Patent Application
20080020474
According to the method of analyzing a biosample using laser ablation and an apparatus therefor by the present invention, they are capable of analyzing the molecules in a biosample, and in the following explanation, a case where a biotissue section is used as the biosample to be analyzed will be described.
It is to be noted that, as a biosample to be analyzed in the method of analyzing a biosample using laser ablation and an apparatus therefor by the present invention, there are a smear sample of blood, saliva, sputum, urine or the like, a cultured cell, or an infection inspection, for example, other than the biotissue section.
The analyzing apparatus 10 has: a vacuum tank 12 that can be set to the vacuum level of 10−8 to 10−6 Torr; a target 14 of a sample being the biotissue section to be analyzed that is arranged in the vacuum tank 12; a time-of-flight mass spectrometer (TOF mass spectrometer) 16 as a mass spectrometer arranged in the vacuum tank 12; a rotational inlet terminal 18 that rotates the target 14; an ultra-short pulse laser generating unit 20 that outputs ultra-short pulse laser beams such as a femto-second laser beam, for example, and irradiates it on the target 14; a microscope unit 22 for observing the target 14; and an image analysis apparatus 24 including a display section 24a, which analyzes the image of the target 14 observed by the microscope unit 22 and displays its analytical result.
It is to be noted that the ultra-short pulse laser beams irradiated from the ultra-short pulse laser generating unit 20 are focused on the target 14 via an optical system such as a focusing lens (not shown) and a mirror (not shown).
Herein, although not shown in detail, the microscope unit 22 may be either an upright-type microscope (upright microscope) unit or an inverted-type microscope (inverted microscope) unit.
Further, both of the observation by the microscope unit 22 and irradiation of the ultra-short pulse laser beams by the ultra-short pulse laser generating unit 20 maybe performed from the same surface of the target 14 or may be performed from different surfaces. In other words, the ultra-short pulse laser beams are irradiated on one surface of the target 14 from the ultra-short pulse laser generating unit 20, and the state of the target 14 may be observed from the one surface by the microscope unit 22. Alternatively, the ultra-short pulse laser beams are irradiated on one surface of the target 14 from the ultra-short pulse laser generating unit 20, and the state of the target 14 may be observed from the other surface different from the one surface by the microscope unit 22, so that the changing state of target 14 caused by the irradiation of the ultra-short pulse laser beams by the ultra-short pulse laser generating unit 20 can be observed in situ in real time.
Specifically, when the upright microscope is used as the microscope unit 22, the objective lens of the upright microscope unit is arranged on the upper surface of the target 14, and the irradiation of the ultra-short pulse laser beams from the ultra-short pulse laser generating unit 20 can be performed from the lower surface of the target 14.
Similarly, when the upright microscope is used as the microscope unit 22, the objective lens of the upright microscope is arranged on the upper surface of the target 14, and the irradiation of the ultra-short pulse laser beams from the ultra-short pulse laser generating unit 20 may be performed from the upper surface of the target 14. Furthermore, in this case, the irradiation of the ultra-short pulse laser beams may be performed through the objective lens of the upright microscope.
On the other hand, when inverted microscope is used as the microscope unit 22, the objective lens of the inverted microscope is arranged on the lower surface of the target 14, and the irradiation of the ultra-short pulse laser beams from the ultra-short pulse laser generating unit 20 can be performed from the upper surface of the target 14.
Similarly, when the inverted microscope is used as the microscope unit 22 the objective lens of the inverted microscope is arranged on the lower surface of the target 14, and the irradiation of the ultra-short pulse laser beams from the ultra-short pulse laser generating unit 20 may be performed from the lower surface of the target 14. Furthermore, in this case, the irradiation of the ultra-short pulse laser beams may be performed through the objective lens of the inverted microscope.
In other words, when the ultra-short pulse laser beams are irradiated from the ultra-short pulse laser generating unit 20 on one surface of the target 14 and the state of the target 14 is observed from the one surface of the target 14 by the microscope unit 22, arrangement may be done in such a manner that the irradiation of the ultra-short pulse laser beams are performed through the objective lens of the microscope unit 22.
Further, as the ultra-short pulse laser generating unit 20, a unit such as a femto-second laser, for example, capable of irradiating the ultra-short pulse laser beams whose pulse time width is 1 femto second or more and 1 pico second or less, and whose peak value output is 1 mega watt or more and 10 giga watts or less can be used.
More particularly, such an ultra-short pulse laser generating unit 20 is constituted of a titanium sapphire laser, for example, and a unit having parameters shown below can be used. Specifically, they are as follows.
Peak width (pulse time width): up to 110 fs (femto seconds)
Output: 50 to 480 μJ (microjoule)
(peak value output: 0.5 to 4 GW (giga watts))
Wavelength: up to 800 nm (nanometers)
Repetition: 1 kHz (kilohertz)
Further, it goes without saying that various types of mass spectrometers such as a quadrupole mass spectrometer may be used as the mass spectrometer instead of the time-of-flight mass spectrometer.
Further, the focal distance of the focusing lens that focuses the ultra-short pulse laser beams outputted from the ultra-short pulse laser generating unit 20 is set to 25 cm, for example.
In the above-described constitution, description will be made for a method of performing mass spectrometry of molecules in the biotissue section by using the above-described analyzing apparatus 10.
Herein, in the mass spectrometry of molecules in the biotissue section by the present invention, mass spectrometry of molecules in the biotissue section is performed by using the ablation by the ultra-short pulse laser beams outputted from the ultra-short pulse laser generating unit 20 such as the femto-second laser and the analysis by the time-of-flight mass spectrometer 16.
In other words, the mass spectrometry of molecules in the biotissue section by the present invention, by irradiating the ultra-short pulse laser beams on the biotissue section to be analyzed and ablating the biotissue section, atomizes the molecules contained in the biotissue section into constituting elements, ionizes the atomized constituting elements, and analyzes the ionized constituting elements.
Specifically, the biotissue section is arranged in the vacuum tank 12 as the target 14, the ultra-short pulse laser beams such as a femto-second laser beam outputted from the ultra-short pulse laser generating unit 20 is irradiated on the biotissue section being the target 14 to perform ablation, and analysis is done by the time-of-flight mass spectrometer 16.
In this occasion, when labels are applied to the molecules in the biotissue section to be analyzed by element labels or the like, the biotissue section containing labeled molecules is arranged in the vacuum tank 12 as the target 14, the ultra-short pulse laser beams such as the femto second laser beam outputted from ultra-short pulse laser generating unit 20 are irradiated on the biotissue section being the target 14 to perform ablation, the labeled elements are measured by the time-of-flight mass spectrometer 16, and thus the molecules in the biotissue section to be analyzed can be detected and analyzed.
Herein, the irradiation position of the ultra-short pulse laser beams such as the femto second laser beam outputted from ultra-short pulse laser generating unit 20 can be determined in advance by observing the target 14 being the biotissue section by the microscope unit 22.
Moreover, an image at each point, where laser irradiation was performed on the target 14 being the biotissue section obtained by the microscope unit 22, is analyzed by the image analysis apparatus 24, the strength of labeled element at each point is displayed on the biotissue section image being the target 14 in a converted state into chromatic display on the display section 24a of the image analysis apparatus 24, a processing to allow the morphologic characteristics of the biotissue section and the measurement result to be simultaneously recognized is conducted, and thus data can be analyzed in the same style as a conventional in situ hybridization method.
Further, before setting the biotissue section as the target 14 on the analyzing apparatus 10 that performs ablation by the ultra-short pulse laser beams, the biotissue section is observed by the microscope unit 22 in advance, a reference point is provided on the biotissue section, a laser irradiation position is positioned by using the point as a reference, and thus an observed image by the microscope unit 22 and an analytical result of molecules measured by the ablation using the ultra-short pulse laser beams can be corresponded with each other.
It is to be noted that, in the present invention, molecules themselves that are labeled by isotopic elements are ionized on an atomic level and labeled elements can be detected, so that an applicable range of mass spectrometry can be dramatically widened. For example, isotopic elements can be used as labels, and the types of label can be increased to as many as 270 that is the number of stable isotopic elements, for example. Thus, amount of information can be dramatically increased comparing to the fluorescence method (2 types) and the radioactive isotopic elements (about 10 types) being conventional labeling methods.
In the following, description will be made for a method of fabricating a biotissue section, which is formed by slicing a mouse brain, as an example of fabricating a biosample.
Animal: As a model animal, 10 to 11 week-old male wild-type CD-1 mice and C57BL/6J purchased from Oriental Yeast Co., Ltd. were used.
Fixative as a reagent when preparing a mouse brain is as shown in the following table 1.
By using the above-described reagent, a paraffin section and a frozen section of the mouse brain were fabricated as biotissue sections by the following method.
<Method>
1) Fabricating a paraffin section
After anesthetizing a mouse by ether to eliminate pain response, an abdominal cavity was opened to expose a heart, 20 ml each of ice-cooled neutral fixative and acidic fixative were sequentially poured into the heart to perform perfusion fixation.
After that, the brain was removed, it was left to stand for 3 days in acidic fixative at 4° C. and fixed.
The fixed tissue was sliced to fabricate a section, and it was adhered on a glass slide. A paraffin section was fabricated by using an automatic in situ hybridization (in situ hybridization) unit manufactured by Ventana Medical Systems Inc.
2) Fabricating a frozen section
The brain was removed after dislocating a spinal cord under ether anesthesia, embedded in OCT and frozen by liquid nitrogen, and a section was created by a slicer manufactured by Leica Microsystems.
The cDNA sequence of a target gene was searched from RIKEN FANTOM clone. Furthermore, cDNA was referred to by the public database of LoucusLink and a target cDNA sequence was selected. The selected base sequence was transformed into an amino-acid sequence, and homology search was conducted by an NCBI Protein BLAST. A cDNA area corresponding to a low homology sequence was identified, and a primer was designed in order to amplify it as a template. Furthermore, depending on needs, a primer was designed in order to amplify a low homology sequence from a genome DNA as a template. Now, Table 2 shows the LoucusLink IDs of genes.
Spectrophotometer (Bio Spec-1600) manufactured by Shimadzu Corporation and GeneAmp PCR System 9700 manufactured by Applied Biosystems were used as equipment, and reagent shown in Table 3 was used, and plasmid purification was performed by the following method.
A plasmid DNA was purified from coliform bacillus by using the QIAprep Spin Miniprep Kit according to the attached protocol, absorbance was measured by the spectrophotometer, and concentration was calculated. PCR was performed by using a primer that was designed by using the Plasmid DNA as a template. Composition of reaction liquid and reaction conditions are shown below. Electrophoresis was applied to a reaction product in 2% agarose gel and confirmed.
1) Fabricating a stable isotopic labeling ssDNA probe
The stable isotopic labeling ssDNA probe was fabricated by the reaction liquid and the reaction condition shown in Table 4. Eu-labeled dUTP, Sm-labeled dUTP, Tb-labeled dUTP and Dy-labeled dUPT manufactured by PerkinElmer, Inc. were used as a labeled dNTP.
Phenol-chloroform extraction was performed to a PCR product and it was purified by the MicroSpin (trademark) column. The purified product was confirmed on agarose gel.
2) Fabricating a DIG-labeled RNA probe
A DIG-labeled RNA probe was fabricated by the reaction liquid and the reaction condition shown in Table 5.
By using the PCR product and a primer containing the recognition sequence of T7 and SP6 polymerase (*T7, SP6 adaptor), PCR was performed again. The reaction liquid composition and the reaction condition are shown on Table 6. Electrophoresis was applied to a reaction product in 2% agarose gel and confirmed.
Phenol-chloroform extraction was performed to a PCR product and it was purified by the MicroSpin (trademark) column.
Next, description will be made for the fabrication of a DIG-labeled RNA antisense probe and sense probe.
In other words, T7 polymerase was used in fabricating the antisense probe, SP6 polymerase was used in fabricating the sense probe, and they were incubated in the composition of reaction liquid shown in Table 7 at 37° C. for 2 hours. Electrophoresis was applied to a reaction product in 1% agarose gel and confirmed.
After the confirmation, 2 μl of DNaseI (10 U/μl) was added to remove DNA, and they were incubated at 37° C. for 30 minutes. Whether or not the DNA was removed was confirmed by performing electrophoresis in 1% agarose gel. When it was removed, 2 μl of 0.2 M EDTA was added to stop reaction, and the probes were purified in a BD CHROMA SPIN Column.
Accurately determining the obtained labeled DNA after the fabrication of probe is extremely important for optimizing the result of ISH and giving reproducibility. Now, diluted standard having a known amount, dilution series of an unknown amount of actually labeled probe, and dilution array of specimen are parallelly spotted, and they were examined by a laser irradiation method and a color development method using an anti-DIG antibody.
In the following, the determination of DIG-labeled probe will be described. First, equipment and reagent to be used will be described.
A shaker, a UV crosslinker and a nylon membrane (Amersham Pharmacia Biotech Hybond-N) were used as equipment, reagent as shown in Table 8 was used, and the determination of the DIG-labeled probe was performed by the following method.
<Method>
Dilution series of control RNA and of the fabricated probe was made on a 96-well plate, and diluted solution of each well was spotted on nylon membrane by 1 μl. After air-drying the membrane, it was processed (120 mJ/cm2) by a UV crosslinker. After the membrane was soaked in solution added with the blocking reagent and shaken by a shaker for 10 minutes, it was shaken by 5000-fold diluted liquid of Anti-DIG-AP for 30 minutes to perform the antibody reaction. After cleaning the membrane by TBS buffer, NBT/BCIP solution was added and shaken to perform color-developing reaction. After color was fully developed, MilliQ water was added and shaken for 10 minutes to stop the color-developing reaction. Then, it was washed by running water and air-dried, hermetically sealed by hybripack, spot signals of the control RNA and the fabricated probe were compared, and the determination of probe was performed.
1) Hybridization of marker gene
As shown in
2) Hybridization using simultaneous multiple probes
As shown in
Map2 marker gene DIG label
Tph2 target gene 1 Sm-labeled NM—173391
MaoB target gene 2 Eu-labeled NM—172778
AADC target gene 3 Dy-labeled NM—016672
Htr1B target gene 4 Tb-labeled NM—010482
3) High-sensitive hybridization
As shown in
In the following, description will be made for a result obtained by using a biotissue section as a biosample, irradiating the ultra-short pulse laser beams on the biotissue section to perform ablation, and analyzing the biotissue section.
In other words, the experiment result of an analysis performed by the analyzing apparatus 10, where the biotissue section of the mouse brain obtained by the method described in the above-described 1 to 4 was used as the target 14, is shown as follows.
This experiment is that the biotissue section fabricated as described above is used as the target 14, the ultra-short pulse laser beams such as the femto-second laser beam from the ultra-short pulse laser generating unit 20 are irradiated to perform ablation, and it is analyzed by the time-of-flight mass spectrometer 16.
More particularly, the target 14 fabricated as described above is installed in the vacuum tank 12, the inside of the vacuum tank 12 is evacuated such that the vacuum level in the vacuum tank 12 becomes 10−6 Torr or less.
Next, the ultra-short pulse laser beams outputted from the ultra-short pulse laser generating unit 20 are focused on the target 14 by using an optical system such as the focusing lens, and a region set on the target 14 is ablated.
Then, the mass of monovalent ions generated by the irradiation of the ultra-short pulse laser beams onto the target 14 is measured by the time-of-flight mass spectrometer 16.
Herein,
By using the position of the marker gene as an origin, regions (1 to 4) surrounded by squares in
Conditions in irradiating the ultra-short pulse laser beams are as follows.
Pulse width: 100 fs (femto seconds)
Laser power: 0.2 mJ (millijoule)
The ultra-short pulse laser beams were irradiated onto each of the regions (1 to 4) on the above-described irradiation conditions, ions generated by the irradiation of the ultra-short pulse laser beams were measured by the time-of-flight mass spectrometer 16, and labeled elements in each region were measured, and the mass spectrum shown in
Specifically,
From the above-described mass spectrum shown in
As shown in
Further,
As shown in
It is to be noted that, in this example, an image for each gene was obtained for each region, that is, 4 images for genes (1 to 4) were obtained for each region of the regions (1 to 4), but it goes without saying that the invention is not limited to this. The expression of a plurality of genes may allowed to be shown on the image of each region by changing the color of gene using color images.
Next, description will be made for detection using a Pt-labeled RNA probe.
The expression of the gene of microtubule-associated protein MAP2, which exists on dendrite in a large volume, in the mouse brain was investigated.
It is to be noted that the preparation of the mouse brain, the design of the primer, and the purification of plasmid were performed in the same manner as “Example 1”.
1) Fabricating Pt-labeled RNA probe
A template DNA was created by the reaction liquid and reaction condition shown in
By using the PCR product and the primer containing the recognition sequence (*T7, SP6 adaptor) of T7 and SP6 polymerase, PCR was performed again. The reaction liquid composition and the reaction condition are as shown in Table 6. Electrophoresis was applied to the reaction product in 2% agarose gel and confirmed.
Phenol-chloroform extraction was conducted to the PCR product and it was purified by a MicroSpin (trademark) column.
Next, by using T7 polymerase in fabricating an antisense probe and by using SP6 polymerase in fabricating a sense probe, and they were incubated in the composition of reaction liquid shown in Table 9 at 37° C. for 2 hours. Electrophoresis was applied to a reaction product in 1% agarose gel and confirmed.
After the confirmation, 2 μl of DNaseI (10 U/μl) was added to remove DNA, and they were incubated at 37° C. for 30 minutes. Whether or not the DNA was removed was confirmed by performing electrophoresis in 1% agarose gel. When it was removed, 2 μl of 0.2M EDTA was added to stop reaction, and the probes were purified in a BD CHROMA SPIN Column.
The purified RNA was labeled by using a ULYSIS Nucleic Acid Labeling Kit (manufactured by Molecular Probe Inc.). After 1/10 volume of 3M NaAcO (pH 5.2) and two-fold volume of ethanol were added to 1 μg of the purified RNA and left it to stand at −70° C. for 30 minutes, it was centrifuged on 12000 rpm at 4° C. for 15 minutes.
After washing a pellet by 70% ethanol and air-drying it, it was dissolved in 20 μl of labeling buffer (Component C). This was incubated at 95° C. for 5 minutes and placed on ice. By spinning it down to collect water droplets on the bottom of a tube, 5 μl of ULS labeling reagent (Alexa Fluor 532) was added, the labeling buffer (Component C) was added to make the total volume of 25 μl. After incubating at 90° C. for 10 minutes, it was placed on ice to stop reaction, and water droplets were collected on the bottom of the tube by spinning it down. 100 μl of TE was added to the labeled sample, and it was purified by a MicroSpin S-400 to form a Pt-labeled RNA probe.
Similar to Example 1, hybridization reaction was performed by using the automatic in situ hybridization (in situ hybridization) unit manufactured by Ventana Medical Systems Inc.
20 μl of the RNA probe was used for the in situ hybridization.
In the case of using the Pt-labeled antisense probe, peaks (mass number 194, 195, 196) derived from Pt are observed, while no peak of Pt was detected in the negative control.
This matches well the result by a conventional staining method, which is shown for comparison, and it was made clear that the analysis using the Pt-labeled antisense probe was useful in analyzing the expression of gene.
It is to be noted that the above-described embodiments can be modified as shown in (1) to (12) below.
(1) In the above-described embodiments, the time-of-flight mass spectrometer that performs mass spectrometry by measuring the time of flight of atoms was used as a mass spectrometer, and mass spectrometry of a plurality of atoms can be performed simultaneously by one-time laser irradiation when the time-of-flight mass spectrometer is used. Further, even in the case where the ion cyclotron Fourier transform mass spectrometer is used as the mass spectrometer, mass spectrometry of a plurality of atoms can be performed simultaneously.
(2) In the above-described embodiments, description was made for mass spectrometry as an analysis method of molecule, but it goes without saying that the invention is not limited to this and the present invention may be used for analysis other than mass spectrometry.
(3) In the above-described embodiments, the rotational inlet terminal 18 that rotates the target 14 was used as the moving means for moving the target 14, but it goes without saying that the invention is not limited to this and appropriate moving means such as a freely movable table capable of mounting the target 14 thereon may be used.
(4) In the above-described embodiments, the target 14 was ablated without omission and duplication by rotating the target 14 with the use of the rotational inlet terminal 18, but it goes without saying that the invention is not limited to this and moving means for moving an irradiation position of the ultra-short pulse laser beams on the target may be provided to ablate the target 14 without omission and duplication.
(5) In the above-described embodiments, hybridization was shown as an example where the nucleic acid probe bonded to a particular target, but it goes without saying that the invention is not limited to this and bond such as aptamer is also acceptable other than hybridization.
The present invention is utilized in the analysis of biotissue in the field of life science such as medical science and biochemistry.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2004-097260 | Mar 2004 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP05/05809 | 3/29/2005 | WO | 00 | 6/28/2007 |
