We thank Yumiko Iwamoto for supporting the production of the video. We are also grateful to Akihiro Isomura for discussion and supports of image analysis, Hitoshi Miyachi for technical supports for generation of transgenic animals, Yuji Shinjo (Olympus Medical Science), Masatoshi Egawa (Olympus Medical Science), Takuya Ishizu (Olympus Medical Science) and Ouin Kunitaki (Andor Japan) for the technical support and discussions of the bioluminescence imaging system. This work was supported by Core Research for Evolutional Science and Technology (JPMJCR12W2) (R.K.), Grant-in-Aid for Scientific Research on Innovative Areas (MEXT 24116705 for H.S. and MEXT 16H06480 for R.K.), Grant-in-Aid for Scientific Research (C) (JSPS 18K06254) (H.S.), Takeda Foundation (R.K. and H.S.), and Platform for Dynamic Approaches to Living System from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

All the procedure including animal subjects have been approved by Institutional Animal Care and Use Committee at the institute for Frontier Life and Medical Sciences, Kyoto University.
1. Bioluminescence reporters
NOTE: The luciferase reporter is suitable for measuring the rapid dynamics of promoter activity by fusing the degradation signal. Moreover, the luciferase fusion reporter enables monitoring of the protein dynamics in the single cell. Both types of reporters ...

<ol>
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The authors have no conflicting financial interest.

The components of Notch signaling show oscillatory expressions in synchrony during somitogenesis but out of synchrony during neurogenesis, leading to the difficulties in capturing the expression dynamics by static analysis in the latter case. Thus, real-time monitoring is required to reveal the expression dynamics of Notch signaling components, such as Hes1 and Dll1. Because the periods of the expressions of Hes1 and Dll1 oscillations are extremely short, approximately 2-3 h, rapid res...

The mammalian brain is composed of a large number of various types of neurons and glial cells. All cells are generated from neural stem/progenitor cells (NPCs), which first proliferate to expand their numbers, then start to differentiate into neurons, and finally give rise to glial cells1,2,3,4,5. Once cells have differentiated into neurons, they cannot proliferate or increase their numbers, and, therefore, the maintenance of NPCs until later stages is important. Notch signaling via cell-cell interactions plays an important role in maintaining NPCs6,7. Notch ligands interact with the membrane protein, Notch, on the surface of neighboring cells and activates the Notch protein. After activation, proteolysis of Notch protein occurs, thereby releasing the intracellular domain of Notch (NICD) from the cell membrane into the nucleus8,9,10. In the nucleus, NICD binds to the promoter regions of Hes1 and Hes5 (Hes1/5) and activates the expression of these genes. Hes1/5 repress the expression of the proneural genes Ascl1 and Neurogenin1/2 (Neurog1/2)11,12,13,14. Because proneural genes induce neuronal differentiation, Hes1/5 play essential roles in maintaining NPCs. Furthermore, as proneural genes can activate the expression of the Notch ligand Delta-like1 (Dll1), Hes1/5 also repress the expression of Dll1. Therefore, the expression of Dll1 leads to neighboring cells being negative for Dll1 via Notch signaling. In this way, cells inhibit adjacent cells from following their same fate, a phenomenon known as the lateral inhibition8. In the developing brain, lateral inhibition plays a role in generating various different cell types.
Real-time imaging at the single cell level reveals dynamic expressions of the components of Notch signaling in NPCs15,16,17. Notch signaling activates the expression of Hes1, but Hes1 protein binds to its own promoter and represses its own expression. Furthermore, Hes1 is an extremely unstable protein, that is degraded by the ubiquitin-proteasome pathway; therefore, the repression of its own promoter is only short lived and then the transcription starts again. In this way, the expression of Hes1 oscillates at both the transcription and translational levels in a 2 h cycle18. The oscillatory expression of Hes1, in turn, induces the oscillatory expression of the downstream target genes, such as Ascl1, Neurog2, and Dll1, via periodic repression15,16,17,19. While proneural genes can induce neuronal differentiation, their oscillatory expression is not sufficient for neuronal differentiation; rather their sustained expression is essential for the neuronal differentiation. The oscillatory expression of proneural genes is important for maintaining NPCs rather than for inducing neuronal differentiation14,15,16. The expression of Dll1 oscillates at both the transcription and translational levels during various morphogenesis, such as neurogenesis and somitogenesis. The dynamic expression of Dll1 is important for the normal morphogenesis and steady expression of Dll1 induces defects in neurogenesis and somitogenesis17. These findings demonstrate the important function that the dynamics of gene expression and protein kinetics have on the regulation of various developmental events (i.e., different expression dynamics produce different outputs in cellular behaviors).
To analyze the dynamics of Notch signaling, the static analysis of tissues and cells are insufficient because they are constantly changing. Real-time imaging of single cells is a powerful tool to reveal the dynamics in gene expression. The dynamic expression of Notch signaling molecules undergo rapid cyclic responses in the period of 2-3 h. This rapid periodic expression presents two difficult problems for the real-time monitoring: (1) the expression of the molecules is suppressed to low levels, and (2) rapid turnover requires fast-response reporters. To overcome these problems, we previously developed a bioluminescence real-time imaging method20. Because the bioluminescence reporter has a higher sensitivity and shorter maturation time than fluorescent reporters, this strategy enables us to monitor the rapid dynamics in living cells. Using real-time visualization, we found that more genes exhibited dynamic expression than we had previously thought. In addition, the number of reports showing expression and protein dynamics in living cells and the significance of these dynamics in various biological events has increased, suggesting a fundamental role of the dynamics in gene expressions21,22.
In this report, we describe a way to visualize the expression of the Notch ligand Dll1 in NPCs in both dissociated cultures and in cortical slice cultures. To monitor the dynamics of Dll1 transcription at single cell levels, we generated dissociated cultures of NPCs derived from the embryonic telencephalon of transgenic mice carrying pDll1-Ub-Fluc reporter, a Dll1 promoter-driven destabilized luciferase reporter. To monitor Dll1 protein dynamics in vivo, we introduced the Dll1-Fluc fusion reporter into NPCs in the cortex and visualized the expression of the reporter in NPCs in cortical slice cultures. Real-time imaging enabled us to capture the various features of gene expression and protein dynamics in living cells at high temporal resolution.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bioluminescence Imaging System</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chilled water circulator (chiller)</td>
			<td>Julabo</td>
			<td>Model: F12-ED</td>
			<td></td>
		</tr>
		<tr>
			<td>Cooled CCD camera</td>
			<td>Andor Technology</td>
			<td>Model: iKon-M 934</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator system</td>
			<td>TOKAI HIT</td>
			<td>Model: INU-ONICS</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Olympus</td>
			<td>Model: IX81</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Olympus</td>
			<td>Model: IX83</td>
			<td></td>
		</tr>
		<tr>
			<td>LED illumination device</td>
			<td>CoolLED</td>
			<td>Model: pE1</td>
			<td></td>
		</tr>
		<tr>
			<td>MetaMorph</td>
			<td>MOLECULAR DEVICES</td>
			<td>Model: 40000</td>
			<td></td>
		</tr>
		<tr>
			<td>Mix gas controller</td>
			<td>Tokken</td>
			<td>Model: TK-MIGM OLO2</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective lens</td>
			<td>Olympus</td>
			<td>Model: UPLFLN 40X O</td>
			<td></td>
		</tr>
		<tr>
			<td>Preparations for Dissection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Nikon</td>
			<td>Model: SMZ-2B</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence stereoscopic microscope</td>
			<td>Leica</td>
			<td>Model: MZ16FA</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>DUMONT</td>
			<td>INOX No.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors, Micro scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ring-shaped forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10-cm plastic petri dish</td>
			<td>greiner</td>
			<td>664160-013</td>
			<td></td>
		</tr>
		<tr>
			<td>35-mm plastic petri dish</td>
			<td>greiner</td>
			<td>627160</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Nacalai Tesque</td>
			<td>14249-24</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>invitrogen</td>
			<td>11039-021</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents for NPC dissociation culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>invitrogen</td>
			<td>12587-010</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>invitrogen</td>
			<td>13256-029</td>
			<td>Stock solution: 1 &#956;g/ml in 0.1% BSA/PBS</td>
		</tr>
		<tr>
			<td>D-luciferin</td>
			<td>Nacalai Tesque</td>
			<td>01493-85</td>
			<td>Stock solution: 100mM in 0.9% saline</td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LK003172</td>
			<td>Stock solution: 1000U/ml in EBSS</td>
		</tr>
		<tr>
			<td>EBSS</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LK003188</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottom dish</td>
			<td>IWAKI</td>
			<td>3910-035</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement (100x)</td>
			<td>invitrogen</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetyl-cystein</td>
			<td>Sigma</td>
			<td>A-9165-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LK003178</td>
			<td>Stock solution: 7U/ml in EBSS</td>
		</tr>
		<tr>
			<td>Penicillin/Streptmycine</td>
			<td>Nacalai Tesque</td>
			<td>09367-34</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine</td>
			<td>Sigma</td>
			<td>P-6281</td>
			<td>40 mg/ml in DW</td>
		</tr>
		<tr>
			<td>Preparations for in utero electroporation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50-ml syringe</td>
			<td>TERUMO</td>
			<td>181228T</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode</td>
			<td>Neppagene</td>
			<td>7-mm</td>
			<td></td>
		</tr>
		<tr>
			<td>Electroporator</td>
			<td>Neppagene</td>
			<td>CUY21 EDIT</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gauzes</td>
			<td>Kawamoto co.</td>
			<td>7161</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro capillary</td>
			<td>Made in-house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Nacalai Tesque</td>
			<td>14249-24</td>
			<td></td>
		</tr>
		<tr>
			<td>Pentbarbital</td>
			<td>Kyoritsuseiyaku</td>
			<td>Somnopentyl</td>
			<td></td>
		</tr>
		<tr>
			<td>Ring-shaped forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors, Micro scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Suture needle</td>
			<td>Akiyama MEDICAL MFG. CO</td>
			<td>F17-40B2</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer</td>
			<td>Seractal</td>
			<td></td>
		</tr>
		<tr>
			<td>Preparations for Slice culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10-cm plastic petri dish</td>
			<td>greiner</td>
			<td>664160-013</td>
			<td></td>
		</tr>
		<tr>
			<td>35-mm plastic petri dish</td>
			<td>greiner</td>
			<td>627160</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture insert</td>
			<td>Millipore</td>
			<td>PICM01250</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>invitrogen</td>
			<td>11039-021</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma</td>
			<td>172012-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>DUMONT</td>
			<td>INOX No.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Gibco</td>
			<td>16050-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro surgical knife</td>
			<td>Alcon</td>
			<td>19 Gauge V-Lance</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-gas incubator</td>
			<td>Panasonic</td>
			<td>MCO-5MUV-PJ</td>
			<td></td>
		</tr>
		<tr>
			<td>N2/B27 media</td>
			<td>Made in-house</td>
			<td></td>
			<td>ref. NPC dissociatioin culture</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Nacalai Tesque</td>
			<td>14249-24</td>
			<td></td>
		</tr>
		<tr>
			<td>Ring-shaped forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors, Micro scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon rubber cutting board</td>
			<td>Made in-house</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

real-time bioluminescence imaging of notch signaling dynamics during murine neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Expressions of the genes Hes1/7 exhibit 2 h oscillation cycle in various cell lines and during somitogenesis. Furthermore, the period of oscillation is very short and both their mRNAs and proteins are extremely unstable with the half-lives of around 20 min. If using a slow response reporter, we cannot trace such rapid dynamics, and if using a stable reporter, it gradually accumulates while the gene expression oscillates. Thus, the reporter must be rapidly degraded to monitor the rapid turnover of such cyclically...

Watch this Scientific Journal Video about Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis at JoVE.com

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

This method can help answer key questions about the significance of gene expression dynamics in cell proliferation and differentiation, particularly the dynamics of Notch signaling molecules in neural stem cells. The main advantage of this technique is that we can monitor gene expression both in vitro and in vivo with a very high precision by live imaging. Demonstrating the procedure will be Hiromi Shimojo, the assistant professor from my laboratory who developed this new technology.For Dll1 F luciferase reporter introduction into NPC, confirm a lack of response to pedal reflex in an embryonic day 12.5 to 14.5 pregnant mouse before making a two-to three-centimeter incision through the abdomen, along the midline. Place PBS-soaked gauze around the incision, and use ring-shaped forceps to carefully extract the right uterine horn. After counting the embryos, use a micro capillary to inject one to two microliters of mixed Dll1 F luciferase reporter DNA into the ventricle of the telencephalon of each embryo.A successful injection will result in the observance of blue dye over the surface of the uterus. For electroporation, wet the uterus and the electrode with PBS, and gently grasp the head of the first embryo. Set the positively charged electrode to the side of the hemisphere in which the DNA was injected, and provide five 30-to 50-volt, 50-millisecond pulses, with a one-second pause between pulses, checking that bubbles are generated from the negatively charged electrode.When all of the embryos have been injected, return the left uterine horn to the abdomen, and use a 17-millimeter needle to close the incision with a 4-0 silk suture, placing warm PBS into the abdominal cavity before completing the closure. To set up a dissociated NPC culture, first place the uterus from an embryonic day 12.5 to 14.5 pregnant Dll1 ubiquitinated luciferase transgenic mouse into a 10-centimeter Petri dish containing 25 milliliters of ice-cold PBS, and use micro scissors and fine forceps to remove the embryos from the uterus. After decapitating the embryos, place the heads into a new 10-centimeter Petri dish containing ice-cold DMEM/F12, and remove the epidermis and cartilage surrounding each brain.Transfer the brains into a 35-millimeter dish under a dissecting microscope containing three milliliters of ice-cold N2B27 medium. Remove the right and left telencephalon from diencephalon. Use fine forceps to remove the meninges covering the surface of telencephalon and to dissect the dorsolateral part of cortex from the telencephalon.When all of the neural tissues of interest have been isolated, use a P1000 pipette to transfer the samples into individual 1.5-milliliter tubes, and use a P200 pipette to aspirate any extra medium from each tube. Add 100 microliters of papain solution per cortex pair, and place the samples at 24 degrees Celsius for 15 minutes. At the end of the incubation, gently pipette the samples 10 times with a new P1000 pipette, and return the tubes to 24 degrees Celsius for an additional 15 minutes.At the end of the incubation, gently pipette the samples again before collecting the digested tissues by centrifugation. Aspirate the supernatants, and gently resuspend each pellet with one milliliter of DMEM/F12 medium. Centrifuge the samples three more times as just demonstrated, gently resuspending each pellet with a fresh milliliter of medium after each centrifugation.After the last centrifugation, resuspend the pellets in 500 microliters of N2B27 medium supplemented with one-millimolar luciferin, and seed one times 10 to the six cells from each sample onto individual poly-L-lysine-coated glass bottom dishes. Then, place the plates in the cell culture incubator for one hour. Once the cells have adhered to the dish, add two milliliters of fresh N2B27 medium plus one-millimolar luciferin to each plate.To visualize luciferase reporter expression in NPC dissociation cultures, select the oil immersion objective, and place the sample dish onto the microscope stage. View the field manually to determine the best position for viewing the cells, and focus on the cells of interest. Click Live to acquire the test image, and use the Multi-Dimensional Acquisition program to run the time-lapse acquisition by two-dimensional luminescence and bright-field acquisitions for 24 hours.To prepare developing cortex slice cultures, one day after reporter injection, harvest the embryonic day 13.5 to 14.5 embryo brains as demonstrated, and place the dish containing the brains onto the stage of a fluorescence stereoscopic microscope. Confirm that each cortex expresses the injected fluorescent reporter under the appropriate excitation light, and transfer the brains to a silicone rubber cutting board filled with 30 milliliters of DMEM/F12 medium, bubbled with 100%oxygen gas. Use a micro surgical knife and fine forceps to cut the border between the medial and lateral portion of the dorsal telencephalon, and separate the tissue into two hemispheres.Then, use the knife to cut the cortex like stripes to obtain cortical slices, transferring the slices in medium from the cutting board into a new 35-millimeter Petri dish as they are acquired. When all of the slices have been collected, place the cut surface of each slice onto a culture insert set into a glass bottom dish containing enriched medium, adjusting the orientation of each slice with fine forceps as necessary. Then, remove any excess medium with a pipette, and place the dish into a multi-gas incubator set to 40%oxygen, 5%carbon dioxide, and 37 degrees Celsius for 30 minutes.At the end of the incubation, add 300 microliters of enriched medium supplemented with one-millimolar luciferin to the culture dish. To visualize the luciferase reporter expression within the slice cultures, select the 40x objective, and place the sample dish onto the microscope stage. Acquire a test image of the fluorescence, and set the position and focus plane to the region of interest under the illumination of the appropriate excitation light.Then, run the time-lapse acquisition by three-dimensional luminescence, fluorescence, and bright-field acquisitions for 24 hours. To monitor the promoter activity of the oscillating gene Dll1, ubiquitinated luciferase, a destabilized luciferase reporter with a half-life of about 10 minutes, can be used. Like a fluorescent reporter, the luciferase reporter can be used to monitor the expression dynamics of a protein by being fused to the gene coding sequence of interest.To visualize reporter expression at the single-cell level in tissue cultures, the reporter gene can be transiently transfected into NPCs via in utero electroporation as demonstrated. In addition, microscope-based imaging enables the acquisition of bright-field, fluorescence, and chemiluminescence images. The reporter of Dll1 promoter activity exhibits an oscillatory expression in NPCs derived from the telencephalon of Dll1 ubiquitinated F luciferase reporter mice, with the destabilized luciferase reporter indicating a sharp up and down regulation of the expression of promoter activity.In adjacent enhanced green fluorescent protein-positive cells carrying Hes1 reporter and mCherry-positive cells expressing Dll1 protein reporter that made contact with each other during observation, Hes1 reporter expression appears to start about 60 minutes after contact, suggesting that the time delay for the transmission of Notch signaling between the adjacent cells is about one hour. Furthermore, during signal transmission, Dll1 protein expression exhibits a dynamic translocation. When acquiring the bioluminescence imaging, keep the microscope room completely dark, as extraneous light can prevent an optimal acquisition.Recent developments in optogenetics allows us to control gene expression with light. With this technique, we can simultaneously introduce various pattern of gene expression and monitor luciferase reporter responses.

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SCIENTISTS IN ACTION

7.5K Görüntüleme.  Kyoto University. Bu yöntem, hücre çoğalması ve farklılaşmasında gen ekspresyonu dinamiğinin önemi, özellikle nöral kök hücrelerdeki Çentik sinyal moleküllerinin dinamikleri hakkında anahtar soruların yanıtlanmasına yardımcı olabilir. Bu tekniğin en büyük avantajı, canlı görüntüleme ile hem in vitro hem de in vivo gen ekspresyonunu çok yüksek hassasiyetle izleyebiliyor olmamızdır. Prosedürü gösteren Hiromi Shimojo, bu yeni teknolojiyi geliştiren laboratuvarımdaki yardımc...