Name: measuring fluxes of mineral nutrients and toxicants in plants with radioactive tracers
Uploaded: 2014-08-22T14:00:00.000+00:00
Duration: 13 min 14 s
Description: Watch this Scientific Journal Video about Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers at JoVE.com

This work was supported by the Natural Sciences and Engineering Council of Canada (NSERC), the Canada Research Chair (CRC) program, and the Canadian Foundation for Innovation (CFI).

<html> 1 공장 문화와 준비 <ol><li> (자세한 내용은 10 참조) 기후의 성장 챔버 7 일 동안 보리 모종 수경 성장. 참고 : 영양 요구 사항이 시대에 따라 변화하므로, 개발 단계의 다양한에서 식물을 검사 고려하는 것이 중요합니다. </li><li> 하루 전에 실험으로, 하나의 복제 (DI에 대한 부당 3 식물, 기업용 번들 당 6 식물)를 만들기 위해 함께 몇 가지 모종을 번들. 촬영의 기초 부분의 주위에 타이곤 튜브의 2 cm 조각을 포장하고, &quot;칼라&quot;를 만들기 위해 테이프로 튜브를 고정하여 번들 모종. 참고 : 부당 식물의 수는 실험 조건의 10,13,14에 따라 달라질 수 있습니다. 번들링 루트 질량 및 / 또는 특정 활동이 낮은 특히, 통계 및 측정 정확도를 개선하기위한 것입니다. </li></ol> 실험 솔루션 / 소재의 2 준비 내용 &quot;&gt; 참고 : 다음은 일반적으로 이전의 실험에 일일 수행됩니다. <ol><li> 및 식물 물질과 비활성 [S O를위한 샘플 바이알 ((공장 샘플의 스핀 - 건조) 원심 분리 튜브 (11 참조, 자세한 내용) 예약 라벨, 라벨 및 탈착 해법; DI를 들어, 다음의 정보가 아래 참조). 에어레이션 모든 솔루션을 섞는다. </li><li> (10 참조, 자세한 내용은) 용출액, 식물 샘플 유출 유입 경로, (식물 샘플의 탈수 용) 원심 분리 튜브 및 샘플 유리 병 (잘 혼합, 폭기 라벨 및 용출 솔루션 및 : 케이트를 들어, 다음의 정보 S 오와 희석 배수의 결정 [D f를, 아래 참조). </li></ol> 3 추적자를 준비 주의 : 다음의 안전 단계가 방사능으로 작업하기 전에주의해야한다. <ol><li> 그 확인의 요구 radioacTIVE 재료 라이센스는 이해하고 따라야한다. 적절한 안전 장비 (즉, 고글, 장갑, 실험실 코트, 납 조끼 / 칼라)와 선량계 (예를 들어, TLD 링과 배지)를 착용 할 것. 차폐 설정 (즉, 플렉시 유리, 납 벽돌)과 뒤에 방사성 작업을 수행합니다. 가이거 뮬러 카운터가 정기적으로 오염을 모니터링하기 위해 존재 함을 확인합니다. </li><li> 42 K의 준비 + <ol><li> 균형 깨끗하고 마른 비커를 놓습니다. 균형을 제로. </li><li> 포장 (분말 형태로 42 K 2 CO 3의 20 mCi의) 추적의 병을 제거하고 비커에 추적을 붓는다. 질량에주의하십시오. </li><li> 비커에 H 2 SO 4 0.07 ㎖의 다음 dH보다 2 O의 피펫 19.93 ML,. 이것은 다음과 같은 화학 반응을 구동한다 : 42 K 2 CO 3 (들) + H 2 SO 4 (리터) + H 2 O (리터) 42 → </sK 2&gt; 최대 SO 4 (리터) + CO 2 (V) + 2H 2 O (리터) </li><li> K 2 CO 3의 질량과 분자량 주어진 방사성 스톡 용액의 농도 및 부피 (20 ㎖)을 계산한다. NOTE : 13 NH 13분의 3 NH 4 + 트레이서을 물 산소 원자의 양성자 충격 통해 싸이클로트론에서 생성되는 작업 경우 (일반적으로 100 ~ 200 mCi의 활동의 결과, 생산 자세한 것은, 12 참조). 14 NH 14분의 3 NH 4 +의 양이 이러한 솔루션에 매우 낮기 때문에, 원액의 N 농도는 무시할 수있다. </li></ol></li></ol> (4) 직접 유입 (DI) 측정 <ol><li> 42 K +의 경우, 라벨 용액에 K +의 원하는 최종 농도에 도달하는 데 필요한 방사성 스톡 용액의 양을 피펫. <ol><li> NH 13분의 3 13 NH 4 +, 피펫 라벨 솔루션으로 소량 (&lt;0.5 mL)을 첨가 하였다. 라벨 솔루션 (폭기를 통해) 잘 혼합 할 수 있습니다. </li></ol></li><li> 샘플 병에 라벨 솔루션의 한 ML의 서브 샘플을 피펫 (총 4 샘플)을 세 번 반복합니다. <ol><li> 감마 카운터를 이용하여, ( &quot;분당 카운트&quot;CPM에서) 튜브의 방사능을 측정한다. (이 같은 수명이 짧은 트레이서 특히 중요하다) 카운터가 노출 당 비용 (CPM) 수치는 동위 원소의 붕괴에 대해 보정되도록 프로그램되어 있는지 확인합니다. </li><li> 4 개의 샘플의 개수 (노출 당 비용 (CPM)을 ML-1) 평균과 (μmol의 ML -1) 용액에 기판의 농도로 나누어 (노출 당 비용 (CPM) μmol의 -1로 표시) S O를 계산합니다. </li></ol></li><li> 볼 (시험 조건 하에서 식물을 미리 평형화, 5 분 동안 미리 라벨링 (비 방사성) 용액에 뿌리를 담금예를 들어, 사전 라벨 시간의 변화에 대한 10,13,14). </li><li> 5 분 동안 라벨 (방사성) 용액에 뿌리를 담가. 참고 : 레이블 시간은 실험 3,4,7-10에 따라 차이가있을 수 있습니다. </li><li> 표면 부착 방사능의 대부분을 제거하기 위해 5 초 동안 탈착 용액에 뿌리를 전송합니다. 세포 외 추적자 더 명확 뿌리에 5 분 동안 탈착 솔루션의 두 번째 비커에 뿌리를 전송합니다. </li><li> 별도의 촬영, 기초 촬영, 뿌리는 해부합니다. </li><li> 낮은 속도에서 30 초 동안 원심 분리기 튜브의 뿌리와 스핀 샘플을 배치, 임상 수준의 원심 분리기 (~ 5000 XG)는 표면과 간질 물을 제거합니다. </li><li> 뿌리 (신선한 무게, FW)를 달아. </li><li> 식물 시료에서 방사능 카운트 (촬영, 기초 촬영, 루트를 스텝 4.2.1 참조). </li><li> 플럭스를 계산합니다. 공식을 사용하여 공장으로 유입을 계산 Φ = Q * / S O의 중량 L 여기서 Φ는 자속은(μmol의는 g-1의 시간 -1), Q * 조직에 축적 추적의 양 (보통 루트, 촬영, 노출 당 비용 (CPM), 그리고 결합 된 기저 촬영), S o를 라벨 솔루션 (노출 당 비용 (CPM) μmol의 특정 활동 - 1), w는 루트 신선 중량 (g)이며, t는 L의 표시 시간 (시간)이다. 참고 :보다 정교한 계산이 CATE에서 얻은 매개 변수를 기반으로, 라벨 및 탈착시 뿌리에서 동시 추적 유출을 설명하기 위해 만들어 질 수있다 (; 자세한 내용은, 4 참조 아래 참조). </li></ol> 추적기 유출 (CATE) 측정에 의해 5 Compartmental 분석 <ol><li> 라벨 솔루션 및 측정 S O를 (- 위, 4.2 단계 4.1 참조) 준비합니다. </li><li> 희석 계수 (D의 F)을 측정한다. NOTE : 종종 감마 카운터에서 검출기 샘플의 상대적인 위치에 영향을 미칠 수있는 수량방사선 측정했다. 자세한 내용은 설명을 참조하십시오. <ol><li> S O를 측정 한 후, 각각의 샘플에 2 O H의 19 ML을 추가 (예 : 그 최종 부피 = 용출액 볼륨 = 20 ㎖). 각 20 ㎖의 시료의 방사능 량 (cpm)을 측정한다 (단계 4.2.1 참조). </li><li> 20 ㎖의 샘플의 평균 CPM으로 1 ㎖의 샘플의 평균 CPM 나누어 D의 F를 계산한다. </li></ol></li><li> 1 시간 동안 라벨링 솔루션에 뿌리를 담근다. </li><li> 모든 루트 물질이 유입 내에 보장 깔때기를 유출하는 라벨링 솔루션 및 전송 식물에서 식물을 제거합니다. 플라스틱 칼라를 통해 테이프의 작은 스트립을 적용하여 유출 깔때기의 측면에 부드럽게 안전한 식물. </li><li> 조심스럽게 깔때기에 첫 번째 용출액을 붓는다. 타이머를 시작 (카운트 업). </li><li> 마개를 열고 15 초 후 샘플 병에서 용출를 수집 (참고 : 용출 시간이 달라집니다; 아래 참조). 마개를 닫습니다. 조심스럽게 깔때기에 다음 용출액을 붓는다. </li><li> 반복 하오첫째의 최종 용출액에 다음과 용출 시리즈의 나머지 t 단계 5.6 : 15 초 (사 회), 20 초 (세 번), 30 초 (2 회), 40 초 (한 번), 50 초 (한 번), 29.5 분의 전체 용출량에 대한 1주기 분 (25 시간), 참고 : 탈착 시리즈는 실험 조건 7-10,13,14에 따라 차이가있을 수 있습니다. </li><li> 용출 프로토콜이 완료되면 수확 식물 (- 위의 4.8, 4.6 단계). </li><li> (5.2 참조, D f를하여 각 용출액에 대한 읽기를 곱) 감마 카운터에서 용출액과 식물 샘플에서 방사능 량 (cpm)을 측정한다. </li><li> 용출 시간의 함수로 플롯 추적 자료 (노출 당 비용 (CPM) g (루트 FW) -1 분 -1). 정상 상태의 경우, 선형 회귀 분석 및 플럭스의 계산, 환율의 반감기 및 풀 크기를 (자세한 내용은 6-9 참조) 수행합니다. </li></ol>

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Natl. Acad. Sci. U.S.A. 93, 10510-10514 (1996).</li><li>Holm, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NH3+and+NH4++permeability+in+aquaporin-expressing+Xenopus+oocytes.">NH3 and NH4+ permeability in aquaporin-expressing Xenopus oocytes.</a> Pflugers Archiv. Eur. J. Physiol. 450, 415-428 (2005).</li><li>Britto, D. T., Kronzucker, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans-stimulation+of+13NH4++efflux+provides+evidence+for+the+cytosolic+origin+of+tracer+in+the+compartmental+analysis+of+barley+roots.">Trans-stimulation of 13NH4+ efflux provides evidence for the cytosolic origin of tracer in the compartmental analysis of barley roots.</a> Funct. Plant Biol. 30, 1233-1238 (2003).</li><li>Malagoli, P., Britto, D. T., Schulze, L. M., Kronzucker, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Futile+Na++cycling+at+the+root+plasma+membrane+in+rice+(Oryza+sativa+L.):+kinetics,+energetics,+and+relationship+to+salinity+tolerance.">Futile Na+ cycling at the root plasma membrane in rice (Oryza sativa L.): kinetics, energetics, and relationship to salinity tolerance.</a> J. Exp. Bot. 59, 4109-4117 (2008).</li><li>Kronzucker, H. J., Britto, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+transport+in+plants:+a+critical+review.">Sodium transport in plants: a critical review.</a> New Phytol. 189, 54-81 (2011).</li></ol>

The authors have nothing to disclose.

위의 예에서 알 수 있듯이, 추적자 방법은 란타 영양소와 독성 물질의 단방향 플럭스를 측정 강력한 수단이다. 1 NH 3 유입 아마도 225 μmol의 g -1의 시간 -1, 초과에 도달 할 수 있음을 보여준다 가장 높은 선의의 횡단 자속 적 공장 시스템 13에보고 있지만 순 플럭스를 측정 한 경우 플럭스의 크기는 볼 수 없습니다. NH (3)의</s...

영양분과 독성 물질의 흡수 및 유통 강하게 식물의 성장에 영향을 미친다. 따라서, 기본 전송 프로세스의 조사가 특히 영양 최적화 및 환경 스트레스의 문맥에서, 식물 생물학 연구 및 농업 과학 1,2의 주요 영역을 구성한다 (예를 들어, 염 스트레스, 암모늄 독성). 식물체에서 플럭스의 측정을위한 방법들 중 최고 상당히 1950 개발 된 방사성 동위 원소 추적자의 사용 오늘날 널리 사용가되고 (도 3 등 참조)이다. 이러한 MIFE (자속 추정 이온 미세 전극)과 SIET (스캔 이온 선택성 전극 기법), 및 사용과 같은 조직에서의 루트 매체 및 / 또는 축적 이온 선택성 진동 미소 전극의 사용으로부터 영양분 고갈의 측정과 같은 다른 방법 이온 - 선택적 형광 염료는 또한 널리 적용되지만, 네트 독감을 검출 할 수있는 능력에 제한이XES (즉, 유입과 유출의 차이). 방사성 동위 원소의 사용은, 다른 한편으로는, 연구원 동역학 매개 변수를 해결하는 데 사용될 수있는 단방향 플럭스를 분리 및 정량화하는 독특한 능력을 허용한다 (예를 들면, K M과 V 최대) 및 용량에 대한 통찰력을 제공 지학, 교통 시스템의 메커니즘 및 규제. 방사성 트레이서 만든 단방향 플럭스 측정은 반대 방향의 자속이 높은 조건에서 특히 유용하고, 세포 풀의 회전율은 4 급이다. 추적 동위 원소는 같은 원소의 다른 동위 원소의 배경에 대해 관찰되어 있기 때문에 또한, 추적자 방법 (아래, &#39;토론&#39;참조) 측정은 많은 다른 기술과는 달리, 상당히 높은 기질 농도에서 수행 할 수 있습니다. 여기서 우리는 단방향 및 N의 방사성 동위 원소 측정을위한 자세한 단계를 제공그대로 식물 등 미네랄 영양소의 플럭스 및 독성 물질. 중점 플럭스 칼륨 (K +)의 측정, 식물 영양소 (5), 암모니아 / 암모늄에 이루어집니다 (NH 3 / NH 4 +), 그러나, 예를 들어, 높은 농도 (에 존재하는 경우 독성이 다른 다량 영양소, 1 10 mM의) 2. 우리는 방사성 동위 원소 (42) K + (t 2 = 12.36 시간)과 13 NH 13분의 3 NH 4 + (t 2 = 9.98 분), 각각 모델 시스템 보리의 손상 모종 (Hordeum의 vulgare의의 L를 사용합니다 .), 두 가지 핵심 프로토콜의 설명 : 추적 유출 (CATE)으로 직접 유입 (DI) 및 compartmental 분석. 우리는이 문서가 단순히 각 프로토콜을 수행하는 데 필요한 단계를 설명합니다 처음부터주의해야한다. 각 기술의 적절한 계산과 이론에 대한 간단한 설명을 제공하지만, 자세한 아르 박람회의 배경과 이론은 주제 4,6-9에 대한 몇 가지 주요 기사에서 찾을 수 있습니다. 중요한 것은,이 프로토콜은 다른 영양소 / 독성 물질의 분석을 플럭스 광범위하게 양도 (예를 들어, 24 나 +, 22 나 +, 86 굽은 +, 13 NO 3 -) 및 다른 식물 종에 대한 몇 가지주의 사항이기는하지만 (아래 참조) . 우리는 또한 방사성 물질로 작업하는 모든 연구자가 기관의 이온화 방사선 안전 규제를 통해 제공 라이센스에 따라 작동해야 중요성을 강조한다.

<table><tbody><tr><td>Gamma counter</td><td>Perkin Elmer</td><td></td><td>Model: Wallac 1480 Wizard 3&quot;</td></tr><tr><td>Geiger-M&amp;uuml;ller counter</td><td>Ludlum Measurements Inc.</td><td></td><td>Model 3 survey meter</td></tr><tr><td>400 ml glass beakers</td><td>VWR</td><td>89000-206</td><td>For pre-absorption, absorption, and desorption solutions</td></tr><tr><td>Glass funnel</td><td>VWR</td><td>89000-466</td><td>For efflux funnel</td></tr><tr><td>Large tubing</td><td>VWR</td><td>529297</td><td>For efflux funnel</td></tr><tr><td>Medium tubing</td><td>VWR</td><td>684783</td><td>For bundling</td></tr><tr><td>Small tubing</td><td>VWR</td><td>63013-541</td><td>For aeration</td></tr><tr><td>Aeration manifold</td><td>Penn Plax Air Tech</td><td>vat 5.5</td><td>To control/distribute pressurized air into solutions</td></tr><tr><td>Glass scintillation vials</td><td>VWR</td><td>66022-128</td><td>For gamma counting</td></tr><tr><td>Glass centrifuge tubes</td><td>VWR</td><td>47729-576</td><td>For spin-drying root samples</td></tr><tr><td>Kimwipes</td><td>VWR</td><td>470173-504</td><td>For spin-drying root samples</td></tr><tr><td>Dissecting scissors</td><td>VWR</td><td>470001-828</td><td></td></tr><tr><td>Forceps</td><td>VWR</td><td>470005-496</td><td></td></tr><tr><td>Low-speed clinical centrifuge</td><td>International Equipment Co.</td><td>76466M-4</td><td>For spin-drying root samples</td></tr><tr><td>1 ml pipette</td><td>Gilson</td><td>F144493</td><td></td></tr><tr><td>10 ml pipette</td><td>Gilson</td><td>F144494</td><td></td></tr><tr><td>1 ml pipette tips</td><td>VWR</td><td>89079-470</td><td></td></tr><tr><td>10 ml pipette tips</td><td>VWR</td><td>89087-532</td><td></td></tr><tr><td>Analytical balance</td><td>Mettler toledo</td><td>PB403-S/FACT</td><td></td></tr></tbody></table>

measuring fluxes of mineral nutrients and toxicants in plants with radioactive tracers

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. Radioisotope tracing is a major technique used to measure such fluxes, both within plants, and between plants and their environments. Flux data obtained with radiotracer protocols can help elucidate the capacity, mechanism, regulation, and energetics of transport systems for specific mineral nutrients or toxicants, and can provide insight into compartmentation and turnover rates of subcellular mineral and metabolite pools. Here, we describe two major radioisotope protocols used in plant biology: direct influx (DI) and compartmental analysis by tracer efflux (CATE). We focus on flux measurement of potassium (K+) as a nutrient, and ammonia/ammonium (NH3/NH4+) as a toxicant, in intact seedlings of the model species barley (Hordeum vulgare L.). These protocols can be readily adapted to other experimental systems (e.g., different species, excised plant material, and other nutrients/toxicants). Advantages and limitations of these protocols are discussed.

그림 1은 높은에서 성장 그대로 보리 모종의 뿌리로 NH 3의 유입에 대한 (13 N)와 DI 기술을 사용하여 찾을 등온선을 보여줍니다 (10 mM)을 NH 4 +, 그리고 하나 (0.02 mM)을 높거나 낮은 (5 mM의 ) K +. (, 용액의 pH 13의 변화에 의해 조정 된 NH 3] 내선) NH 3 플럭스 외부 NH 3 농도의 함수로 플롯 할 때 등온선은 미카엘리스 - 멘?...

Watch this Scientific Journal Video about Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers at JoVE.com

Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

In planta measurement of nutrient and toxicant fluxes is essential to the study of plant nutrition and toxicity. Here, we cover radiotracer protocols for influx and efflux determination in intact plant roots, using potassium (K+) and ammonia/ammonium (NH3/NH4+) fluxes as examples. Advantages and limitations of such techniques are discussed.

방사성 트레이서와 식물에 미네랄 영양 성분 및 독성 물질의 플럭스를 측정

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. ...

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Research

JoVE Journal

Environment

11.3K 조회수.  University of Toronto. In planta measurement of nutrient and toxicant fluxes is essential to the study of plant nutrition and toxicity. Here, we cover radiotracer protocols for influx and efflux determination in intact plant roots, using potassium (K+) and ammonia/ammonium (NH3/NH4+) fluxes as examples. Advantages and limitations of such techniques are discussed.

비디오: Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes and soil organic matter formation to small-scale stable-isotope biomarker probing. Coupling multiple stable isotopes such as 13C with 15N, 18O or&#160;2H has the potential to reveal even more information about complex stoichiometric relationships during biogeochemical transformations. Isotope labeled plant material has been used in various studies of litter decomposition and soil organic matter formation1-4. From these and other studies, however, it has become apparent that structural components of plant material behave differently than metabolic components (i.e. leachable low molecular weight compounds) in terms of microbial utilization and long-term carbon storage5-7. The ability to study structural and metabolic components separately provides a powerful new tool for advancing the forefront of ecosystem biogeochemical studies. Here we describe a method for producing 13C and 15N labeled plant material that is either uniformly labeled throughout the plant or differentially labeled in structural and metabolic plant components.
Here, we present the construction and operation of a continuous 13C and 15N labeling chamber that can be modified to meet various research needs. Uniformly labeled plant material is produced by continuous labeling from seedling to harvest, while differential labeling is achieved by removing the growing plants from the chamber weeks prior to harvest. Representative results from growing Andropogon gerardii Kaw demonstrate the system&#39;s ability to efficiently label plant material at the targeted levels. Through this method we have produced plant material with a 4.4 atom%13C and 6.7 atom%15N uniform plant label, or material that is differentially labeled by up to 1.29 atom%13C and 0.56 atom%15N in its metabolic and structural components (hot water extractable and hot water residual components, respectively). Challenges lie in maintaining proper temperature, humidity, CO2 concentration,&#160;and light levels in an airtight 13C-CO2 atmosphere for successful plant production. This chamber description represents a useful research tool to effectively produce uniformly or differentially multi-isotope labeled plant material for use in experiments on ecosystem biogeochemical cycling.

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

This method explains how to build and operate a continuous 13C and 15N isotope labeling chamber for uniform or differential plant tissue labeling. Representative results from metabolic and structural labeling of Andropogon gerardii are discussed.

연속의 설계 및 운영<sup&gt; 13</sup&gt; C 및<sup&gt; 15</sup통일 또는 차등을위한&gt; N 레이블 상공 회의소, 대사 및 구조, 식물 동위 원소 라벨링

Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes ...

연구

환경과학

Measuring Carbon-based Contaminant Mineralization Using Combined CO2 Flux and Radiocarbon Analyses

A method is described which uses the absence of radiocarbon in industrial chemicals and fuels made from petroleum feedstocks which frequently contaminate the environment. This radiocarbon signal &#8212; or rather the absence of signal &#8212; is evenly distributed throughout a contaminant source pool (unlike an added tracer) and is not impacted by biological, chemical or physical processes (e.g., the 14C radioactive decay rate is immutable). If the fossil-derived contaminant is fully degraded to CO2, a harmless end-product, that CO2 will contain no radiocarbon. CO2 derived from natural organic matter (NOM) degradation will reflect the NOM radiocarbon content (usually &#60;30,000 years old). Given a known radiocarbon content for NOM (a site background), a two end-member mixing model can be used to determine the CO2 derived from a fossil source in a given soil gas or groundwater sample. Coupling the percent CO2 derived from the contaminant with the CO2 respiration rate provides an estimate for the total amount of contaminant degraded per unit time. Finally, determining a zone of influence (ZOI) representing the volume from which site CO2 is collected allows determining the contaminant degradation per unit time and volume. Along with estimates for total contaminant mass, this can ultimately be used to calculate time-to-remediate or otherwise used by site managers for decision-making.

Measuring Carbon-based Contaminant Mineralization Using Combined CO2 Flux and Radiocarbon Analyses

A protocol is described wherein CO2 mineralized from organic contaminant (derived from petroleum feedstocks) biodegradation is trapped, quantified, and analyzed for 14C content. A model is developed to determine CO2 capture zone's spatial extent. Spatial and temporal measurements allow integrating contaminant mineralization rates for predicting remediation extent and time.

측정 탄소 기반의 오염 물질 무기화 결합 된 CO를 사용하여<sub&gt; 2</sub&gt; 플럭스 및 방사성 탄소 분석

A method is described which uses the absence of radiocarbon in industrial chemicals and fuels made from petroleum feedstocks which frequently contaminate ...

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

방사성 핵 종 형광 취재 원 유전자 설치류 종양 모델에서 종양의 진행을 추적 하기 위해 이미징

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

암 연구학

Fluorescently Labeled Bacteria as a Tracer to Reveal Novel Pathways of Organic Carbon Flow in Aquatic Ecosystems

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial communities has many limitations, and determining a predator, prey, and predatory rates is often difficult. Presented here is an optimized method based on the addition of fluorescently labelled prey as a tracer, which allows for reliable quantitation of the grazing rates in aquatic predatory eukaryotes and estimation of nutrient transfer to higher trophic levels.

Presented here is a protocol for a single-cell, epifluorescence microscopy-based technique to quantify grazing rates in aquatic predatory eukaryotes with high precision and taxonomic resolution.

수생 생태계에서 유기 탄소 흐름의 새로운 경로를 밝히기 위해 추적자로 형광으로 분류 된 박테리아

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial ...

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. A stable isotope approach is necessary to follow and quantify the fate of fertilizer derived N (FDN) through the soil-crop system. The purpose of this paper is to describe a small-plot research&#160;design utilizing non-confined 15N enriched microplots for multiple soil and plant sampling events over two growing seasons and provide sample collection, handling, and processing protocols for total 15N analysis. The methods were demonstrated using a replicated study from south-central Minnesota planted to corn (Zea mays L.). Each treatment consisted of six corn rows (76 cm row-spacing) 15.2 m long with a microplot (2.4 m by 3.8 m) embedded at one end. Fertilizer-grade urea was applied at 135 kg N&#8729;ha-1 at planting, while the microplot received urea enriched to 5 atom % 15N. Soil and plant samples were taken several times throughout the growing season, taking care to minimize cross-contamination by using separate tools and physically separating unenriched and enriched samples during all procedures. Soil and plant samples were dried, ground to pass through a 2 mm screen, and then ground to a flour-like consistency using a roller jar mill. Tracer studies require additional planning, sample processing time and manual labor, and incur higher costs for 15N enriched materials and sample analysis than traditional N studies. However, using the mass balance approach, tracer studies with multiple in-season sampling events allow the researcher to estimate FDN distribution through the soil-crop system and estimate unaccounted-for FDN from the system.

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

A microplot design for 15N tracer research is described to accommodate multiple in-season plant and soil sampling events. Soil and plant sample collection and processing procedures, including grinding and weighing protocols, for 15N analysis are put forth.

15개의질소 분석을 위한 마이크로플롯 설계 및 식물 및 토양 샘플 준비

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. ...

Two-Dimensional Visualization and Quantification of Labile, Inorganic Plant Nutrients and Contaminants in Soil

We describe a method for two-dimensional (2D) visualization and quantification of the distribution of labile (i.e., reversibly adsorbed) inorganic nutrient (e.g., P, Fe, Mn) and contaminant (e.g., As, Cd, Pb) solute species in the soil adjacent to plant roots (the &#8216;rhizosphere&#8217;) at sub-millimeter (~100 &#181;m) spatial resolution. The method combines sink-based solute sampling by the diffusive gradients in thin films (DGT) technique with spatially resolved chemical analysis by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The DGT technique is based on thin hydrogels with homogeneously distributed analyte-selective binding phases. The variety of available binding phases allows for the preparation of different DGT gel types following simple gel fabrication procedures. For DGT gel deployment in the rhizosphere, plants are grown in flat, transparent growth containers (rhizotrons), which enable minimal invasive access to a soil-grown root system. After a pre-growth period, DGT gels are applied to selected regions of interest for in situ solute sampling in the rhizosphere. Afterwards, DGT gels are retrieved and prepared for subsequent chemical analysis of the bound solutes using LA-ICP-MS line-scan imaging. Application of internal normalization using 13C and external calibration using matrix-matched gel standards further allows for the quantification of the 2D solute fluxes. This method is unique in its capability to generate quantitative, sub-mm scale 2D images of multi-element solute fluxes in soil-plant environments, exceeding the achievable spatial resolution of other methods for measuring solute gradients in the rhizosphere substantially. We present the application and evaluation of the method for imaging multiple cationic and anionic solute species in the rhizosphere of terrestrial plants and highlight the possibility of combining this method with complementary solute imaging techniques.

This protocol presents a workflow for sub-mm 2D visualization of multiple labile inorganic nutrient and contaminant solute species using diffusive gradients in thin films (DGT) combined with mass spectrometry imaging. Solute sampling and high-resolution chemical analysis are described in detail for quantitative mapping of solutes in the rhizosphere of terrestrial plants.

토양의 2차원 시각화 및 래빌, 무기 식물 영양소 및 오염 물질의 정량화

We describe a method for two-dimensional (2D) visualization and quantification of the distribution of labile (i.e., reversibly adsorbed) inorganic ...

Automated 90Sr Separation and Preconcentration in a Lab-on-Valve System at Ppq Level

A quick, automated and portable system for the separation and determination of radiostrontium in aqueous samples, using Sr-resin and multi sequential flow injection analysis, has been developed. The concentrations of radioactive strontium were determined by flow scintillation counting, allowing for on-line and also on-site determination. The proposed system can determine radioactive strontium at industrial relevant levels without further modification using overall analysis time of less than 10 min per aqueous sample. The limit of the detection is 320 fg&#183;g-1 (1.7 Bq/g).

Automated 90Sr Separation and Preconcentration in a Lab-on-Valve System at Ppq Level

Here, we present the portable autoRAD platform for quick radiometric separation and determination of 90Sr, an important fission product highly pertinent to nuclear waste.

자동화 된 90Sr 분리 및 Ppq 수준에서 실험실에 밸브 시스템 Preconcentration

A quick, automated and portable system for the separation and determination of radiostrontium in aqueous samples, using Sr-resin and multi sequential flow ...

화학

Workflow Based on the Combination of Isotopic Tracer Experiments to Investigate Microbial Metabolism of Multiple Nutrient Sources

Studies in the field of microbiology rely on the implementation of a wide range of methodologies. In particular, the development of appropriate methods substantially contributes to providing extensive knowledge of the metabolism of microorganisms growing in chemically defined media containing unique nitrogen and carbon sources. In contrast, the management through metabolism of multiple nutrient sources, despite their broad presence in natural or industrial environments, remains virtually unexplored. This situation is mainly due to the lack of suitable methodologies, which hinders investigations.
We report an experimental strategy to quantitatively and comprehensively explore how metabolism operates when a nutrient is provided as a mixture of different molecules, i.e., a complex resource. Here, we describe its application for assessing the partitioning of multiple nitrogen sources through the yeast metabolic network. The workflow combines information obtained during stable isotope tracer experiments using selected 13C- or 15N-labeled substrates. It first consists of parallel and reproducible fermentations in the same medium, which includes a mixture of N-containing molecules; however,a selected nitrogen source is labeled each time. A combination of analytical procedures (HPLC, GC-MS) is implemented to assess the labeling patterns of targeted compounds and to quantify the consumption and recovery of substrates in other metabolites. An integrated analysis of the complete dataset provides an overview of the fate of consumed substrates within cells. This approach requires an accurate protocol for the collection of samples&#8211;facilitated by a robot-assisted system for online monitoring of fermentations&#8211;and the achievement of numerous time-consuming analyses. Despite these constraints, it allowed understanding, for the first time, the partitioning of multiple nitrogen sources throughout the yeast metabolic network. We elucidated the redistribution of nitrogen from more abundant sources toward other N-compounds and determined the metabolic origins of volatile molecules and proteinogenic amino acids.

This protocol describes an experimental procedure to quantitatively and comprehensively investigate the metabolism of multiple nutrient sources. This workflow, based on a combination of isotopic tracer experiments and an analytical procedure, allows the fate of consumed nutrients and the metabolic origin of molecules synthetized by microorganisms to be determined.

여러 영양소 소스의 미생물 대사를 조사 하기 위해 동위 원소 추적 실험의 조합에 따라 워크플로

Studies in the field of microbiology rely on the implementation of a wide range of methodologies. In particular, the development of appropriate methods ...

생체공학

Hydroponics: A Versatile System to Study Nutrient Allocation and Plant Responses to Nutrient Availability and Exposure to Toxic Elements

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several crops, including lettuce and tomato. Within the plant research community, numerous hydroponic systems have been designed to study plant responses to biotic and abiotic stresses. Here we present a hydroponic protocol that can be easily implemented in laboratories interested in pursuing studies on plant mineral nutrition.
This protocol describes the hydroponic system set up in detail and the preparation of plant material for successful experiments. Most of the materials described in this protocol can be found outside scientific supply companies, making the set up for hydroponic experiments less expensive and convenient.
The use of a hydroponic growth system is most advantageous in situations where the nutrient media need to be well controlled and when intact roots need to be harvested for downstream applications. We also demonstrate how nutrient concentrations can be modified to induce plant responses to both essential nutrients and toxic non-essential elements.

Here, we present an easy-to-follow protocol to establish a successful hydroponic system for plant nutrition studies. This protocol has been extensively tested in Arabidopsis and can easily be adapted to other plant species to study specific nutritional requirements or the effect of non-essential elements on plant growth and development.

수경법, • 다목적 시스템은 유해 요소에 영양소 가용성 및 노출에 영양 할당 및 플랜트 응답을 공부하기

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several ...

생물학

Neutron Radiography and Computed Tomography of Biological Systems at the Oak Ridge National Laboratory's High Flux Isotope Reactor

Neutrons have historically been used for a broad range of biological applications employing techniques such as small-angle neutron scattering, neutron spin echo, diffraction, and inelastic scattering. Unlike neutron scattering techniques that obtain information in reciprocal space, attenuation-based neutron imaging measures a signal in real space that is resolved on the order of tens of micrometers. The principle of neutron imaging follows the Beer-Lambert law and is based on the measurement of the bulk neutron attenuation through a sample. Greater attenuation is exhibited by some light elements (most notably, hydrogen), which are major components of biological samples. Contrast agents such as deuterium, gadolinium, or lithium compounds can be used to enhance contrast in a similar fashion as it is done in medical imaging, including techniques such as optical imaging, magnetic resonance imaging, X-ray, and positron emission tomography. For biological systems, neutron radiography and computed tomography have increasingly been used to investigate the complexity of the underground plant root network, its interaction with soils, and the dynamics of water flux in situ. Moreover, efforts to understand contrast details in animal samples, such as soft tissues and bones, have been explored. This manuscript focuses on the advances in neutron bioimaging such as sample preparation, instrumentation, data acquisition strategy, and data analysis using the High Flux Isotope Reactor CG-1D neutron imaging beamline. The aforementioned capabilities will be illustrated using a selection of examples in plant physiology&#160;(herbaceous plant/root/soil system)&#160;and biomedical applications&#160;(rat femur and mouse lung).

This manuscript describes a protocol for neutron radiography and computed tomography of biological samples using a High Flux Isotope Reactor (HFIR) CG-1D beamline to measure a metal implant in a rat femur, a mouse lung, and an herbaceous plant root/soil system.

Oak Ridge National Laboratory의 High Flux Isotope Reactor에서 생물학적 시스템의 중성자 방사선 촬영 및 컴퓨터 단층 촬영

Neutrons have historically been used for a broad range of biological applications employing techniques such as small-angle neutron scattering, neutron ...

방사성 트레이서와 식물에 미네랄 영양 성분 및 독성 물질의 플럭스를 측정

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