MapAnalyzer-脑细胞荧光显微系统-安捷来国际股份（香港）有限公司

脑细胞荧光显微系统：

Mapping FluorescenceMicrophotometry System

MapAnalyzer

Bringing new techniques to your research.

In addition to conventionalobservation and photomicrographic techniques, the quantitative andqualitative analysis of specimen through microphotometry is moreａnd more in demand. In the medical and biological fields,microphotometry is established technique for quantitative analysisof various intercellular substances. MapAnalyzer was developed for quantitative microanalysis ofdistribution of various substances, such as neurotransmitters andneuromodulators, in the large tissue slice. With Yamato ScientificCo.fs MapAnalyzer, the possibilities of your research will beextended.

- Application -

(1)	Quantitative analysis of immunohistochemical fluorescence Distribution of substances in animal and human slices Detection of abnormal changes of substances in diseased tissues Detection of effect of drugs on the central nervous system and others in the animal experiments Elimination of non-specific autofluorescence Comparison analysis among various substances in the same slice
(2)	Quantitative analysis of enzyme-labelled immunohistochemistryａnd various histochemistry
(3)	Measurement of fluorescence chelating agent or fluorescenceligand
(4)	Quantitative analysis of DNA or RNA
(5)	Quantitative analysis of autoradiographs or X-ray film
(6)	Cytotoxicity testing

Quantitative immunohistochemicaldistribution
of calmodulin-dependent protein kinase II in the
coronal slice of a rat brain.

An image of the quantitativedistribution of a substance can be obtained as follows: (1) thetarget substance labeled by immunofluorescent staining in amicroarea of a slice is illuminated by a fine excitation beam (theminimum diameter on a slice is 5 µm) which is narrowed by a fielddiaphragm and aperture diaphragm; (2) the fluorescence in this areais collected into the photometer by the objective lens through aphotometry diaphragm, and its intensity is measured; (3) the sliceis moved by a two-dimensional scanning stage (the maximum stagemotion is 140 mm x 140 mm), and the fluorescence intensity in thenext microarea is measured; (4) the measured fluorescence intensityin each microarea is collected in a host computer, where it isanalyzed for reconstruction of an image of the entire scanned area;ａnd (5) the image can be viewed as a close-up, in full or at anangle, and displayed quantitatively as a colored or monochromaticimage. The actual intensity in a specific region is displayed whenthat region is selected by a cursor on the image on a TV monitor.

A second halogen lamp and a band-pass filter of variouswavelengths in the visible region are mounted under the scanningstage. Thus, slices stained for histochemical or enzyme-labeledimmunohistochemical analysis as well as films such asautoradiographs, can be analyzed quantitatively.

Relationship between fluorescence intensity and quinine sulfateconcentration. Fluorescence intensity originating from 0.0005 to 50mM quinine sulfate in 0.1 N sulfuric acid was finely measured usinga MapAnalyzer or image analyzer (Hamamatsu, C1966, Japan) combinedwith an SIT camara (Hamamatsu, TH9659). Working curves from theMapAnalyzer at each photomultiplier voltage and from the imageanalyzer are indicated by solid lines (numbers indicatephotomultiplier voltage) and a dotted line, respectively. Themeasurement conditions were as follows: excitation range, 385 to425 nm; band-pass interference filter, 470 nm; and objective lens,20x/0.75 (magnification/ numerical aperture).
(See, Folia Pharmacol. Jpn. 91: 173-180, 1988)

Quantitativeimmunohistochemical
Distribution of tyrosine hydroxylase
in the coronal slice of a rat brain.

The quantitative linearity, sensitivity and resolution ofMapAnalyzer surpass those of image analyzers used with TV cameras,ａnd the sensitivity, reproducibility and facility of this methodare greater than those of the HPLC method. Also, the measuring areaof this analyzer is far larger than that of laser confocalmicroscopes.
(See, J. Neurosci. Methods 85: 161-173, 1998)


Quantitative immunohistochemicaldistribution of tyrosine hydroxylase in the coronal slice of a ratbrain. The stained slice was measured at 20-µm intervals, and thedistribution of the intensity obtained from approximately 250,000microareas is displayed. Markedly intense tyrosine hydroxylase-likeimmunoreactivity was distributed in the dorsolateral area of theneostriatum, nucleus accumbens, olfactory tubercle and motorcortex.
	Quantitative immunohistochemical distributions of glutamatedecarboxylase (left side) and substance P (right side) in the sameslices of a rat brain. Anterior (upper) and posterior (bottom)regions of the brain were analyzed. Double-stained slice wasmeasured at 20-µm intervals. Markedly intense glutamatedecarboxylase-like and substance P-like immunoreactivities weredistributed in the ventral pallidum and substantianigra.

Elimination of autofluorescence from the human brain slice
A: Nonspecific autofluorescence
B: Immunohistochemical fluorescence superimposed on nonspecificautofluorescence
C: Quantitative immunohistochemical distribution of cholineacetyltransferase in the
normal human brain slice, i.e., A-B

Pure immunohistochemical fluorescence intensities can beobtained automatically from human brains containing variousautofluorescences using MapAnalyzer.
Measuring points: approximately six millions at 50-µmintervals.
(See, J. Neurosci. Methods 85: 161-173, 1998)

Quantitative immunohistochemical distribution ofsubstance P in a brain slice of an ***** normal human (male, age50). Data were obtained from approximately six million regions inthe brain at 50-µm intervals. Conspicuously intense substanceP-like immunoreactivity was observed in the internal segment of theglobus pallidus. The immunoreactive intensity in the internalsegment of the globus pallidus was approximately twice as high asthat in the external segment of the globus pallidus.
(See, Neurosci. Res. 35: 339-346,1999)

Quantitative distribution of Nissl bodies in ahuman brain slice. The slice was stained with cresyl violet, andthe distribution in the globus pallidus and putamen area wasanalyzed through measurement of the transmission densities. GPe,external segment of globus pallidus; GPi, internal segment ofglobus pallidus; Pt, putamen.
(See, Neurosci. Res. 35: 339-346, 1999)

Quantitative distribution of bone calcification ofspontaneously hypertensive rats (SHR) and normotensive Wistar Kyotorats (WKY). X-ray film was reversed and the degree of blackeningwas measured. A higher level of calcification is observed in thevarious bones of SHR compared with those of the WKY.
(See, Brain Res. Bull. 30: 107-113, 1993)

-Specification -

Microscope	Epi-fluorescence microscope
Photometry	Fluorescence and Transmittance mode
Measuring spot	Min. diameter: 20 µm on a slice (standard) 5 µm on a slice (option)
Light source	Halogen lamp
Detector	Photomultiplier tube
Scanning stage	Motor-driven X-Y stage Min. stepping movement: 1 µm, Max. stage motion: 140x140 mm Scanning speed: 10 mm/s
Dark box	Desktop type
Computer	OS: Windows NT or Windows 2000
Software	Newly developed software for automatic analysis Save mode: CSV format or BMP file Graphics: two-dimensional and three-dimensional display

-REFERENCES -

Technical report:

Sutoo D, Akiyama K, Maeda I. The development of a highsensitivity and high linearity fluorescence microphotometry systemfor distribution analysis of neurotransmitter in the brain. FolPharmacol Jpn 91:173-180, 1988.

Sutoo D, Akiyama K, Yabe K. Quantitative mapping analyzer fordetermining the distribution of neurochemicals in the human brain.J Neurosci Methods 85:161-173, 1998.

Animal experiment:

Sutoo D, Akiyama K, Geffard M. Central dopamine-synthesisregulation by the calcium-calmodulin-dependent system. Brain ResBull 22:565-569, 1989.

Sutoo D, Akiyama K, Imamiya S. A mechanism of cadmiumpoisoning: the cross effect of calcium and cadmium in thecalmodulin-dependent system. Arch Toxicol 64:161-164, 1990.

Sutoo D, Akiyama K, Takita H. Behavioral changes incold-stressed mice related to a centralcalcium-dependent-catecholamine synthesizing system. PharmacolBiochem Behav 40:423- 428, 1991.

Sutoo D, Akiyama K, Yabe K, Kohno K. Multiple analysis oftyrosine hydroxylase and calmodulin distributions in the forebrainof the rat using a microphotometry system. Brain Res Bull26:973-982, 1991.

Akiyama K, Yabe K, Sutoo D. Quantitative immunohistochemicaldistributions of tyrosine hydroxylase and calmodulin in the brainsof spontaneously hypertensive rats, Kitasato Arch. Exp.Med. 65:199-208, 1992.

Sutoo D, Akiyama K, Takita H. The effect of convulsions on therectification of central nervous system disorders in epilepticmice. Physiol Behav 52:865-872, 1992.

Sutoo D, Akiyama K, Matsukura T, Nakamoto RK. Decrease ofcentral dopamine level in the ***** spontaneously hypertensive ratsrelated to the calcium metabolism disorder. Brain Res Bull30:107-113, 1993.

Calmodulin-dependent protein kinase II

Sutoo D. Disturbances of brain function by exogenous cadmium.In: Isaacson RL, Jensen KF, editors. The vulnerable brain andenvironmental risks, vol 3, toxins in air and water. New York:Plenum Press. pp 281-300, 1994.

Nakamura M, Fujimura Y, Yato Y, Watanabe M, Yabe Y. Changes incholine acetyltransferase activity and distribution followingincomplete cervical spinal cord injury in the rat. Neuroscience75:481-494, 1996.

Sutoo D, Akiyama K. Regulation of blood pressure withcalcium-dependent dopamine synthesizing system in the brain and itsrelated phenomena. Brain Res Rev 25: 1-26, 1997.

Nakagawasai O, Tadano T, Hozumi S, Tan-No K, Niijima F, KisaraK. Immunohistochemical estimation of brain cholineacetyltransferase and somatostatin related to the impairment ofavoidance learning induced by thiamine deficiency. Brain Res Bull52:189-196, 2000.

Hanawa M, Asano T, Akiyama K, Yabe K, Tsunoda K, Tadano T,Sutoo D. Effect of Zena F-III^R, a liquid nutritive andtonic drug, on the neurochemical changes elicited by physicalfatigue in mice. Pharmacol Biochem Behav 66: 771-778, 2000.

Human brain analysis:

Sutoo D, Akiyama K, Yabe K, Kohno K. Quantitative analysis ofimmunohistochemical distributions of cholinergic andcatecholaminergic systems in the human brain. Neuroscience58:227-234, 1994.

Sutoo D, Yabe K, Akiyama K. Quantitative imaging of substance Pin the human brain using a brain mapping analyzer. Neurosci Res35:339 -346, 1999.

Sutoo D, Akiyama K, Yabe K. Quantitative maps of GABAergic andglutamatergic neuronal systems in the human brain. Hum Brain Map11:93-103, 2000.

Sutoo D, Akiyama K, Yabe K. Quantitative imaging of tyrosinehydroxylase and calmodulin in the human brain. J Neurosci Res63:369-376, 2001.

Tyrosine hydroxylase