JP4985214B2 - Biological substance detection method using laser - Google Patents

Description

  The present invention relates to a biological material detection method and the like. More specifically, the present invention relates to a method for detecting a biological material using multiphoton excitation.

  Conventionally, detection of biological materials in tissues and cells has been performed using liquid high-speed chromatograph (hereinafter referred to as “HPLC”), ELISA (Enzyme-Linked ImmunoSorbent Assay) and the like. In these methods, first, a part of a tissue is extracted to prepare a homogenate, or a cultured cell is collected to prepare a lysate, and then the tissue homogenate or cell lysate is centrifuged to extract the target biological material. To prepare.

  Thereafter, in the method using HPLC, the obtained extract is fractionated, and the biological material fractionated into a specific fraction is detected by an ultraviolet absorption detector. In ELISA, an antibody specific for a biological substance is used, and the biological substance in the extract is detected by an antigen-antibody reaction.

Non-Patent Document 1 describes a method for detecting and measuring a glycated protein as a biological material using HPLC. Non-Patent Document 2 describes a method for detecting and measuring a glycated protein using ELISA.
J Clin Chem Clin Biochem. 1981 Feb; 19 (2): 81-87 Clin Chim Acta. 1989 Nov; 185 (2): 157-164

  In the conventional biological material detection method using HPLC or ELISA described above, it is necessary to prepare a tissue or cell extract or a biological material fraction, which is complicated. In addition, in the conventional method, since tissues and cells must be lysed, it is impossible to detect biological materials inside the tissues and cells while they are alive.

  Therefore, the main object of the present invention is to provide a biological material detection method that can perform measurement with a simpler operation than conventional methods and that can also detect biological materials in biological tissues and living cells.

In order to solve the above problems, the present invention uses multiphoton excitation to advance a predetermined reaction in a biological substance, and detects fluorescence from an autofluorescent substance that is a reaction product of the reaction. A method for detecting biological material is provided.
The biological substance may be a substance that exists in the living body.
Glycated proteins such as glycated hemoglobin, glycated albumin, and glycated globulin can be applied as the biological substance, and a terminal glycation product can be applied as the autofluorescent substance.
Moreover, aromatic amino acid-containing proteins such as hemoglobin, albumin, globulin, chromogranin, trypsin, and chymotrypsin may be used as the biological substance, and reaction products of these aromatic amino acid-containing proteins may be applied as the autofluorescent substance.
Furthermore, catecholamine or serotonin can be applied as the biological substance, and a reaction product of catecholamine or serotonin as the autofluorescent substance.
The present invention also provides a method for determining a disease state or determining a physiological function based on the fluorescence detection value obtained from the biological material detection method.

  Here, the definition of each term in this invention is demonstrated.

  “Biological substance” refers to a chemical substance present in a biological tissue or cell. Biological substances widely include amino acids and peptides, proteins, nucleotides and nucleosides, nucleic acids, sugars and lipids, vitamins and hormones, proteins containing metal elements and metal elements, and the like.

  “Multiphoton excitation” means that a single molecule simultaneously absorbs a plurality of photons (multiphoton absorption) and makes a transition to a state higher than the first electronically excited state. Multiphoton excitation can be induced by focusing femtosecond laser light having a pulse width of femtoseconds to picoseconds (sub-picoseconds) onto a target molecule with an objective lens.

  Excited molecules are highly reactive due to their high energy state and have a higher reaction rate than the original energy state. For this reason, even if the reaction proceeds only slowly in the original energy state, the reaction can proceed rapidly in the excited state.

  The excited molecules generate fluorescence when returning to their original energy state. By capturing this fluorescence, it is possible to detect molecules in the specimen.

  In multiphoton excitation, since excitation is performed by a plurality of photons, a laser having a long wavelength with lower energy than that of conventional one-photon excitation can be used. Also, the multiphoton excitation process occurs only when a plurality of photons reach the molecule almost simultaneously, so it is induced only near the focal point of the laser. Furthermore, since a long-wavelength laser excellent in deep reachability is used, it is possible to excite target molecules in the deep part from the specimen surface.

  “Aromatic amino acid-containing protein” means a protein containing tyrosine, phenylalanine and tryptophan (aromatic amino acid) having an aromatic ring in the chemical structure in the amino acid sequence. Aromatic amino acids have autofluorescence derived from an aromatic ring. For example, tryptophan emits autofluorescence near a wavelength of 360 nm when irradiated with excitation light near a wavelength of 270 nm. Therefore, aromatic amino acid-containing proteins have autofluorescence, but further, autofluorescence is remarkably enhanced and fluorescence wavelength is changed by reactions such as oxidation, reduction, cyclization, cross-linking, and dimer formation. It is known.

  According to the biological material detection method of the present invention, it is possible to detect a biological material with a simple operation, and it is also possible to detect a biological material in a biological tissue or a living cell.

  DESCRIPTION OF EXEMPLARY EMBODIMENTS Hereinafter, preferred embodiments for carrying out the invention will be described with reference to the drawings. In addition, embodiment described below shows an example of typical embodiment of this invention, and, thereby, the range of this invention is not interpreted narrowly.

  FIG. 1 is a conceptual diagram showing a procedure of a biological material detection method according to the present invention. This method uses a multiphoton excitation to advance a predetermined reaction to a biological substance to be detected (A) (indicated by symbol (A) in FIG. 1), and from the generated autofluorescent substance. A step (B) (symbol (B)) of detecting the generated fluorescence. Multiphoton excitation is utilized in both step (A) and step (B).

  First, in step (A), multiphoton excitation is performed on a target biological material. Since the excited biological substance is in a highly reactive state, the reaction rate is higher than that in the original energy state. Thereby, a predetermined reaction is rapidly advanced to the biological material, and the biological material is converted into an autofluorescent material which is a reaction product.

  Next, in step (B), multiphoton excitation is performed on the generated autofluorescent material. The excited autofluorescent material generates fluorescence when returning to its original energy state. By detecting this fluorescence, an autofluorescent substance is detected. Step (A) and step (B) need not be performed continuously as long as the autofluorescent material is held in a non-diffusing state.

  Here, since the autofluorescent substance is generated from the biological substance in step (A), the detection of the autofluorescent substance in step (B) means the detection of the biological substance present at the beginning of the predetermined reaction. .

  In addition, the fluorescence detection value (hereinafter also referred to as “fluorescence intensity”) detected from the autofluorescent material in step (B) depends on the amount of biological material that originally existed. Therefore, it is possible to measure the amount of the biological substance by measuring the fluorescence intensity of the autofluorescent substance.

  Thus, the biological material detection method according to the present invention is characterized in that the biological material is detected by detecting fluorescence from the autofluorescent material that is a reaction product thereof. Therefore, any biological substance that can generate an autofluorescent substance by a predetermined reaction can be widely detected.

  Examples of such biological substances include glycated proteins, aromatic amino acid-containing proteins, catecholamines, and serotonin. The glycated protein generates an advanced glycation end product (AGE), which is an autofluorescent substance, by a reaction called Maillard reaction. In addition, aromatic amino acid-containing proteins generate reaction products having autofluorescence by reactions such as oxidation, reduction, cyclization, crosslinking, and dimer formation. Similarly, catecholamines produced from the aromatic amino acid tyrosine and serotonin produced from tryptophan also produce autofluorescent substances by reactions such as oxidation, reduction, cyclization, crosslinking, and dimer formation. .

  Hereinafter, the method for detecting a biological material according to the present invention will be described in more detail with reference to an example in which a glycated protein is detected as the target biological material.

  In the present invention, “glycated protein” refers to a protein to which sugar has been added by a non-enzymatic reaction. Typical glycated proteins include glycated hemoglobin (HbA1c), glycated albumin, and glycated globulin. These are important diagnostic markers as an index of a hyperglycemic state in a diabetic state. This point will be described in detail later.

  A non-enzymatic glycosylation reaction that produces a glycated protein is generally called a glycation or Maillard reaction.

  FIG. 2 is a diagram for explaining the Maillard reaction. The Maillard reaction consists of an early reaction that generates an Amadori rearrangement product (indicated by symbol (A) in the figure) and a late reaction that changes the Amadori rearrangement product into an advanced glycation end product (AGE). (B)).

  In the first reaction (A), the aldehyde group of the reducing sugar reacts with the N-terminal amino group of the protein or the ε-amino group of the lysine residue to form a Schiff base, and then the Amadori rearrangement product is further produced by the Amadori rearrangement of the Schiff base Produces.

  The formation of a Schiff base by the reaction of reducing sugar and protein is a reversible reaction and the reaction proceeds rapidly. In contrast, the Amadori rearrangement is an irreversible reaction and proceeds extremely slowly. The above-mentioned glycated hemoglobin (HbA1c), glycated albumin, and glycated globulin are Amadori rearrangement products generated by this irreversible reaction.

  Furthermore, in the late reaction (B), the Amadori rearrangement product changes to AGE through complicated reactions such as dehydration, oxidation, condensation, and rearrangement.

  Since AGE is an autofluorescent substance that emits strong autofluorescence, the Amadori rearrangement product can be detected by detecting the fluorescence generated from this AGE.

  That is, first, by performing multiphoton excitation on the Amadori rearrangement product, the Maillard reaction proceeds rapidly, and the Amadori rearrangement product is converted to AGE. Next, multiphoton excitation is performed on the generated AGE, and fluorescence from the excited AGE is detected. Thereby, the Amadori rearrangement product which existed initially is detected. Furthermore, if the fluorescence intensity from AGE is measured at this time, the amount of Amadori rearrangement product can be measured. This method is applied when it is desired to detect and measure glycated hemoglobin (HbA1c), glycated albumin, and glycated globulin which are Amadori rearrangement products.

  Detection and measurement of aromatic amino acid-containing proteins, catecholamines, and serotonin can be performed according to the same principle.

  As described above, since aromatic amino acids have autofluorescence derived from aromatic rings, catecholamines produced from proteins containing aromatic amino acids, tyrosine, which is an aromatic amino acid, and serotonin produced from triprophane are self-generated. Has fluorescence. However, aromatic amino acid-containing proteins and catecholamines may significantly increase the fluorescence intensity of the reaction products or change the fluorescence wavelength due to reactions such as oxidation, reduction, cyclization, crosslinking, and dimer formation. It has been known.

  Therefore, by performing multiphoton excitation on aromatic amino acid-containing proteins or catecholamines, reactions such as oxidation, reduction, cyclization, cross-linking, dimer formation, etc. can proceed rapidly, and these reaction products can be obtained. If the fluorescence generated from the multiphoton excitation of the reaction product is detected (see FIG. 1B), the aromatic amino acid-containing protein or catecholamine and serotonin can be detected.

  Of the aromatic amino acid-containing proteins such as hemoglobin, albumin, globulin, chromogranin, trypsin, and chymotrypsin, chromogranin, especially in patients with neuro / endocrine tumors (brown cell types and pituitary tumors), has a markedly increased blood concentration. It is known to be used as a tumor marker. Moreover, chromogranin secreted from salivary glands or adrenal glands is also used as a mental stress marker. Globulin secreted into saliva is known to have a rhythm of the day, and is also used as a biorhythm marker.

Since trypsin and chymotrypsin are secreted from the pancreas, they serve as markers for the secretion ability of pancreatic juice. Hemoglobin and albumin are known to have a total rhythm of serum protein (TP) and secreted from the lacrimal gland and have a diurnal rhythm, and are also used as biorhythm markers.
Catecholamine is also a diagnostic marker for brown cell types in blood and urine concentrations. Furthermore, since catecholamine and serotonin are neurotransmitters, nerve activity can be monitored.

  Since trypsin, chymotrypsin, chromogranin, catecholamine, and serotonin are present at high concentrations in secretory vesicles until their function is expressed in the living body, it is considered that the above reaction can easily occur. Therefore, by measuring autofluorescence after the above reaction, the physiological function of the organ secreting trypsin, chymotrypsin, chromogranin, catecholamine, and serotonin can be monitored.

  Next, a method for measuring the amount of the target biological substance based on the fluorescence intensity of the autofluorescent substance in the present invention will be described.

    The amount (H) of the biological material can be expressed by the following formula (1).

(A fluorescence intensity obtained by multi-photon excitation of .Fn internal standard that represents the correction coefficient, Fn ₀ .Fh representing the fluorescence intensity obtained when no multi-photon excitation of the internal standard (back ground Wound value) is Fluorescence intensity derived from the autofluorescent material generated from the biological material by multiphoton excitation, Fh ₀ represents the fluorescence intensity (background value) obtained when the biological material is not subjected to multiphoton excitation, where the fluorescence intensity is In other words, it can be paraphrased as an increase in fluorescence per hour.

  The measurement of Fh is performed when all the biological material is changed to an autofluorescent material by multiphoton excitation and the fluorescence intensity obtained from the autofluorescent material is stabilized.

  Here, the internal standard substance is for calibrating the amount (H) of the biological substance. As the internal standard substance, an autofluorescent substance that exists universally in living tissues and living cells is used. For example, NADPH (nicotinamide adenine dinucleotide phosphate) which is a coenzyme is used.

The correction coefficient a is a coefficient for associating the amount of biological material measured by an existing method with the fluorescence ratio ((Fh−Fh ₀ ) / (Fn−Fn ₀ )). The correction coefficient a is calculated by performing statistical processing by converting a biological material whose amount has been measured in advance by an existing method into an autofluorescent material by multiphoton excitation, and obtaining the fluorescence intensity from the generated autofluorescent material. The correction coefficient a is set to a different numerical value for each biological substance to be detected and measurement conditions (laser light intensity, laser light irradiation time, etc.).

  Near-infrared femtosecond laser light having a pulse width of femtoseconds to picoseconds (sub-picoseconds) is used for multiphoton excitation of the target biological material or autofluorescent material. For the laser beam, a mode-locked laser is used to realize the above pulse width. As the mode-locked laser, titanium sapphire, an erbium-doped fiber laser medium, or the like is employed.

  Near-infrared femtosecond laser light (hereinafter also simply referred to as “laser light”) has a wavelength in the range of 650 nm to 1100 nm, and from this range, it matches the absorption wavelength of the biological substance and autofluorescent substance to be detected. It is selected appropriately. More specifically, for example, at a wavelength of 830 nm, the pulse width is 200 fs or less and the repetition frequency is 80 MHz. The output stability is about ± 0.5%, and the average light output is about 2W.

  This near-infrared femtosecond laser light is condensed to a focal point where a biological substance and an autofluorescent substance to be measured exist using a condenser lens (objective lens). An infrared transmissive lens is used as the condenser lens. The magnification of the lens is not particularly limited, but about 20 to 40 times is appropriate.

  The biological material and the autofluorescent material simultaneously absorb a plurality of photons of the focused laser beam near the focal point (the site where the biological material and the autofluorescent material are present), leading to a multiphoton excitation process. The biological material that has transitioned to the excited state is quickly converted into an autofluorescent material by a predetermined reaction. In addition, the autofluorescent substance that has transitioned to the excited state generates fluorescence when returning to the original energy state. By capturing this fluorescence, it becomes possible to detect and measure biological substances.

  The fluorescence is detected by collecting the fluorescence generated by the multiphoton excitation of the autofluorescent substance with a condenser lens and guiding it to the photoelectric conversion element. The photoelectric conversion element detects fluorescence, converts its intensity into data, and outputs the data to a connected computer.

  The biological material to be measured can widely include biological materials that can generate an autofluorescent material by a predetermined reaction. Under in vitro conditions, tissue or cell extracts or biological material fractions prepared according to conventional methods can be used. Biomaterials are detected and measured by performing multiphoton excitation on the biological materials in the extract and the biological material fraction solution. Note that the biological material detection method according to the present invention is capable of detecting not only the measurement under such in vitro conditions but also the biological material in living tissues and living cells (in vivo conditions). Have

  Hereinafter, biological substance detection under in vivo conditions will be described. The characteristics of the near-infrared femtosecond laser light are that it has a longer wavelength and lower energy than the conventional laser light used for one-photon excitation. In addition, since near-infrared femtosecond laser light is excited by a plurality of photons, only molecules near the focal point can be excited. Therefore, the multiphoton excitation by the near-infrared femtosecond laser beam has an advantage that a high depth reachability can be obtained and a long-time measurement can be performed while suppressing fluorescence fading and damage other than the focal point of the specimen.

  From these advantages, the biological material detection method using multiphoton excitation can be said to be an optimal method for detecting biological material in biological tissue or living cells. The feature that a high depth reachability is obtained means that a biological material existing inside (deep part) of a biological tissue can be detected. Further, the feature that fluorescence fading and damage other than the focal point of the specimen can be suppressed enables long-time measurement without damaging tissues or cells (non-invasively). Therefore, unlike the conventional method, the biological material detection method using multiphoton excitation can directly detect the biological material in the living tissue and living cells without preparing tissue homogenate or cell lysate.

  Hereinafter, taking the above-described glycated hemoglobin (HbA1c) as an example of the biological material, a specific method for carrying out the biological material detection method according to the present invention will be described.

  Glycated hemoglobin is obtained by binding sugar (such as glucose) in blood to erythrocyte hemoglobin by non-enzymatic reaction (Maillard pre-reaction). There are stable glycated hemoglobin in which glucose and hemoglobin form a Schiff base, and unstable glycated hemoglobin generated by Amadori rearrangement of stable glycated hemoglobin. HbA1c currently used as a diagnostic marker for diabetes is this unstable glycated hemoglobin (Amadori rearrangement product). HbA1c is considered to reflect the average blood glucose level over the past 1-2 months.

  FIG. 3A is a schematic diagram showing a method for measuring HbA1c in erythrocytes.

  In FIG. 3A, symbol S represents the skin surface, symbol V represents a blood vessel, and symbol E represents red blood cells in the blood vessel V. The measurement site is preferably a site where the skin surface S is smooth, the body hair is small, and the epidermis is thin. The blood vessel V is preferably a venule or capillary near the skin surface S. For example, venules or capillaries from the back of the lower arm to the wrist are suitable. By setting it as such a site | part, absorption and scattering of the laser beam on the skin surface can be prevented, and it is possible to detect fluorescence efficiently. Furthermore, the measurement accuracy can be further improved by applying water, oil, or the like to the skin surface to prevent scattering of laser light on the surface of the living body.

  Near-infrared femtosecond laser light (indicated by a broken line in the figure) is condensed by a condensing lens 28a to a point (focal point) in red blood cells E where HbA1c to be measured exists. At this time, the near-infrared femtosecond laser beam is highly absorbed by water and blood in the skin tissue and scattered by the tissue, and exhibits a high depth reachability. Moreover, since the invasiveness to the skin is extremely low, there is almost no skin damage.

  During measurement, the measurement site is shielded so that light from the environment does not enter. The irradiated laser beam particularly excites a region (an elliptical region surrounded by a dotted line in the figure) in which the XY plane has a diameter of about 500 μm and the Z-axis direction is about 1 mm. Excitation occurs deeper than the 5 mm skin surface at the working distance of the condenser lens L.

  The Amadori rearrangement product, HbA1c, is excited by multiphotons by the irradiated laser beam, promptly causing dehydration, oxidation, condensation, and rearrangement (late Maillard reaction) reaction, and converted to AGE. Further, this AGE is subjected to multiphoton excitation, and the intensity of the obtained fluorescence is measured. At the same time, autofluorescence obtained by multiphoton excitation of NADPH in red blood cells E is also measured.

  It is desirable to measure the fluorescence intensity of AGE and NADPH with the same erythrocytes in order to eliminate measurement errors. For this reason, the red blood cell image in the blood vessel V is acquired in real time by the CCD camera having infrared sensitivity, and the red blood cell E is tracked. By the image processing, the position of the red blood cell E in the visual field is sequentially measured, and the laser beam is continuously applied to the same part of the same red blood cell based on this position information.

In the above formula (1), Fn represents the fluorescence intensity obtained by multi-photon excitation of NADPH, and Fn ₀ represents the fluorescence intensity (background value) obtained when NADPH is not subjected to multi-photon excitation. Fh represents the fluorescence intensity derived from the AGE generated from HbA1c by multiphoton excitation, Fh ₀ is the fluorescence intensity obtained when no multi-photon excitation of HbA1c (background value).

  For the measurement of Fh, fluorescence at a wavelength of 350 to 400 nm is measured for 33 milliseconds from 1 millisecond after the start of laser irradiation. As for NADPH, fluorescence with a wavelength of 450-500 nm is measured for 33 milliseconds. The fluorescence intensity can be rephrased as the amount of increase in fluorescence during this period.

  For multiphoton excitation of HbA1c, an optical filter having a central wavelength of 520 nm and a wavelength region of 50 nm is used. Since AGE emits autofluorescence at wavelengths of 330-550 nm and its maximum is known to be at 350-400 nm, an optical filter with a central wavelength of 375 nm and a wavelength range of 50 nm is used to obtain the fluorescence intensity of AGE. . For obtaining NADPH fluorescence intensity, an optical filter having a central wavelength of 475 nm and a wavelength region of 50 nm is used.

  Alternatively, other glycoproteins such as glycated albumin and glycated globulin can be measured. Saccharified albumin is produced by binding sugar (such as glucose) in blood to serum albumin by a non-enzymatic reaction (Maillard pre-reaction). In addition, glycated globulin is produced by reacting sugar (such as glucose) with serum globulin. Serum albumin and serum globulin are collectively referred to as fructosamine. As with HbA1c, these are used as diagnostic markers that are indicators of hyperglycemia.

  When glycated albumin, glycated globulin, and fructosamine are measured, laser light is irradiated with the red blood cell E non-existing site in the blood vessel V as a focal point, as shown in FIG. At this time, the blood flow in the blood vessel V needs to be temporarily stopped.

  Glycated albumin and glycated globulin present near the focal point are converted to AGE by multiphoton excitation. The amount of glycated albumin or glycated globulin can be measured by subjecting this AGE to multiphoton excitation in the same manner as HbA1c and measuring the fluorescence intensity.

  At this time, as the optical filter to be used and the correction coefficient a in the above formula (1), an optimal one is appropriately selected according to the glycoprotein to be detected.

  As described above, the method for measuring the amount of the biological substance in the living tissue has been described by taking HbA1c in the erythrocyte as an example. The biological tissue that can be a measurement site in the present invention is not limited to blood vessels, and includes not only skin but also nails, ears, fingertips, lips, retina, hair, and the like, depending on the site where the target biological material is present. Further, the present invention is not limited to the tissues exposed on the body surface, and can be applied to internal organs such as the liver, brain, kidney, and muscle.

  For example, when the chromogranin is used as a neuro-endocrine tumor marker, the adrenal gland, thyroid gland, and hypothalamus are used as measurement sites. Moreover, when using also as a mental stress marker, a mandibular gland duct part becomes a suitable measurement site | part. For catecholamines and serotonin, the adrenal gland, sympathetic ganglion, and brain are used as measurement sites.

  In order to detect a biological substance in a body organ tissue, an optical fiber is used to guide laser light to a measurement site of the body organ and to collect fluorescence. Measurement using an optical fiber may be applied to detect a biological substance in the affected part (surgical part) tissue at the time of endoscopy or laparotomy.

  FIG. 4 is a schematic diagram showing a method for detecting a biological substance in a living cell.

  In FIG. 4, symbol C represents a cultured cell. In this figure, the cultured cells C are shown as being cultured in a petri dish. In addition to petri dishes, cell culture is not particularly limited, such as flasks and chamber slides, and any material that transmits laser light can be used.

  Near-infrared femtosecond laser light (indicated by a broken line in the figure) is condensed into a cultured cell C, which is a site where a biological material exists, by a condenser lens 28a. At this time, by collecting light on specific intracellular organelles such as nuclei, mitochondria, and Golgi apparatus, it is possible to detect biological substances present in these intracellular organelles. During measurement, light from the environment is shielded from contamination.

  Subsequently, an apparatus used for the biological material detection method according to the present invention will be described. FIG. 5 is a schematic diagram showing an example. As the basic structure of the apparatus, a known near-infrared multiphoton excitation upright microscope system can be used. The structure will be briefly described below with reference to the drawings.

  In FIG. 5, reference numeral 11 denotes a laser light source that oscillates with a near-infrared pulse. Laser light emitted from the laser light source 11 (shown by a broken line in the drawing) passes through a laser light diameter adjuster 21 for adjusting the laser light diameter, and functions as a galvanometer mirror for scanning the laser light on the specimen 4. 22 is reflected.

  Subsequently, the laser light enters the light branching unit 23. The light branching unit 23 includes an optical system using a dichroic filter, a dichroic mirror, and the like. The band-pass filter 24 in the figure transmits laser light and absorbs a specific wavelength of reflected light (shown by a dotted line in the figure) from the specimen 4 irradiated with light having a wavelength of 600 to 950 nm from the light source 61. After attenuation, the light is reflected to a CCD camera unit 32 described later.

  The laser light transmitted through the band pass filter 24 is guided to the light branching unit 25. Similar to the light branching unit 23, the light branching unit 25 is also composed of an optical system using a dichroic filter, a dichroic mirror, and the like. The illustrated dichroic mirror 26 transmits the laser light from the laser light source 11. Further, the fluorescence from the specimen 4 is selected to reflect a specific wavelength and reflected to a fluorescence detection unit 31 described later, and the reflected light is transmitted to the light branching unit 23.

  The laser light that has passed through the dichroic mirror 26 is condensed at the focal position 4 a of the specimen 4 by the condenser lens 28 a attached to the focal position adjusting mechanism 27. As the condenser lens 28a, the laser beam diameter adjuster 21, the band pass filter 24, and the dichroic mirror 26, those having infrared transmission properties are used. This applies to other optical filters unless otherwise specified. The magnification of the condensing lens 28a is not particularly limited, but about 20 to 40 is appropriate.

  The condensed laser light induces a multiphoton excitation process of the target biological material and the autofluorescent material at the focal position 4a. The condensing lens 28 a is attached in the focal position adjustment mechanism 27 so that the position in the vertical (height) direction in the figure can be adjusted. Thereby, the position in the vertical (depth) direction of the focal position 4a in the sample 4 can be adjusted.

  The condensing lens 28a condenses the fluorescent light and the reflected light emitted from the focal position 4a (indicated by a one-dot broken line and a dotted line in the figure, respectively) and guides it to the fluorescent detecting unit 31 and the CCD camera unit 32. Also works. The condensed fluorescence is first reflected by the dichroic mirror 26 at a specific wavelength. The reflected fluorescence is further transmitted through the short pass filter 29 and guided to the fluorescence detection unit 31. The short pass filter 29 has a characteristic of blocking a specific wavelength or longer.

  The fluorescence detection unit 31 has a function of selecting and receiving a specific wavelength, and uses one or more. The fluorescence detection unit 31 includes a photoelectric conversion element for detecting fluorescence. The fluorescence detector 31 is connected to a computer (not shown) for accumulating and processing data from the photoelectric conversion element. The photoelectric conversion element detects fluorescence, converts its intensity into data, and outputs the data to a connected computer. From this data (fluorescence measurement value), the amount (H) of the biological material is calculated according to the above equation (1).

  Further, the reflected light (shown by a dotted line in the figure) collected by the condensing lens 28a and transmitted through the dichroic mirror 26 of the light branching unit 25 is reflected by the bandpass filter 24 of the light branching unit 23, and the CCD camera unit 32. A long pass filter 33 is provided in front of the CCD camera unit 32. The long pass filter 33 has a characteristic of blocking below a specific wavelength.

  An image display means (not shown) is connected to the CCD camera unit 32. As a result, the normal white light source 61 is illuminated by the epi-illumination optical system, and the portion to be irradiated with the laser light is confirmed while the specimen 4 is confirmed on the monitor by the CCD camera unit 32 and the image display means which are also sensitive in the infrared region. It can be determined. The epi-illumination optical system includes a light source 61, a light diameter adjuster 71, and a long-pass filter 72 in FIG. The incident light from the light source 61 to the specimen 4 is not shown.

  Since the CCD camera unit 32 is also sensitive in the infrared region, for example, when the specimen 4 is skin, the red blood cells in the blood vessel can be observed using the skin permeability of infrared light. Is possible.

  As a result, as described above, the red blood cell image in the blood vessel can be acquired in real time and the red blood cell can be tracked. That is, it is possible to sequentially measure the position of red blood cells in the field of view by image processing of the acquired image, and continue to irradiate the same site of the same red blood cells based on this position information.

  The near-infrared femtosecond laser light used for the laser light source has a wavelength in the range of 650 nm to 1100 nm, and is appropriately selected in accordance with the absorption wavelength of the substrate and the substrate metabolite to be used from this range. More specifically, for example, at a wavelength of 830 nm, the pulse width is 200 fs or less and the repetition frequency is 80 MHz. The output stability is about ± 0.5%, and the average light output is about 2W.

  In this case, the fluorescence detected by the fluorescence detection unit 31 has a wavelength of 500 to 600 nm, for example, and the wavelength of the reflected light detected by the CCD camera unit 32 is 600 to 950 nm.

  The wavelength of the laser light and the wavelength of the fluorescence detected by the fluorescence detection unit 31 vary depending on the biological material and autofluorescent material to be detected. The respective characteristics of the band pass filter 24, the dichroic mirror 26, and the short pass filter 29 are appropriately optimized according to the target biological material and autofluorescent material.

  The specimen 4 includes a wide range of tissues exposed to the body surface such as skin, nails, ears, fingertips, lips, retina, and hair (see also FIG. 3). Alternatively, the specimen 4 can be a cultured cell, and the biological material in the living cell can be detected as shown in FIG.

  The biological material detection apparatus shown in FIG. 6 is characterized in that it includes an optical fiber 28c for transmitting laser light, fluorescence, and reflected light. This apparatus is intended to be applied as a specimen 4 to internal organs such as the liver, brain, kidney, and muscle. The optical fiber 28c is designed to transmit laser light and irradiate these internal organs.

  The laser light emitted from the laser light source 11 passes through the dichroic mirror 26 of the light branching section 25, is then guided into the optical fiber 28c by the condensing lens 28b, is transmitted through the optical fiber 28c, and is collected into the condensing lens (objective). Lens) 28a. Subsequently, the laser light is condensed at the focal position 4a of the specimen 4 by the condensing lens 28a, and the multi-photon excitation process of the biological material and the autofluorescent material is induced here.

  The fluorescence (and reflected light) generated from the focal position 4a by the multiphoton excitation process of the autofluorescent substance is collected by the condenser lens 28a, transmitted through the optical fiber 28c, and sent to the fluorescence detection unit 31 and the CCD camera unit 32. Led.

  It is assumed that the detection of the biological material in the organ tissue is performed at the time of endoscopy or laparotomy. For this reason, it is desirable to make the condenser lens 28a and the optical fiber 28c as small as possible.

  Finally, a method for determining a disease state or physiological function based on the fluorescence detection value obtained by the biological material detection method according to the present invention will be described.

  In the present invention, the biological material to be measured can include any biological material under in vitro and in vivo conditions, as already described. Here, as a biological material, the appearance or a large amount of accumulation of the biological material in biological tissue and living cells is measured by measuring the biological material associated with a specific disease or physiological function. It becomes possible to judge.

  Preferable examples include the above-mentioned glycated protein. Glycated proteins such as glycated hemoglobin, glycated albumin, glycated globulin, and fructosamine are used as diagnostic markers as an indicator of hyperglycemia under diabetes.

  In the conventional method, for example, in the case of HbA1c, it is necessary to first vote from a patient, separate and lyse red blood cells, fractionate hemoglobin by HPLC, and measure the fractionated HbA1c.

  In contrast, if the method for detecting a biological material according to the present invention is used, the amount of HbA1c in erythrocytes can be directly measured without performing such analysis using HPLC. It is possible to grasp the blood sugar state. This makes it possible to evaluate the risk of developing diabetes, prognosis determination, treatment results, etc. in a short time without giving a physical burden to the patient.

  In addition, the above-mentioned chromogranin, catecholamine, and serotonin can be directly measured in sympathetic ganglia, adrenal medulla, and brain without using HPLC or ELISA methods for the concentration in plasma or saliva, If the amount of chromogranin is measured in the mandibular gland duct, it is possible to easily evaluate the autonomic nervous function that defines the stress state.

  The biological material detection method according to the present invention can be used, for example, for detection of biological materials in biological tissues and living cells in pharmacological tests and safety tests in the field of drug discovery. Moreover, the biological substance detection method according to the present invention can be used for evaluation of risk of developing diseases such as diabetes, prognosis determination, treatment results, and the like.

It is a conceptual diagram which shows the procedure of the biological material detection method which concerns on this invention. It is a figure explaining a Maillard reaction. It is the schematic diagram which showed the method for measuring HbA1c in erythrocytes. It is the schematic diagram which showed the method for detecting the biological material in a living cell. It is a schematic diagram showing an example of the apparatus used for the biological material detection method which concerns on this invention. It is a schematic diagram showing the other example of the apparatus used for the biological material detection method which concerns on this invention.

Explanation of symbols

S Skin surface V Blood vessel E Red blood cell C Cell 11 Laser light source 21 Laser light diameter controller 22 Galvano mirror 23 Optical branching unit 24 Bandpass filter 25 Optical branching unit 26 Dichroic mirror 27 Focusing mechanism 28a Condensing lens (objective lens)
28b Condensing lens 28c Optical fiber 29 Short pass filter 31 Radiation wave detection unit 32 CCD camera unit 33 Barrier filter 4 Sample 4a Focal position 61 Light source 71 Light diameter adjuster 72 Long pass filter

Claims (3)

Concentrate near-infrared femtosecond laser light on red blood cells in blood vessels and use multiphoton excitation,
The glycated hemoglobin in the erythrocyte undergoes a reaction that changes to a terminal glycation product ,
By detecting autofluorescence from the terminal glycation product, which is the reaction product of the reaction,
A method for detecting glycated hemoglobin in a living body . The method according to claim 1, wherein the amount of the glycated hemoglobin is measured based on the intensity of the autofluorescence according to the following formula (I).
(In the formula, a represents a correction coefficient. Fn represents fluorescence intensity obtained by multiphoton excitation of the internal standard substance , and Fn ₀ represents fluorescence intensity obtained when the internal standard substance is not multiphoton excited. Fh represents multiphoton. (The fluorescence intensity derived from the terminal glycation product generated from glycated hemoglobin by excitation, and Fh ₀ represents the fluorescence intensity obtained when glycated hemoglobin is not multiphoton excited.)   Acquires images of red blood cells in blood vessels in real time, sequentially measures the position of red blood cells by image processing, and irradiates the same site of the same red blood cells with the near-infrared femtosecond laser light based on the obtained position information The method of claim 2.