Name: a novel technique for generating and observing chemiluminescence in a biological setting
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The authors thank Prof. Jan Grimm and Mr. Travis Shaffer for their helpful discussions and Mr. David Gregory for editing the manuscript. Technical services provided by the MSK Animal Imaging Core Facility, supported in part by NIH Cancer Center Support Grant P30CA008748-48, are gratefully acknowledged. The authors thank the NIH (K25 EB016673 and R21 CA191679, T.R. and 4R00CA178205-02, B.M.Z.), the MSK Center for Molecular Imaging and Nanotechnology (T.R.), the Tow Foundation (B.C.), and the National Science Foundation Integrative Graduate Education and Research Traineeship (IGERT 0965983 at Hunter College for B.C. and T.M.S.) for their generous support. The research reported in this publication was supported by funding from the King Abdullah University of Science and Technology.

Etik deyimi: onaylanmış bir protokole ve Memorial Sloan Kettering Kanser Merkezi (MSK) Kurumsal Hayvan Bakımı ve Kullanımı Komitesi (IACUC) etik kuralları uyarınca göre yapıldı tarif edilen in vivo hayvan deneylerinde tümü. Bir püskürtme cihazı 1. İnşaat <ol><li> Iki vidayı (x 25 mm 2 4) kullanılarak kısım B (12.7 x 10.7 x 1.8 cm 3) merkezinde dik ahşap parça A (12.5 x 2.5 x 1.8 cm 3) takın. Ahşap parça C takın (11 × 2....

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Burada, optik kemilüminesan muhabir tarafından oluşturulan fotonların emisyon yoluyla doku ortaya koymaya yeteneğine sahip bir teknoloji sundu. Diğer aksine, daha bu kemilüminesans muhabir sistemi radyoaktif olmayan ve çok yüksek hassasiyet seviyelerinde tespitini kolaylaştırır bir görüntüleme probu istihdam, teknoloji 4, 5, 6, 7, 8,...

Son yıllarda, görüntüleme teknolojileri doktorlar hastalığı teşhis ve monitör yol devrim var. Bu görüntüleme teknolojileri, ancak bu tür pozitron emisyon tomografisi (PET), tek foton emisyon bilgisayarlı tomografi (SPECT), bilgisayarlı tomografi (BT) ve manyetik rezonans görüntüleme (MRI) gibi tüm vücut görüntüleme sistemleri, büyük ölçüde sınırlı olmuştur. Özellikle dikkat kanseri ödenmiştir ve teknolojik görüntüleme devrimler büyük ölçüde bu hastalığın teşhis ve tedavi edilir şekilde düzeldi. ameliyathane: Bu ilerlemelere rağmen, bu görüntüleme teknolojileri sadece uymaz bir yer var. Tüm vücut görüntüleme teknikleri, cerrahi planlamada yardımcı olabilir, bunlar genellikle tümör dokusunun tüm kaldırıldı veya artık tümör dokusu cerrahi sınırların 1&#39;de gizli kalır olsun hekimler gerçek zamanlı olarak belirlemeye yardımcı olmak için yeterince yüksek uzaysal çözünürlük yoksundur. Hiçbir infiltratif emin yapmaTümör marjları geride en önemli cerrahi hedeflerinden biridir ve cerrahlar titiz ve tedbirli doku rezeksiyonu arasında sıkı-ip yürümek gerekir edilir. Çok fazla çıkarılırsa, hastada istenmeyen yan etkiler arttırılmış olur çok az kaldırılan ise, nüks oranları, 3 2 artmıştır. Nedenle, doğru tümör marjları tasvir için çok önemlidir ve biz kemilüminesan intraoperatif görüntüleme aksi kurulan teknikleri ile tespit edilmemiş kalabileceği malign doku görselleştirmek için cerrahlara yardımcı olarak tümör marjlarının belirlenmesi doğruluğunu geliştirmek için yardımcı olabilir inanıyoruz. Şu anda intraoperatif görüntüleme sistemleri gibi olası yarar için araştırılan birçok görüntüleme teknolojileri vardır. Bu β- ve γ-radyasyon yayan sondalar 4, optik floresan 5, Raman spektroskopisi 6 dahil </sup&gt;, 7 ve Cherenkov ışıma 8, 9. Ancak bugüne kadar, bunların hiçbiri, standart klinik araçlar olarak kullanılır hale gelmiştir. Optik floresan görüntüleme şimdiye kadar bu tekniklerin en umut verici olduğu kanıtlanmıştır ve en çok araştırdı nedenle etti. Zaten birçok uygulama için değerli bir araç olarak gösterilmiş olsa da, bu sınırlamalar olmadan değildir. Gerçekten de, asıl dezavantajı doğal autofluorescent biyolojik doku tarafından üretilen arka plan floresan olduğunu. Bu arka plan otofloresan sinyali, floresan sinyalinin üretimi için gerekli olan, harici bir ışık kaynağı tarafından, florofor ilave olarak, çevre doku uyarılmasının bir ürünüdür. Pratik açıdan bakıldığında, bu otofloresans potansiyel ameliyathanede bu teknolojinin faydasını sınırlayabilir düşük sinyal-gürültü oranları, yol açabilir. Müdürfloresan görüntüleme üzerinden Kemiluminesan görüntüleme avantajı uyarma ışık gerekli olmasıdır. Bunun bir sonucu olarak, herhangi bir arka plan otofloresansı yoktur. Kemiluminesan görüntülemede, uyarma enerjisi yerine kimyasal oluşturulur. Bu işlem, daha yüksek bir sinyal-ses oranlarına neden olabilir, bu nedenle herhangi bir istenmeyen arka plan sinyali üretir ve. Bu sonuçta cerrahi sınırların daha hassas ve doğru algılama neden olabilir. Biraz şaşırtıcı bir ameliyat görüntüleme tekniği olarak bu yaklaşımın yarar 10 keşfedilmemiş kalmıştır. Gerçekten de, bu tekniğin en yakın örnek, farelerde 11, 12, 13, miyeloperoksidaz tarafından luminol oksitlenmesidir. merhaba ile düşük arka plan sinyali sonuçlanan (1) en az otofloresans: kemilüminesans biyomedikal görüntüleme aşağıdaki avantajları sunabilir araştırma oldukça keşfedilmemiş alan nedenleGher sinyal-gürültü oranları; (2) görünür gelen yakın-kızılötesi değişen kemilüminesan emisyonların ayarlanabilir dalga boyları; ve (3), bir bağlayıcı teknolojileri ile bir araya getirilmiş ve zaten mevcut biyomoleküllerin hedef olduğunda, hedef moleküler görüntüleme sondaları 14 tüm kütüphanelerin erişim sağlayan fonksiyonalitesine sahip kimyasal olarak ışık veren kompleksleri. Bu proof-of-prensibi çalışma rutenyum tabanlı görüntüleme maddesi kullanarak biyomedikal ortamda kemilüminesan görüntülemenin potansiyel yarar göstermektedir. Bu bileşiğin kemilüminesan özellikleri iyi 1960&#39;ların ortalarında 15 kadar uzanan araştırmaları ile incelenmiştir. Kimyasal aktivasyonu üzerine, madde, tıbbi görüntüleme amacıyla uygundur 600 nm 16 ° ışık üretir. Aktivasyon enerjisi uyarılmış durumda su 17 -foll 650 ns ömrü vardır yol açan bir redoks reaksiyonu sonucu elde edilenBu heyecanlı devletin gevşeme üzerine foton kuşağının borçlu. Bir özel tasarlanmış uzaktan nebulizatör kullanımı sayesinde, biz bileşik hem ex vivo ve in vivo tespit başardık. İlk deneylerin sonuçları bu teknolojinin daha fazla araştırılması düşündüren, oldukça ümit vericidir.

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			<td height="144" style="height:144px;width:215px;">Wood part A (12.5&#215;2.5&#215;1.8 cm)</td>
			<td style="width:119px;">&#160;Woodcraft</td>
			<td style="width:87px;">131404</td>
			<td style="width:121px;">Cut from a 3/4&#8221; x 24&#8221; x 30&#8221; birch plywood sheet</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:215px;">Wood part B (12.7&#215;10.7&#215;1.8cm)</td>
			<td style="width:119px;">Woodcraft</td>
			<td style="width:87px;">131404</td>
			<td style="width:121px;">Cut from a 3/4&#8221; x 24&#8221; x 30&#8221; birch plywood sheet</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:215px;">Wood part C (11&#215;2.5&#215;1.8cm)</td>
			<td style="width:119px;">Woodcraft</td>
			<td style="width:87px;">131404</td>
			<td style="width:121px;">Cut from a 3/4&#8221; x 24&#8221; x 30&#8221; birch plywood sheet</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Screws (4&#215;25 mm)</td>
			<td style="width:119px;">Screwfix</td>
			<td style="width:87px;">79939</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:215px;">Harmon Face Values 3oz mini sprayer</td>
			<td style="width:119px;">Bed, Bath and Beyond</td>
			<td style="width:87px;"></td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">stainless steel rod (10 cm of 1/16&#8221; steel)</td>
			<td style="width:119px;">Metals Depot Int. Inc.</td>
			<td style="width:87px;">2192</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Pencil Classic HB</td>
			<td style="width:119px;">Papermate</td>
			<td style="width:87px;">58592</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Paper clip</td>
			<td style="width:119px;">Office Depot</td>
			<td style="width:87px;">221720</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">speaker cable</td>
			<td style="width:119px;">RCA Inc.</td>
			<td style="width:87px;">AH1650SN</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:215px;">Energizer 9V alkaline battery</td>
			<td style="width:119px;">Energizer Holdings Inc.</td>
			<td style="width:87px;">EN22</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:215px;">Hitech HS-82MG Micro Servo Motor, 3.4kg/cm output torque @ 6V</td>
			<td style="width:119px;">Hitech RCD USA Inc.</td>
			<td style="width:87px;">32082S</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">Name</td>
			<td style="width:119px;">Company</td>
			<td style="width:87px;">Catalog Number</td>
			<td style="width:121px;">Comments</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:215px;">28 cm plastic cable ties</td>
			<td style="width:119px;">General Electric Inc.</td>
			<td style="width:87px;">50725</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Duct tape</td>
			<td style="width:119px;">3M Inc.</td>
			<td style="width:87px;">3939</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">littleBits w1 wire</td>
			<td style="width:119px;">littleBits Inc.</td>
			<td style="width:87px;">w1 wire</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">littleBits p1 power</td>
			<td style="width:119px;">littleBits Inc.</td>
			<td style="width:87px;">p1 power</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:215px;">littleBits i2 toggle switch</td>
			<td style="width:119px;">littleBits Inc.</td>
			<td style="width:87px;">i2 toggle switch</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">littleBits 011 servo</td>
			<td style="width:119px;">littleBits Inc.</td>
			<td style="width:87px;">011 servo</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:215px;">20 cm plastic covered wire twist ties</td>
			<td style="width:119px;">Four Star Plastics</td>
			<td style="width:87px;">71TIE8000</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="112">
			<td height="133" rowspan="2" style="height:133px;width:215px;">Tris(2,2&#8242;-bipyridyl)dichlororuthenium(II) hexahydrate</td>
			<td rowspan="2" style="width:119px;">Sigma-Aldrich Inc.</td>
			<td rowspan="2" style="width:87px;">224758</td>
			<td rowspan="2" style="width:121px;"></td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">Ammonium cerium(IV) nitrate</td>
			<td style="width:119px;">Sigma-Aldrich Inc.</td>
			<td style="width:87px;">22249</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:215px;">Isofluorane</td>
			<td style="width:119px;">Baxter Healthcare</td>
			<td style="width:87px;">1001936060</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">PBS</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:87px;">PBS1</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Ethanol</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:87px;">2854</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">Triethylamine</td>
			<td style="width:119px;">Sigma-Aldrich Inc.</td>
			<td style="width:87px;">T0886</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="97">
			<td height="97" style="height:97px;width:215px;">Water</td>
			<td style="width:119px;"></td>
			<td style="width:87px;"></td>
			<td style="width:121px;">Water was purified using a Milipore Mili-Q (R &#8805; 18 M&#937;)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Female nude (outbred) mice</td>
			<td rowspan="2" style="width:119px;">Jackson Laboratories</td>
			<td rowspan="2" style="width:87px;">1929</td>
			<td rowspan="2" style="width:121px;">age 5 - 6 weeks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Strain C57BL/6J&#160;&#160;</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:215px;">NU/J male mice at&#160;</td>
			<td style="width:119px;">Jackson Laboratories</td>
			<td style="width:87px;">2019</td>
			<td style="width:121px;">age 6 &#8211; 8 weeks</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:215px;">IVIS 200 bioluminescence reader</td>
			<td style="width:119px;">Caliper Live Science</td>
			<td style="width:87px;"></td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Live Image 4.2 software</td>
			<td style="width:119px;">Perkin-Elmer</td>
			<td style="width:87px;">128165</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Microscope slides</td>
			<td style="width:119px;">ThermoScientific</td>
			<td style="width:87px;">4951PLUS4</td>
			<td style="width:121px;"></td>
		</tr>
	</tbody>
</table>

a novel technique for generating and observing chemiluminescence in a biological setting

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are quickly moving into the operating room. Here, we present a new approach that utilizes a technique not yet explored for intraoperative imaging: chemiluminescent imaging. This method employs a ruthenium-based chemiluminescent reporter along with a custom-built nebulizing system to produce ex vivo or in vivo images with high signal-to-noise ratios. The ruthenium-based reporter produces light following exposure to an aqueous oxidizing solution and re-reduction within the surrounding tissue. This method has allowed us to detect reporter concentrations as low as 6.9 pmol/cm2. In this work, we present a visual guide to our proof-of-concept in vivo studies involving subdermal and intravenous injections in mice. The results suggest that this technology is a promising candidate for further preclinical research and might ultimately become a useful tool in the operating room.

Protokol 1. bölümde tarif edilen vaporizatör, sistemin düşük bir maliyetle kolayca mevcut malzemelerden yapılabilir. Uzak tetikli bir biyolojik olarak ışık veren okuyucu içinde indirgeme / oksitleme maddesinin püskürtülmesi (Şekil 1) için bir ilave olacak şekilde tasarlanmıştır. Tasarım lensten 14 cm mesafeden biyoparlaklık okuyucunun içinde nebulizer güvenli çalışması için izin verir. lensin Hiçbir sisleme veya bulanıklaştırma işlemi s?...

Watch this Scientific Journal Video about A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting at JoVE.com

A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting

This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed.

Biyolojik ortamda oluşturuluyor ve Gözlem Kemilüminesans için Yeni Bir Teknik

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are ...

The overall goal of this protocol is to offer a new preclinical, interoperative, chemiluminescent imaging technique that can provide benefits over existing optical techniques in terms of signal-to-noise ratios and detections limits. This method can help answer key questions that have not been accessible by traditional optical imaging techniques in many fields related to the biological sciences. The main advantages of this technique are that it has a high signal-to-noise ratio and that it requires neither instant light nor radiation.So what we found is that the implications of this technique really extends towards interoperative imaging of multiple disease states including cancer because of the high signal-to-noise ratios that this technique has to offer. As incident light is not needed for this technique there are no issues with incident light absorption. Further, there is a large chemical tool box available to functionalize the chemiluminescent core molecule.To begin the procedure prepare 100 microliter standard solutions of tris(bypiridine)ruthenium(II)chloride in reverse osmosis water. Then, combine each standard with 100 microliters of a 25 millimolar solution of ammonium cerium nitrate in reverse osmosis water on a microscope slide. Open the bioluminescence reader imaging software and click initialize.In imaging mode, ensure that luminescent and photograph are checked and florescent is unchecked. Set the luminescent exposure time, binning, f/stop and emission filter parameters. Set the photograph exposure time, binning and f/stop parameters.Then set the subject height accordingly to the sample to be imaged. Set the field of view to B for a 14-centimeter distance between the camera and the sample stage. Cover the floor of the bioluminescence reader imaging chamber in the sample stage with black construction paper.Then place the first slide with the droplet of standard mixture on the sample stage with the droplet centered in the green lightbox crosshair. Next, fill the three-ounce spray bottle of a custom-built nebulizer with the 1:3 solution of triethylamine in a 50/50 solution of water and ethanol. Ensure that there are no air bubbles in the aspiration pipe.Place the nebulizer assembly in the imaging chamber with the spray nozzle pointed towards the droplet to be imaged without obstructing the camera view of the droplet. Ensure that the nebulizer power switch and power indicator are on, the magnetic toggle switch is off and the power nebulizer and remote cords are not tangled. Cover hot spots such as marks on the slide with small pieces of black construction paper.Place at least 40 centimeters of the nebulizer remote cord in the imaging chamber. Gently close the chamber door without crimping the nebulizer cords. In the instrument software, click acquire, enable autosaving and set the data file path.Initiate the imaging sequence. When the shutter opens with an audible click, switch the nebulizer toggle three times to spray three bursts of triethylamine solution onto the sample. Repeat the procedure with each standard slide to determine the minimum detectable concentration of the ruthenium imaging agent.To begin the in vivo imaging procedure, prepare 100 microliters of a solution containing between 8 and 33 nanomoles of tris(bypiridine)ruthenium(II)chloride in phosphate-buffered saline. Prepare a 25-millimolar aqueous solution of ammonium cerium nitrate. Next, intravenously inject 100 microliters of the ruthenium imaging agent into the tail vein of healthy mice.Sacrifice the mice 10 minutes after injection by carbon dioxide asphyxiation. Remove the skin from the torso with a Y cut and then remove the costal arch to expose the heart and lungs. Cut an outlet in the right atrium and inject 20 milliliters of PBS through a 24-gauge needle into the left ventricle.Cut through the belly skin to expose the kidney and liver. If the organ of interest is the liver, kidney or spleen make a visible longitudinal cut through the organ. Prepare a new imaging sequence in the bioluminescent reader software.For whole abdomen imaging, place the mouse in the imaging chamber with the open abdomen facing the camera and the head pointing towards the back of the instrument. Center the organ of interest in the green lightbox crosshair. Thoroughly wash out the spray bottle and nozzle from the nebulizer assembly and then fill the bottle with the ammonium cerium nitrate solution.Place the nebulizer in the imaging chamber with the spray nozzle directed at the imaging site. Ensure that the camera is unobstructed, the cords are not tangled and at least 40 centimeters of the remote cord are in the chamber. Close the chamber and start the imaging sequence.As the shutter opens with a click, switch the toggle three times to spray three bursts of the oxidizing agent onto the imaging site. After imaging, remove the nebulizer assembly and thoroughly rinse the spray nozzle to prevent crystallization of the ammonium cerium nitrate. For subsequent individual organ imaging and quantification, remove the mouse from the instrument and excise the inner organs starting from the open body cavity.Cut through the hind leg skin to excise muscle tissue samples. If the organ of interest is the liver, kidney or spleen cut the organ in half longitudinally. Then place the organ on a Petri dish or piece of black construction paper.Image each organ in the same way as the whole animal, rinsing the spray nozzle after each sequence. In this procedure, mice were injected with a ruthenium-based imaging agent and the tissue of interest sprayed with an oxidizer to induce chemiluminescence. The minimum detectable concentration of the imaging agent was determined to be 6.9 picomoles per centimeter squared in these conditions.Chemiluminescence in white light images of the organs of interest were obtained with a bioluminescence reader. The chemiluminescence signal was predominantly observed in the kidneys following intravenous injection indicating renal elimination of the hydrophilic tris(bypiridine)ruthenium(II)cation. The signal-to-noise ratio of the imaging agent versus PBS alone was 27:1 in the kidneys and 21:1 in the liver.Mouse popliteal lymph nodes were also evaluated with a subcutaneous injection of a more concentrated solution of the ruthenium-based imaging agent. The lymph nodes containing the imaging agent showed a significantly greater radiance than the lymph nodes containing PBS alone. Once mastered, this technique can be done in two hours if it is properly performed.While attempting this procedure, it's important to remember to exclude ambient light and to avoid contamination with ruthenium tris bpy. After its development, this technique will pave the way for researchers in the field of biomolecular imaging to explore disease states such as cancer in mice. Though this method can provide insight into questions that were not accessible by traditional optical imaging techniques, any question that could be investigated with traditional techniques potentially can be addressed with chemiluminescence.

Research

JoVE Journal

Bioengineering

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Biyomühendislik

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Biyolojik Sistemleri İyon Konsantrasyon Ölçümü için Floresan Nanopartiküller

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Protein ve hücre Etrafının İki Boyutlu Desenli Yüzeyler Oluşturma

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