing resin
particles used for pathological staining, the reactive functional
group-containing fluorescent dye-containing resin particles
described below in the section of "Mode of Fluorescent
Dye-containing Resin Particles", particularly those comprising a
molecule that is likely to form a bond based on affinity
interaction such as biotin, streptavidin and avidin, are often
employed. Therefore, it is preferred that the buffer used in the
present invention have a pH in a range that does not cause
degeneration of such a molecule. Further, in pathological staining
with such fluorescent dye-containing resin particles, since the
fluorescent dye-containing resin particles may be subjected to the
staining in a state of being diluted with the medium of the present
invention, it is preferred that the buffer have a pH in a range
that is suitable for pathological staining. From these standpoints,
the buffer used in the present invention preferably has a pH in a
range of 6.0 to 8.0, more preferably in a range of 6.9 to 7.6.
Examples of a preferred buffer type include phosphate-buffered
physiological saline (PBS), Tris-HCl buffer, phosphate buffer
(excluding PBS), and a combination of two or more of these
buffers.
[0049] Other Components
[0050] In the medium of the present invention, in addition to the
buffer, protein and surfactant, other component(s) such as a
preservative may also be incorporated, as long as the functions of
fluorescent dye-containing resin particles are not impaired and
aggregation of fluorescent dye-containing resin particles can be
inhibited. Examples of the preservative include sodium azide
(NaN.sub.3).
[0051] It is desired that the preservative be incorporated in the
buffer at a concentration of 0.015 N or less.
[0052] Production Method
[0053] The medium of the present invention can be obtained by
dissolving the protein and the surfactant as well as the "other
component(s)", which is/are optionally added, in the buffer in
accordance with a conventional method.
[0054] The combination and composition ratio of the constituents,
namely the protein and the medium, and the "other component(s)"
that is/are optionally added vary depending on the type and the
like of the fluorescent dye-containing resin particles to be stored
and are, therefore, not uniformly and strictly specified here.
However, for the adjustment of the combination and composition
ratio, reference can be made to the results of the below-described
Examples and Comparative Examples.
[0055] In the aggregation of fluorescent dye-containing resin
particles, it is believed that the electrostatic relation between
the fluorescent dye-containing resin particles and/or the
electrostatic relation between the medium and the fluorescent
dye-containing resin particles are also involved. Accordingly, for
the determination of the combination and composition ratio of the
constituents, reference can also be made to the zeta potential of
the fluorescent dye-containing resin particles in the medium. The
zeta potential of the fluorescent dye-containing resin particles
can be measured using a common zeta potential-measuring device
(such as "Zetasizer Nano" manufactured by Malvern Instruments Ltd.)
and adjusted with the protein and preservative as well as,
depending on the case, the surfactant. For example, when the medium
of the present invention is set to contain a buffer of pH 6.0 to
8.0, the medium of the present invention can be prepared by
adjusting the combination and composition ratio of the constituents
and/or finely adjusting the pH of the buffer within a range of 6.0
to 8.0 such that the fluorescent dye-containing resin particles
have a zeta potential of 0 mV to -10 mV in the resulting medium of
the present invention.
(Fluorescent Dye-Containing Resin Particles to be Stored)
[0056] The term "fluorescent dye-containing resin particles to be
stored" using the medium of the present invention refers to a
substance having a structure in which plural fluorescent dye
molecules are immobilized in a state of being encapsulated in a
resin particle by chemical or physical action, and the form thereof
is not particularly restricted.
[0057] Examples of the fluorescent dye-containing resin particles
of interest in the present invention include conventionally known
fluorescent dye-containing resin particles, and their resin may be
composed of a thermosetting resin such as a melamine resin or a
thermoplastic resin such as a polystyrene resin. However, when the
fluorescent dye-containing resin particles are used for
pathological staining, clearing with an organic solvent such as
xylene may be performed in the process of pathological staining.
Therefore, from the standpoint of inhibiting elution of the
fluorescent dye in the clearing step using an organic solvent such
as xylene, fluorescent dye-containing resin particles whose resin
is composed of a thermosetting resin capable of immobilizing a
fluorescent dye inside its fine cross-linked structure, such as a
melamine resin, are preferred.
[0058] The size of the fluorescent dye-containing resin particles
is not particularly restricted as long as it is suitable for the
intended application such as immunostaining of a tissue section;
however, it is usually 10 nm to 500 nm, preferably 40 nm to 200 nm,
more preferably 50 nm to 200 nm. Further, the variation
coefficient, which represents the variation in the particle size,
is also not particularly restricted; however, it is usually 20% or
less, preferably 5 to 15%. Fluorescent dye-containing resin
particles having such a particle size can be obtained by, for
example, the below-described production method.
[0059] The size of a fluorescent dye-containing resin particle can
be determined by taking an electron micrograph thereof using a
scanning electron microscope (SEM), measuring the cross-sectional
area of the fluorescent dye-containing resin particle and then
determining the particle size as the diameter of a circular area
corresponding to the measured value (area-equivalent circle
diameter). With regard to the average particle size (average
particle diameter) and the variation coefficient of a group of
fluorescent dye-containing resin particles, after measuring the
particle size (particle diameter) for a sufficient number (for
example, 1,000) of the fluorescent dye-containing resin particles
in the above-described manner, the average particle size is
calculated as the arithmetic mean of the measured values and the
variation coefficient is calculated by the following equation:
100.times.(standard deviation of particle size)/(average particle
size).
[0060] Fluorescent Dye
[0061] The fluorescent dye constituting the fluorescent
dye-containing resin particles to which the present invention is
applied is not particularly restricted and may be a conventionally
known fluorescent dye.
[0062] Fluorescent dyes that are generally available or can be
prepared may be classified into, for example, rhodamine-based dye
molecules, squarylium-based dye molecules, cyanine-based dye
molecules, aromatic hydrocarbon-based dye molecules, oxazine-based
dye molecules, carbopyronine-based dye molecules,
pyrromethene-based dye molecules, Alexa Fluor (registered
trademark, manufactured by Invitrogen)-based dye molecules, BODIPY
(registered trademark, manufactured by Invitrogen)-based dye
molecules, Cy (registered trademark, manufactured by GE
Healthcare)-based dye molecules, DY (registered trademark,
manufactured by Dyomics GmbH)-based dye molecules, HiLyte
(registered trademark, manufactured by AnaSpec Inc.)-based dye
molecules, DyLight (registered trademark, manufactured by Thermo
Fisher Scientific K.K.)-based dye molecules, ATTO (registered
trademark, manufactured by ATTO-TEC GmbH)-based dye molecules, and
MFP (registered trademark, manufactured by Mobitec Co., Ltd.)-based
dye molecules. The generic names of these dye molecules are
designated based on the main structure (skeleton) or registered
trademark of the respective compounds; therefore, those of ordinary
skill in the art should be able to properly understand the scope of
the fluorescent dyes belonging to the respective generic names
without having to bear undue trial and error. It is noted here that
N,N'-bis(2,6-diisopropylphenyl)-1,6,7,12-tetraphenoxyperylene-3-
,4:9,10-tetracarboxdiimide used in the below-described Examples
corresponds to an aromatic hydrocarbon-based dye molecule.
[0063] Further, the fluorescent dye may be subjected to a
solubilization treatment for the purposes of, for example,
improving the emission intensity of the fluorescent dye and
increasing the Stokes shift. This solubilization treatment is not
particularly restricted as long as it is a technique capable of
solubilizing the fluorescent dye, that is, improving the solubility
of the fluorescent dye in water. Specific examples of the
solubilization treatment include methods in which a fluorescent dye
is treated and allowed to react with an acid (e.g., concentrated
sulfuric acid, concentrated hydrochloric acid, acetic acid or
formic acid) or an aldehyde (e.g., formaldehyde or acetaldehyde),
among which an acid treatment is preferred since generally shows an
excellent effect.
[0064] Further, the emission wavelength of the fluorescent dye can
be selected as desired in accordance with the intended application.
For example, in pathological diagnosis, when such an application
where staining with eosin or the like for morphological observation
and immunostaining with a fluorescent dye are simultaneously
performed is postulated, it is preferred that the fluorescent dye
have an emission wavelength in the infrared to near-infrared range
so that the light emitted from the fluorescent dye can be visually
observed and the emission wavelength of the fluorescent dye does
not overlap with that of fluorescence-emitting eosin. For example,
a fluorescent dye having its maximum excitation wavelength in a
range of 555 to 620 nm and maximum emission wavelength in a range
of 580 to 770 nm is preferred.
[0065] Resin
[0066] The resin constituting the fluorescent dye-containing resin
particles to which the present invention is applied may be a
thermosetting resin or a thermoplastic resin. For example, from the
standpoint of inhibiting elution of the fluorescent dye in the
clearing step using an organic solvent such as xylene, a resin
comprising a thermosetting resin such as a melamine resin, which is
capable of immobilizing the fluorescent dye inside its fine
cross-linked structure, is preferred. In a preferred mode of the
present invention, the resin constituting the fluorescent
dye-containing resin particles to which the present invention is
applied is a thermoplastic resin, more specifically a resin
consisting of only a thermosetting resin such as a melamine
resin.
[0067] Examples of the thermosetting resin include those which
contain a structural unit formed from at least one monomer selected
from the group consisting of melamine, urea, guanamines (including
benzoguanamine, acetoguanamine and the like), phenols (including
phenol, cresol, xylenol and the like), xylene and derivatives
thereof. Any one of these monomers may be used individually, or two
or more thereof may be used in combination. If desired, one or more
co-monomers other than the compounds may also be used in
combination.
[0068] Specific examples of the thermosetting resin include
melamine-formaldehyde resins, urea-formaldehyde resins,
benzoguanamine-formaldehyde resins, phenol-formaldehyde resins and
metaxylene-formaldehyde resins.
[0069] As a starting material of these thermosetting resins, in
addition to the above-described monomers per se, a prepolymer
obtained by allowing such a monomer to react with formaldehyde and
other compound such as a cross-linking agent in advance can also be
used. For example, in the production of a melamine-formaldehyde
resin, generally, methylol melamine prepared by condensation
between melamine and formaldehyde under an alkaline condition is
used as a prepolymer, and this compound may further be subjected to
alkyl-etherification. Examples of the alkyl-etherification of
methylol melamine include methylation for improvement of the
stability in water, and butylation for improvement of the
solubility in an organic solvent.
[0070] Further, in the thermosetting resin, at least some of the
hydrogens contained in the structural unit may be substituted with
a charged substituent or a substituent capable of forming a
covalent bond. Such a thermosetting resin can be synthesized by
using, as a starting material, a monomer in which at least one
hydrogen is substituted with the substituent (derivatized monomer)
by a known method. Normally, melamine resins, urea resins,
benzoguanamine resins and the like naturally contain an amino group
or a cation generated from a moiety originated from an amino group,
and phenol resins, xylene resins and the like naturally contain a
hydroxyl group or an anion generated from a moiety originated from
a hydroxyl group.
[0071] Such a thermosetting resin can be synthesized in accordance
with a known method. For example, a melamine-formaldehyde resin can
be synthesized by heating and polycondensing methylol melamine
prepared in advance in the above-described manner, with an addition
of, as required, a reaction accelerator such as an acid.
[0072] Meanwhile, examples of the thermoplastic resin include those
which contain a structural unit formed from at least one
monofunctional monomer selected from the group consisting of
styrene, (meth)acrylic acid, alkyl esters thereof, acrylonitrile
and derivatives thereof (a monomer having one group involved in
polymerization reaction in one molecule, which group is a vinyl
group in the above-described case). Any one of these monomers may
be used individually, or two or more thereof may be used in
combination. If desired, one or more co-monomers other than the
compounds may also be used in combination.
[0073] Specific examples of the thermoplastic resin include
polystyrenes, styrene-based resins composed of styrene and other
monomer(s), polymethylmethacrylates, acrylic resins composed of
(meth)acrylic acid, an alkyl ester thereof and other monomer(s),
polyacrylonitriles, AS resins (acrylonitrile-styrene copolymers)
and ASA resins (acrylonitrile-styrene-methyl acrylate copolymers),
and acrylonitrile-based resins composed of acrylonitrile and other
monomer(s).
[0074] The thermoplastic resin may also contain, for example, a
structural unit formed from a polyfunctional monomer such as
divinylbenzene (a monomer having two or more groups involved in
polymerization reaction in one molecule, which groups are vinyl
groups in the above-described case), that is, a cross-linked
moiety. Examples of such a thermoplastic resin include cross-linked
polymethyl methacrylates.
[0075] In the thermoplastic resin, at least some of the hydrogens
contained in the structural unit may be substituted with a charged
substituent or a substituent capable of forming a covalent bond.
Such a thermoplastic resin can be synthesized by using, as a
starting material, a monomer in which at least one hydrogen is
substituted with the substituent (derivatized monomer), such as
4-aminostyrene.
[0076] Further, the thermoplastic resin may also contain a
structural unit comprising a functional group used for surface
modification of the resulting fluorescent dye-containing resin
particles. For example, by using an epoxy group-containing monomer
such as glycidyl methacrylate as a starting material, fluorescent
dye-containing resin particles on which epoxy groups are oriented
on the surface can be prepared. These epoxy groups can be converted
into amino groups by allowing them to react with an excess amount
of aqueous ammonia. In the thus formed amino groups, various
biomolecules can be incorporated in accordance with a known method.
As required, the incorporation of various biomolecules into the
amino groups can be carried out through molecules that serve as
linkers.
[0077] Mode of Fluorescent Dye-Containing Resin Particles
[0078] The fluorescent dye-containing resin particles to which the
present invention is applied comprise the fluorescent dye and resin
and may be subjected to surface modification.
[0079] The medium of the present invention can be particularly
preferably used for storing fluorescent dye-containing resin
particles that are used for pathological staining such as
immunostaining. It is preferred that the fluorescent dye-containing
resin particles to which the present invention is applied further
comprise a reactive functional group so that the fluorescent
dye-containing resin particles are easily bound with a
molecule-recognizing substance (e.g., an antibody) capable of
recognizing a biological substance to be detected by pathological
staining (more specifically, a biological substance that can be an
antigen). Examples of the reactive functional group include
chemical functional groups, such as a carboxyl group, an amino
group, an aldehyde group, a thiol group and a maleimide group; and
molecules that are likely to form a bond based on affinity
interaction, such as biotin, streptavidin and avidin. In the
fluorescent dye-containing resin particles, a linker or spacer
having an appropriate chain length may exist between the main part
of the fluorescent dye-containing resin particles (that is, the
part of the fluorescent dye-containing resin particles that
excludes the reactive functional group and optional linker or
spacer).
[0080] Method of Producing Fluorescent Dye-Containing Resin
Particles
[0081] The fluorescent dye-containing resin particles to which the
present invention is applied can be produced in accordance with a
polymerization step known for various resins.
[0082] Fluorescent dye-containing resin particles whose resin is
composed of a thermosetting resin can be produced in accordance
with a known emulsion polymerization method. For example, the
polymerization step of the fluorescent dye-containing resin
particles whose resin is composed of a thermosetting resin may be a
step of generating fluorescent dye-encapsulating resin particles by
heating a reaction mixture which contains a fluorescent dye and a
resin material (a monomer, an oligomer or a prepolymer), and
preferably an appropriate known surfactant and an appropriate known
polymerization reaction accelerator, and thereby allowing
polymerization reaction of the resin to proceed. In this case, the
order of adding the components contained in the reaction mixture is
not particularly restricted.
[0083] The conditions of the polymerization reaction (e.g.,
temperature and time) can be set as appropriate taking into
consideration the type of the resin, the composition of the
material mixture and the like. For the synthesis of a thermosetting
resin such as a melamine resin, the reaction temperature is usually
70 to 200.degree. C. and the reaction time is usually 20 to 120
minutes. Here, it is appropriate that the reaction temperature be a
temperature at which the performance of the fluorescent dye is not
reduced (within the range of the heat resistant temperature). The
heating can be performed in a plurality of steps and, for example,
the material mixture may first be allowed to react for a certain
time at a relatively low temperature and then heated and allowed to
further react for a certain time at a relatively high temperature.
After the completion of the polymerization reaction, impurities
such as unreacted resin material, fluorescent dye and surfactant
are removed, and the thus generated fluorescent dye-containing
resin particles can be recovered and purified.
[0084] Further, in the production of fluorescent dye-containing
resin particles whose resin is composed of a thermosetting resin,
after the polymerization step, as required depending on the
intended application of the fluorescent dye-containing resin
particles, a modification step can also be performed as a step of
introducing the reactive functional group described above in the
section of "Mode of Fluorescent Dye-containing Resin Particles" to
the surface of the fluorescent dye-containing resin particles. The
introduction of the reactive functional group can be appropriately
performed by a conventional method.
[0085] Meanwhile, fluorescent dye-containing resin particles whose
resin is composed of a thermoplastic resin can be produced in the
same manner as the fluorescent dye-containing resin particles whose
resin is composed of a thermosetting resin, except that, as a
polymerization step, a step of generating fluorescent
dye-encapsulating resin particles by heating a reaction mixture
which contains a fluorescent dye, a resin material and a
polymerization initiator (e.g., benzoyl peroxide or
azobis-isobutyronitrile) and thereby allowing polymerization
reaction of the resin to proceed is performed in accordance with a
conventional method.
(Application)
[0086] The above-described medium of the present invention can be
suitably used for storing fluorescent dye-containing resin
particles, particularly fluorescent dye-containing resin particles
used for pathological staining. From a different perspective, the
method of storing fluorescent dye-containing resin particles can be
seen as a method which comprises adding the fluorescent
dye-containing resin particles to the medium of the present
invention. The fluorescent dye-containing resin particles can be
stored usually under refrigeration (for example, at 4 to 5.degree.
C.)
[0087] Specific examples of the pathological staining include
immunostaining.
EXAMPLES
[0088] Examples and Comparative Examples according to the present
invention will now be described referring to the drawings.
[0089] The fluorescent dye-containing resin particles of Examples
and Comparative Examples were measured or evaluated by the
following methods.
(Method of Measuring Average Particle Size of Fluorescent
Dye-Containing Resin Particles)
[0090] A photograph of the fluorescent nanoparticles of interest
was taken under a scanning electron microscope (SEM), the
cross-sectional area was measured for a sufficient number of
particles, and the particle size was determined as the diameter of
a circular area corresponding to the respective measured values. In
the below-described Synthesis Examples, the arithmetic mean of the
particle sizes of 1,000 particles was defined as the average
particle size.
Synthesis Examples 1-1 to 1-7
Preparation of Fluorescent Dye-Containing Resin Particles
[0091] As fluorescent dye-containing resin particles of Synthesis
Examples 1-1 to 1-7, using a conventionally known method,
fluorescent dye-containing resin particles A1 to A7 having an
average particle size of 40, 60, 80, 100, 150, 200 and 250 nm,
respectively, were each prepared.
[0092] As an example of the method of producing the fluorescent
dye-containing resin particles, the method of producing the
fluorescent dye-containing resin particles A5 is described
below.
Synthesis Example 1-5
[0093] By treating
N,N'-bis(2,6-diisopropylphenyl)-1,6,7,12-tetraphenoxyperylene-3,4:9,10-te-
tracarboxdiimide with concentrated sulfuric acid, a sulfo group was
introduced to give a corresponding sulfonic acid. This sulfonic
acid was converted into a corresponding acid chloride by a
conventional method.
[0094] After adding 14.4 mg of the thus obtained acid chloride to
22.5 mL of water, the resultant was heated at 70.degree. C. for 20
minutes on a hot stirrer and 0.65 g of a melamine resin Nikalac
MX-035 (manufactured by Nippon Carbide Industries Co., Ltd.) was
added thereto, followed by heating of the resulting mixture with
stirring for another 5 minutes. Then, 100 .mu.L of formic acid was
further added, and the resultant was heated with stirring at
60.degree. C. for 20 minutes and subsequently cooled to room
temperature. Thereafter, the resulting reaction mixture was placed
in a centrifugal tube and centrifuged at 12,000 rpm for 20 minutes,
followed by removal of the resulting supernatant. The precipitates
were washed with ethanol and water.
[0095] Then, 0.1 mg of the thus obtained particles was dispersed in
1.5 mL of EtOH (ethanol), and 2 .mu.L of
aminopropyltrimethoxysilane LS-3150 (manufactured by Shin-Etsu
Chemical Co., Ltd.) was added thereto. The resultant was allowed to
react for 8 hours so as to perform a surface amination
treatment.
[0096] The thus obtained dye-containing nanoparticles were adjusted
with PBS (phosphate-buffered physiological saline) containing 2 mM
of EDTA (ethylenediamine tetraacetic acid) to a concentration of 3
nM, and this solution was mixed with SM (PEG) 12 (manufactured by
Thermo Fisher Scientific K.K.;
succinimidyl-[(N-maleomidopropionamid)-dodecaethylene glycol]ester)
to a final concentration of 10 mM and allowed to react for 1 hour.
The thus obtained mixture was centrifuged at 10,000 G for 20
minutes, and the resulting supernatant was removed. Then, PBS
containing 2 mM of EDTA was added to disperse the precipitates, and
the resulting dispersion was centrifuged again. The precipitates
were washed three times by the same procedure to give fluorescent
dye-containing resin particles (fluorescent particles) A5 having a
maleimide group at a terminal.
[0097] When the particle size was measured for the thus obtained
fluorescent dye-containing resin particles A5 under an electron
microscope by the above-described method, the average particle size
was found to be 150 nm.
Synthesis Examples 1-1 to 1-4, 1-6 and 1-7
[0098] Fluorescent dye-containing resin particles A1 to A4, A6 and
A7 of Synthesis Examples 1-1 to 1-4, 1-6 and 1-7, which had
different particle sizes from the fluorescent dye-containing resin
particles A5 of Synthesis Example 1-5, were also each synthesized
in the same manner as in Synthesis Example 1-5, except that the
amount of the resin was changed as appropriate while maintaining
the dye/added resin amount in the synthesis constant.
[0099] For reference, FIG. 7 shows the relationship of the average
particle size of the resulting fluorescent dye-containing resin
particles with respect to the amount of the added resin material
(the melamine resin in Synthesis Examples 1-1 to 1-7) in a case
where fluorescent dye-containing resin particles are synthesized
under the same conditions as in Synthesis Example 1-5.
[0100] It is noted here that, in the following descriptions, in
order to distinguish the fluorescent dye-containing resin particles
A1 to A7 from the below-described streptavidin-modified fluorescent
dye-containing resin particles, the fluorescent dye-containing
resin particles A1 to A7 may be referred to as "maleimide
group-modified fluorescent dye-containing resin particles A1 to
A7", respectively, and these resin particles may be collectively
referred to as "maleimide group-modified fluorescent dye-containing
resin particles".
Synthesis Examples 2-1 to 2-7
Synthesis of Streptavidin-Modified Fluorescent Dye-Containing Resin
Particles
[0101] The maleimide group-modified fluorescent dye-containing
resin particles A1 to A7 were each modified with streptavidin in
the below-described manner to give streptavidin-modified
fluorescent dye-containing resin particles S1 to S7,
respectively.
[0102] A thiol group addition treatment was performed for
streptavidin (manufactured by Wako Pure Chemical Industries, Ltd.),
by allowing the streptavidin to react with
N-succinimidyl-S-acetylthioacetate (SATA), and then subjecting the
resultant to a known hydroxylamine treatment for deprotection of
S-acetyl group. Then, by filtering the resultant through a gel
filtration column, a solution of streptavidin capable of binding to
fluorescent dye-containing resin particles was obtained.
[0103] The thus obtained streptavidin solution was mixed with 1 mL
of a liquid containing fluorescent dye-containing resin particles
which was obtained by diluting the maleimide group-modified
fluorescent dye-containing resin particles with PBS containing 2 mM
of EDTA to a concentration of 1 nM, and the resulting mixture was
allowed to react at room temperature for 1 hour, whereby the
fluorescent dye-containing resin particles were bound with
streptavidin. The resultant was then centrifuged and washed with
PBS containing 2 mM of EDTA, and only streptavidin-modified
fluorescent dye-containing resin particles were recovered.
[0104] The thus obtained streptavidin-modified fluorescent
dye-containing resin particles were subjected to various
evaluations in a state of being once diluted with 1% BSA-containing
PBS buffer.
Examples 1 to 12 and Comparative Examples 1 to 16
Medium and Fluorescent Dye-Containing Resin Particles
[0105] As a medium, a Tris buffer containing 0.6% .alpha.-casein,
0.6% .beta.-casein, 3% BSA, 0.1% Tween (registered trademark) 20
and 0.015 N NaN.sub.3 (pH=6.9) was employed in Examples 1 to 6 and
Comparative Example 1; a PBS buffer containing 10% BSA, 0.1% Tween
(registered trademark) 20 and 0.05 N NaN.sub.3 (pH=7.6) was
employed in Examples 7 to 12 and Comparative Example 2; a PBS
buffer containing 1% BSA (pH=7.2) was employed in Comparative
Examples 3 to 9; and a polymeric surfactant (0.1% DISPERBYK-194:
pH=7.0) was employed in Comparative Examples 10 to 16.
[0106] Further, in Examples and Comparative Examples, as
fluorescent dye-containing resin particles, the
streptavidin-modified fluorescent dye-containing resin particles S1
to S7 were each used as shown in Table 1 below.
[0107] The fluorescent dye-containing resin particles were
subjected to the following storage and evaluations using the
medium.
(Storage of Fluorescent Dye-Containing Resin Particles)
[0108] The respective streptavidin-modified fluorescent
dye-containing resin particles contained in a 1% BSA/PBS solution
were subjected to removal of supernatant, substitution with the
medium and then a filtering treatment (0.65 .mu.m, manufactured by
Merck Millipore Corporation). Thereafter, the resultant was
adjusted by dilution with the medium to an intended concentration
of the streptavidin-modified fluorescent dye-containing resin
particles (0.2 nM), thereby giving a medium containing the
respective fluorescent dye-containing resin particles.
[0109] The fluorescent dye-containing resin particles were stored
in this form of being contained in the medium in a refrigerator at
4.degree. C.
(Evaluation of Precipitation and Aggregation of Fluorescent
Dye-Containing Resin Particles)
[0110] Precipitation and aggregation of fluorescent dye-containing
resin particles were evaluated using TURBISCAN (trademark)
(TURBISCAN Lab) manufactured by Formulaction SA.
[0111] Specifically, for the respective streptavidin-modified
fluorescent dye-containing resin particles of immediately after the
synthesis, a medium containing the fluorescent dye-containing resin
particles was prepared in accordance with the method described
above in the section of "Storage of Fluorescent Dye-containing
Resin Particles" and, for this medium containing the fluorescent
dye-containing resin particles, the backscatter intensity
(transmitted light) was measured by TURBISCAN using a light source
emitting an infrared radiation of 880 nm in wavelength. The
measurement was continued for 24 hours while sampling at 30-minute
intervals.
[0112] With the backscatter intensity (transmitted light) measured
at the center of the height immediately after the start of the
measurement (which corresponds to the "backscatter intensity
(transmitted light) measured at the center of the height . . .
immediately after the addition") being defined as "I'.sub.0" and
the backscatter intensity (transmitted light) measured at the
center of the height after allowing the medium containing the
fluorescent dye-containing resin particles to stand for 24 hours
after the start of the measurement being defined as "I'.sub.24",
the rate of change in the backscatter intensity (transmitted light)
at the center of the height, D' (%), was calculated as follows.
D'=(I'.sub.24-I'.sub.0)/I'.sub.0.times.100
[0113] Table 1 shows the rate of change (D') determined for the
respective Examples and Comparative Examples. For example, in
Example 5, based on the start of the measurement, the backscatter
intensity (transmitted light) showed a -0.9% change at 24 hours
after the start of the measurement.
(Staining with Fluorescent Dye-Containing Resin Particles)
[0114] In order to evaluate the performance of each medium, using
the respective streptavidin-modified fluorescent dye-containing
resin particles immediately after the synthesis and after one month
of storage in the medium, the following immunostaining,
morphological staining and observation were performed.
[0115] As a tissue cell slide, a breast cancer tissue array
manufactured by US Biomax, Inc. (model: BR243 Series (24-core);
core diameter=1.5 mm) was employed.
[0116] Immunostaining
[0117] The tissue cell slide was deparaffinized in accordance with
a conventional method and then washed by substitution with water.
The thus washed tissue cell slide was subjected to a 5-minute
autoclave treatment at 121.degree. C. in 10 mM citrate buffer (pH
6.0), thereby performing an antigen activation treatment.
[0118] After the activation treatment, the tissue cell slide was
washed with PBS buffer and then subjected to a 1-hour blocking
treatment with 1% BSA-containing PBS buffer in a moist chamber.
After the blocking treatment, an anti-HER2 rabbit monoclonal
antibody (4B5, manufactured by Ventana Medical Systems, Inc.)
diluted with 1% BSA-containing PBS buffer to a concentration of
0.05 nM was allowed to react with the tissue cell slide for 2
hours. After washing this tissue cell slide with PBS buffer, the
tissue cell slide was further allowed to react for 30 minutes with
a biotin-labeled anti-rabbit monoclonal antibody that would bind to
4B5 and had been diluted with 1% BSA-containing PBS buffer to a
concentration of 2 .mu.g/mL.
[0119] After the reaction with the biotin-labeled anti-rabbit
monoclonal antibody, the tissue cell slide was stained with the
fluorescent dye-containing resin particles of interest.
[0120] It is noted here that, for staining with the fluorescent
dye-containing resin particles immediately after the synthesis
thereof, the tissue cell slide was allowed to react for 3 hours
with the fluorescent dye-containing resin particles of immediately
after the synthesis that had been diluted with 1% BSA-containing
PBS buffer to a concentration of 0.2 nM, in a neutral pH
environment (pH 6.9 to 7.4) at room temperature. Prior to the
dilution of the fluorescent dye-containing resin particles to a
concentration of 0.2 nM, the solvent was substituted with the
medium by repeating appropriate times the operations of
centrifugation, removal of supernatant, dilution with the medium
and redispersion by ultrasonication, and the resultant was
subsequently subjected to a filtering treatment (0.65 .mu.m,
manufactured by Merck Millipore Corporation).
[0121] Meanwhile, staining with the fluorescent dye-containing
resin particles after one month of storage in the medium was also
performed in the same manner, except that the fluorescent
dye-containing resin particles that had been stored for one month
in the medium were used in place of the phosphor-containing resin
particles of immediately after the synthesis that were diluted to
0.2 nM. In this case, the fluorescent dye-containing resin
particles that had been stored in the form of the above-described
medium containing the fluorescent dye-containing resin particles
were subjected to pipetting (stirring) and then directly used for
staining without being diluted. In Examples 1 and 7, however, the
fluorescent dye-containing resin particles were directly used for
staining without being subjected to pipetting since no
precipitation thereof was observed. In the present specification,
unless otherwise specified, the term "pipetting" means stirring of
a liquid of interest that is performed by repeating the operations
of sucking up and discharging the liquid using a pipette.
[0122] In any of the above-described cases, after the reaction with
the fluorescent dye-containing resin particles, the tissue cell
slide was washed with PBS buffer.
[0123] Morphological Staining
[0124] The tissue cell slides subjected to the immunostaining were
each further subjected to morphological staining.
[0125] Specifically, the immunostained tissue cell slide was
subjected to hematoxylin staining (HE staining) for 1 minute using
Mayer's hematoxylin solution. Then, the tissue cell slide was
washed with running water of about 45.degree. C. for 3 minutes.
Next, an operation of immersing the tissue cell slide in pure
ethanol for 5 minutes was repeated four times to perform washing
and dehydration. Subsequently, an operation of immersing the tissue
cell slide in xylene for 5 minutes was repeated four times to
perform clearing. Lastly, the tissue section was mounted with a
mounting medium ("Entellan New", manufactured by Merck KGaA) to
give a sample slide for observation.
[0126] Observation
[0127] The tissue section on the sample slide that had been
subjected to the immunostaining and morphological staining was
allowed to emit fluorescence by irradiating thereto a prescribed
excitation light. The tissue section in this state was observed and
photographed under a fluorescence microscope (BX-53, manufactured
by Olympus Corporation). It is noted here that the observation and
photographing were performed in 10 visual fields for each core (a
single tissue spot) on the sample slide. In this process, an
objective lens of .times.40 magnification and an ocular lens of
.times.10 magnification were used. Further, the bright spots were
measured by ImageJ FindMaxima method.
[0128] The excitation light was set to have a wavelength of 575 to
600 nm through an optical filter. In addition, the wavelength range
(nm) of the fluorescence to be observed was also set at 612 to 682
nm through an optical filter.
[0129] The conditions of the excitation wavelength in the
microscope observation and image acquisition were set such that the
intensity of the irradiation light in the vicinity of the center of
the visual field was 900 W/cm.sup.2 for excitation at 580 nm. In
the image acquisition process, a photograph was taken by
arbitrarily setting the exposure time such that the image
brightness was not saturated (for example, the exposure time was
set at 4,000 .rho.s).
[0130] The evaluation results are shown in Table 1 below. For
reference, FIGS. 1, 3 and 5 show the stained images obtained using
the phosphor-containing resin particles immediately after the
synthesis in Examples 5 and 11 and Comparative Example 7,
respectively; and FIGS. 2, 4 and 6A show the stained images
obtained using the phosphor-containing resin particles after one
month of storage in Examples 5 and 11 and Comparative Example 7,
respectively.
[0131] As for the determination of the presence or absence of
coarse aggregates, an evaluation of "x" (presence of coarse
aggregates) was given when about 10 visual fields were observed for
each tissue cell slide and three or more aggregates having an
apparent size of 1 to 2-mm square (that is, an actual size
equivalent to 2.5 to 5-.mu.m square) or larger were observed under
a microscope. For example, in the case of Comparative Example 7, on
the stained image shown in FIG. 6A which was obtained using the
phosphor-containing resin particles after one month of storage,
three coarse aggregates were confirmed as illustrated in the sketch
of FIG. 6B.
TABLE-US-00001 TABLE 1 Evaluation of storage performance After 24
hours Used fluorescent Staining Rate of change in dye-containing
results the backscatter After one month resin particle immediately
intensity (transmitted of storage Particle after Composition light)
at the center Staining Pre-staining Type size (nm)
synthesis.sup..dagger. of medium of the height, D' (%)
results.dagger-dbl. operation Note Example 1 S1 40 .smallcircle.
(0.6% .alpha.-casein + -0.1 .smallcircle. none Example 2 S2 60
.smallcircle. 0.6% .beta.-casein + -0.2 .smallcircle. pipetting
Example 3 S3 80 .smallcircle. 3% BSA + 0.1% -0.4 .smallcircle.
pipetting Example 4 S4 100 .smallcircle. Tween 20 + 0.015N -0.8
.smallcircle. pipetting Example 5 S5 150 .smallcircle.
NaN.sub.3)/Tris buffer -0.9 .smallcircle. pipetting Example 6 S6
200 .smallcircle. -1 .smallcircle. pipetting Comparative S7 250 x
-2 x pipetting *1 Example 1 Example 7 S1 40 .smallcircle. (10% BSA
+ 0.1% 0 .smallcircle. none Example 8 S2 60 .smallcircle. Tween 20
+ 0.05N 0.1 .smallcircle. pipetting Example 9 S3 80 .smallcircle.
NaN.sub.3)/PBS buffer 0.3 .smallcircle. pipetting Example 10 S4 100
.smallcircle. 0.5 .smallcircle. pipetting Example 11 S5 150
.smallcircle. 0.8 .smallcircle. pipetting Example 12 S6 200
.smallcircle. -0.9 .smallcircle. pipetting Comparative S7 250
.smallcircle. -2.4 x pipetting *1 Example 2 Comparative S1 40
.smallcircle. 1% BSA/PBS -1.2 .DELTA. pipetting *1 Example 3 (prior
art) Comparative S2 60 .smallcircle. -1.4 .DELTA. pipetting *1
Example 4 Comparative S3 80 .smallcircle. -1.8 .DELTA. pipetting *1
Example 5 Comparative S4 100 .smallcircle. -2.4 .DELTA. pipetting
*1 Example 6 Comparative S5 150 .smallcircle. -2.6 .DELTA.
pipetting *1 Example 7 Comparative S6 200 .smallcircle. -3.2
.DELTA. pipetting *1 Example 8 Comparative S7 250 x -4.1 xx
pipetting *2 Example 9 Comparative S1 40 x Polymeric -1.3 xx
pipetting *2 Example 10 surfactant Comparative S2 60 x (0.1% -1.5
xx pipetting *2 Example 11 DISPERBYK-194) Comparative S3 80 x -1.9
xx pipetting *2 Example 12 Comparative S4 100 x -2.4 xx pipetting
*2 Example 13 Comparative S5 150 x -2.8 xx pipetting *2 Example 14
Comparative S6 200 x -3.5 xx pipetting *2 Example 15 Comparative S7
250 x -5.5 xx pipetting *2 Example 16 <.sup..dagger.Evaluation
of staining immediately after synthesis> .smallcircle.: The
tissue cell slide was stained. x: The tissue cell slide could not
be stained. <.sup..dagger-dbl.Evaluation of staining after one
month of storage> .smallcircle.: In comparison to the staining
performed immediately after the synthesis, 80% of the bright spots
were maintained. .DELTA.: At least 80% of the bright spots were
maintained; however, coarse aggregates were observed. x: Not more
than 80% of the bright spots were maintained and coarse aggregates
were observed. xx: The tissue cell slide could not be stained and
coarse aggregates were observed. <Note> *1: Stainable even
after one month when ultrasonication, solvent substitution and
filtering treatment were performed prior to the staining. *2: Not
stainable even when ultrasonication, solvent substitution and
filtering treatment were performed prior to the staining.
* * * * *