우리는 깨끗한 객실 시설이 비디오의 필름 부분에 우리를있게 위해 MIT에서 마이크로 시스템즈 기술 연구소 감사하고 싶습니다.

준비 1. 마스크 만들기 <blockquote> CAD 소프트웨어를 사용하여 투명성에 대한 고해상도 인쇄를위한 채널을 디자인합니다. 이것은 &quot;마스크&quot;가 될 것입니다. </blockquote> 클린 룸에서 : 2. 웨이퍼를 청소 구워 아세톤과 함께 첫째, 물총 웨이퍼 후 신속하게 메탄올과 다음 이소프로판올와 함께. 마지막으로, 질소를 사용하여 웨이퍼를 건조. 5 분 오븐 (130 ° C)에서 웨이퍼를 구워. 3. 코팅 웨이퍼에게 스핀 - 코팅 기계의 중앙에 웨이퍼를 놓습니다. 웨이퍼에 병에서 부어 포토 레지스트 (SU - 8). SU - 8의 흐름을하자 ~ 10 S.를위한 휴식 스핀 - coater 켜고 0에서 5의 500 rpm을하기 위해 속도를 램프, 10 S 500 rpm으로 유지, 10 S 이상의 최종 속도를 최대 30 진입로 S.에 대한 최종 속도 유지 최종 속도는 사용 대상 코팅 두께와 SU - 8에 따라 달라집니다. 자세한 사항은에서 찾을 수 있습니다 <a href="http://www.microchem.com/">http://www.microchem.com/</a> 4. 소프트 - 베이크 코팅 후 웨이퍼는 ° C 그리고 95에서 65 먼저 구워 ° C. 베이킹 시간은 사용되는 포토 레지스트의 타겟 두께​​와 종류에 따라 다릅니다. 그런 다음, 웨이퍼 적어도 5 분 실온에서 앉아 보자. 5. 노출 웨이퍼 위에 마스크를 놓고 SU - 8 사용 설명서에서 권장하는 시간 자외선에 웨이퍼를 노출. 6. 포스트 노출 베이킹 65 웨이퍼를 구워 ° C 그리고 95 ° C SU - 8 사용 설명서의 지침에 따라. 7. &quot;마스터&quot;(금형)를 얻기 위해 웨이퍼를 개발 개발자 (PMMA)로 가득 찬 비커를 준비합니다. 포토 레지스트의 unexposed 부분이 씻겨 때까지 매우 부드럽게 비커를 진동하면서 비커에 웨이퍼를 잠그다. 우리가 실험실에서 : 8. PDMS를 준비하고 웨이퍼에 그것을 붓다 한잔에 10시 1분 비율은 치료 요원과 함께 PDMS를 섞는다. 휘저어하고 homogenously 혼합 :이 거품 많이 생성하고 혼합물이 불투명 보이게됩니다. &quot;마스터&quot;의 혼합물을 부어. 9. 진공 챔버의 데 - 거품 거품을 제거하려면 모든 거품들이 갈 때까지 진공 챔버로를 덮고있는 마스터 및 PDMS 혼합물을 배치. 10. 오븐에 베이킹 65 ° C 강화 PDMS에 오븐에 최소한 12 시간 동안 구워. 11. 펀치 구멍 채널 인레츠 및 상점에 대한 마스터 및 펀치 구멍에서 PDMS 보지. 청정실에서 (표시되지 않음) 12. 플라즈마 결합 채널은 PDMS 층 1 분 산소 플라즈마와 함께 유리 슬라이드를 모두 치료 후 유리 슬라이드에 접착됩니다. 실험 : EXP # 1 : 마이크로 규모의 영양 레이어 해양 미생물의 chemotactic 응답을 조사 1) 실험을 설정 <ol><li> 유리 주사기로 생물과 기판을 추가 </li><li> 현미경 단계에 microfluidic 채널을 장소와 적절한 인레츠 및 콘센트에 튜브 첨부 </li><li> 저수지 낭비 튜빙을 연결합니다. 튜브가 완전히 압력 oscillations을 피하기 위해 폐기물 저수지에있는 액체에 빠져들되어 있는지 확인 </li><li> 플레이스 주사기 펌프에 주사기 및 밸브 및 튜브에 연결 </li><li> 조명 조건, 배율 등 : 현미경을 설정 </li><li> 채널 적절한 위치에 초점을 </li><li> 인공 해수로 가득 큰 주사기를 사용하는 데 - 버블 채널 </li><li> 주사기 펌프에 적절한 유량을 설정합니다. 이 경우에는 채널에 220 μm의 S -1의 의미 유속에 해당하는 2 ML / 분, </li></ol> 2) 실험을 실행 <ol><li> 채널 영양소 그라디언트를 구축하기 위해 주사기 펌프를 시작합니다 </li><li> 흐름이 안정하고 영양소의 밴드가 개발되면, 주사기 펌프의 흐름을 중지하고이 시점에서 시간을 기록 시작 </li><li> 영양 밴드가 옆으로 확산 시작 </li><li> 정기적인 시간 간격으로 &#39;영화&#39;를 만드는 프레임의 시퀀스를 기록 이미지 분석 소프트웨어를 사용 </li><li> 오직 움직이는 물체 지금 시각되도록 두 후속 프레임 사이의 시간 차이 이미지를 복용 우리가 아닌 이동 입자와 배경 잡음의 운동성이있는 세포를 차별함으로써 수영 생물을 차별 </li><li> P 참조와 채널 세포의 위치를​​ 결정하기 위해 영화를보세요영양 패치 osition </li><li> 10-20 분 정기적으로 기록 영화는 위치와 생물의 수영 패턴을 분석하는 </li><li> 다른 프레임 (각 픽셀에 대한 동영상의 기간 동안 해당 픽셀에 기록된 최대 빛의 강도를 지정)에서 생물의 위치를​​ 추가 하시려면 이미지 분석 소프트웨어를 사용함으로써, 우리는 수영 세포에 대한 궤도 정보를 얻을 수 있습니다 </li></ol> EXP # 2 : vortexZ에서 수영 해양 박테리아에 전단의 효과를 조사 <ol><li> 다른 채널 기하학 사용하여, 우리는 다른 전단 속도로 소용돌이에서 수영 박테리아의 행동을 관찰할 수 </li></ol>

<ol>
	<li>Azam, F., Malfatti, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+structuring+of+marine+ecosystems.">Microbial structuring of marine ecosystems.</a> Nature Reviews Microbiology. 5, 782-791 (2007).</li><li>Blackburn, N., Azam, F., Hagstrom, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+explicit+simulations+of+a+microbial+food+web.+.">Spatially explicit simulations of a microbial food web. .</a> Limnology and Oceanography. 42, 613-622 (1997).</li><li>Blackburn, N., Fenchel, T., Mitchell, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+nutrient+patches+in+plankton+habitats+shown+by+chemotactic+bacteria.">Microscale nutrient patches in plankton habitats shown by chemotactic bacteria.</a> Science. 282, 2254-2256 (1998).</li><li>Keymer, J. E., Galajda, P., Muldoon, C., Park, S., Austin, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+metapopulations+in+nanofabricated+landscapes.">Bacterial metapopulations in nanofabricated landscapes.</a> Proceedings of the National Academy of Science. 103, 17290-17295 (2006).</li><li>Mao, H., Cremer, P. S., Manson, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive,+versatile+microfluidic+assay+for+bacterial+chemotaxis.">A sensitive, versatile microfluidic assay for bacterial chemotaxis.</a> Proceedings of the National Academy of Science. 100, 5449-5454 (2003).</li><li>Marcos, R., Stocker, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microorganisms+in+vortices:+a+microfluidic+setup.">Microorganisms in vortices: a microfluidic setup.</a> Limnology and Oceanography: Methods. 4, 392-398 (2006).</li><li>Park, S., Wolanin, P. M., Yuzbahyan, E. A., Lin, H., Darnton, N. C., Stock, J. B., Silberzan, P., Austin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+topology+on+bacterial+social+interaction.">Influence of topology on bacterial social interaction.</a> Proceedings of the National Academy of Sciences. 100, 13910-13915 (2003).</li><li>Whitesides, G. M., Ostuni, E., Takayama, S., Jiang, X., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+lithography+in+biology+and+biochemistry.">Soft lithography in biology and biochemistry.</a> Annual Review of Biomedical Engineering. 3, 335-373 (2001).</li></ol>

해양 미생물들이 지역의 화학 및 물리적 환경과 상호 작용하는 방법을 이해하는 바다의 영양분과 탄소 순환 (Azam 및 Malfatti 2007)에서 planktonic 미생물의 역할을보다 완벽하고 정확한 인식을 위해 필수적입니다. 그러나 많은 중요한 미생물의 상호 작용이 이루어지는있는 동안 작은 비늘 (&lt;㎜)로 인해, 기술적 제한이 이종 바이오 physico - 화학 풍경 내에서 미생물의 동작에 대한 자세한 시험을 예방할 필요하면 바다...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<tr>
<td>PDMS, Sylgard 184</td>
<td>Silicone Elastomer Kit</td>
<td>Dow Corning, Midland, MI, USA</td>
<td>&nbsp;</td>
<td>http://www.ellsworth.com/sylgard.html</td>
</tr>
<tr>
<td>SU8-2100</td>
<td>Photoresist</td>
<td>MicroChem, Newton, MA, USA</td>
<td>&nbsp;</td>
<td>www.microchem.com
</td>
</tr>
<tr>
<td>Nikon Eclipse TE2000-E inverted microscope</td>
<td>Microscope</td>
<td>Nikon, Japan</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PEEK tubing (0.762 mm ID, 1.59 mm OD)</td>
<td>Tool</td>
<td>Upchurch Scientific, Oak Harbor, WA, USA</td>
<td>&nbsp;</td>
<td>www.upchurch.com</td>
</tr>
<tr>
<td>Syringes (Luer-Lok Tip)</td>
<td>Tool</td>
<td>BD, Franklin Lakes, NJ, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fitting Part P-704-01</td>
<td>Tool</td>
<td>Upchurch Scientific, Oak Harbor, WA, USA</td>
<td>&nbsp;</td>
<td>To connect tubing to Luer-Lok Tip Syringes</td>
</tr>
<tr>
<td>Syringe Pump (PHD 2000 Programmable)</td>
<td>Equipment</td>
<td>Harvard Apparatus, Holliston, MA, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CCD Camera (PCO 1600)</td>
<td>Equipment</td>
<td>Cooke, Romulus, MI, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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chemotactic response of marine micro-organisms to micro-scale nutrient layers

planktonic의 미생물이 microscale 자원 패치를 악용 수있는 정도 해양 trophodynamics 및 biogeochemical 플럭스에 대한 상당한 영향을 것입니다. 그러나, 수영 미생물이 패치의 가용성과 그들을 찾을 박테리아의 능력을 제한합니다 분자 확산과 난류 전단을 포함한 물리적인 세력의 영향을 극복한다, 바다에 영양 패치를 활용할 수 있습니다. 최근까지, 방법 론적 한계 곳곳에 서식지와 현실적인 소규모 흐름 조건 내에서 미생물의 행동을 직접 시험을 배제했습니다. 따라서 많은 바다 미생물 행동에 관한 현재의 지식은 이론적 예측에서 procured되었습니다. 우리가 만드는 데 사용해야이 microfluidic 장치 (전) 해양 프로세스와 관련된 치수와 잘 퍼지는 특성과 영양 패치를 microscale 그리고 (ii) microscale 개발을 소프트 리소그래피 제조 기술을 적용 바다에 미생물의 식량 약탈을 배워 행동에 새로운 정보를 얻으려면 바다에서 예상되는 것과 해당 전단 속도 vortices. 이러한 microfluidic 장치는 이기종 및 동적 씨스 케이프 내에서 미생물 수영 chemotactic 행동의 첫번째 직접 시험을 허용했습니다. 개인 미생물의 수영장 행동 이외에, 인구 수준 aggregative 반응을 관찰하면서 epifluorescence와 위상 콘트라스트 현미경의 통합 사용, 물리적 치수 및 영양소 패치 잘 퍼지는 특성을 직접 시험을 수 있습니다. 이러한 실험은 식물성 플랑크톤, heterotrophic 박테리아와 phagotrophic protists의 일부 수종은 매우 짧은 시간 프레임 내에 diffusing microscale 자원 패치를 찾기 및 활용에 숙달되는 밝혀있다. 우리는 또한 전단 속도를 중간 그런 나타날, 해양 박테리아는 자신의 협정에서 자신의 환경을 통해 흐름과 수영을 싸울 수 있습니다. 그러나, 임계값 높은 전단 수준을 넘어, 박테리아는 전단 흐름에 부합하고 흐름의 방해없이 수영을 덜 수 있습니다. Microfluidics는 수생 미생물 생태학을 공부를위한 소설과 저렴한 접근을 대표하고, 정확하게 microscale에서 현실적인 흐름 필드와 기판 그라디언트를 만들에 대한 적합성으로 인해, 상호 작용의 가장 작은 비늘에 미생물의 행동 검사에 이상적으로 적용됩니다. 따라서 microfluidics가 바다에 미생물의 생태에 대한 이해를위한 유용한 도구를 대표하는 것이 좋습니다.

Watch this Scientific Journal Video about Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers at JoVE.com

Meiosis  | Biology Video Collection

Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers

전단 흐름 내의 곳곳에 영양 씨스 케이프와 박테리아의 수영 동작 이내에 해양 미생물의 chemotactic 식량 약탈을 배워 동작을 공부를위한 microfluidic 채널과 실험에서 구현의 제조가 설명되어 있습니다.

마이크로 스케일 영양소 레이어에 해양 미생물의 Chemotactic 응답

The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and ...

chemotactic-response-marine-micro-organisms-to-micro-scale-nutrient

Research

JoVE Journal

Biology

13.2K 조회수.  MIT - Massachusetts Institute of Technology. 전단 흐름 내의 곳곳에 영양 씨스 케이프와 박테리아의 수영 동작 이내에 해양 미생물의 chemotactic 식량 약탈을 배워 동작을 공부를위한 microfluidic 채널과 실험에서 구현의 제조가 설명되어 있습니다.

비디오: Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers

Layers of Symbiosis - Visualizing the Termite Hindgut Microbial Community

Jared Leadbetter takes us for a nature walk through the diversity of life resident in the termite hindgut - a microenvironment containing 250 different species found nowhere else on Earth.  Jared reveals that the symbiosis exhibited by this system is multi-layered and involves not only a relationship between the termite and its gut inhabitants, but also involves a complex web of symbiosis among the gut microbes themselves.

Jared Leadbetter takes us for a nature walk through the diversity of life resident in the termite hindgut - a microenvironment containing 250 different species found nowhere else on Earth. Jared reveals that the symbiosis exhibited by this system is multi-layered and involves not only a relationship between the termite and its gut inhabitants, but also involves a complex web of symbiosis among the gut microbes themselves.

공생의 레이어 - 마이트의 H​​indgut의 미생물 커뮤니티를 떠올리

Jared Leadbetter takes us for a nature walk through the diversity of life resident in the termite hindgut - a microenvironment containing 250 different ...

연구

생물학

Micro-scale Engineering for Cell Biology

세포 생물학을위한 미세 규모 공학

In vivo Imaging of Deep Cortical Layers using a Microprism

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size and have a reflective coating on the hypotenuse to allow internal reflection of excitation and emission light.  The microprism probe simultaneously images multiple cortical layers with a perspective typically seen only in slice preparations.  Images are collected with a large field-of-view (~900 &mu;m).  In addition, we provide details on the non-survival surgical procedure and microscope setup.  Representative results include images of layer V pyramidal neurons from Thy-1 YFP-H mice showing their apical dendrites extending through the superficial cortical layer and extending into tufts.  Resolution was sufficient to image dendritic spines near the soma of layer V neurons.  A tail-vein injection of fluorescent dye reveals the intricate network of blood vessels in the cortex.  Line-scanning of red blood cells (RBCs) flowing through the capillaries reveals RBC velocity and flux rates can be obtained. This novel microprism probe is an elegant, yet powerful new method of visualizing deep cellular structures and cortical function in vivo.

In vivo Imaging of Deep Cortical Layers using a Microprism

Right-angle microprisms inserted into the mouse neocortex allows for deep imaging of multiple cortical layers with a viewpoint typically found in slice. One-millimeter microprisms offer a wide field-of-view (~900 &mu;m) and spatial resolutions sufficient to resolve dendritic spines. We demonstrate layer V neuronal imaging and neocortical vascular imaging using microprisms.

Microprism를 사용하여 딥 두피 레이어의 생체내 이미징에서

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

zebrafish의 시각 기능을 연구하기 위해 optokinetic 응답을 사용하여

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

Ionising 방사선에 대한 응답으로 γH2AX Foci의 부량

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short lifespan, ease of culture, and parthenogenesis that enables clonal culture. However, caution is required when measuring their survival time as it is affected by maternal age and maternal feeding conditions. Here we provide a protocol for powerful and reproducible measurement of the survival time in Brachionus rotifers following a careful synchronization of culture conditions over several generations. Empirically, poor synchronization results in early mortality and a gradual decrease in survival rate, thus resulting in weak statistical power. Indeed, under such conditions, calorie restriction (CR) failed to significantly extend the lifespan of B. plicatilis although CR-induced longevity has been demonstrated with well-synchronized rotifer samples in past and present studies. This protocol is probably useful for other invertebrate models, including the fruitfly Drosophila melanogaster and the nematode Caenorhabditis elegans, because maternal age effects have also been reported in these species.

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

Rotifers are microscopic zooplankton used as models in ecotoxicological and aging studies. Here we provide a protocol for powerful and reproducible measurement of survival time in Brachionus rotifers. Synchronization of culture conditions over several generations is of particular importance because maternal condition affects life history of offspring.

생존 시간의 측정에<em&gt; Brachionus</em&gt; 로티퍼 : 산모 조건의 동기화

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short ...

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 ...

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH distribution and calcium ions can be observed using live microscopy over time. This allows identification of the life stage and the tissue with the feature of interest for further electron microscopy studies. Life stage and tissues of interest are typically higher in pH and Ca signals. 
Here, using H. elegans, we present a protocol to characterize the presence of calcium carbonate structures in a biological specimen on the scanning electron microscope (SEM), using energy-dispersive X-ray spectroscopy (EDS) to visualize elemental composition, using electron backscatter diffraction (EBSD) to determine the presence of crystalline structures, and using transmission electron microscopy (TEM) to analyze the composition and structure of the material. In this protocol, a focused ion beam (FIB) is used to isolate samples with dimension suitable for TEM analysis. As FIB is a site specific technique, we demonstrate how information from the previous techniques can be used to identify the region of interest, where Ca signals are highest.

We demonstrate the use of various microscopy methods that are useful in observing the calcification of a tubeworm, Hydroides elegans, as well as locating and characterizing the first calcified material. Live microscopy and electron microscopy are used together to provide functional and material information that are important in studying biomineralization.

해양 tubeworm 인 라이브 광학 및 전자 현미경 기술을 사용하여 석회화 이벤트의 특성

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH ...

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent fusion proteins, such as green fluorescent protein (GFP), to determine their expression, localization, and function. The subcellular localization of target proteins is important for identification, characterization, and functional analyses. Internalization is one of the predominant mechanisms controlling G protein-coupled receptor (GPCR) signaling to ensure the appropriate cellular responses to stimuli. Here, we describe an experimental method to detect the subcellular localization and internalization of GPCR in HEK293 cells with confocal microscopy. In addition, this experiment provides some details about cell culture and transfection. This protocol is compatible with a variety of widely available fluorescent markers and is applicable to the visualization of the subcellular localization of a majority of proteins, as well as of the internalization of GPCR. This technique should enable researchers to efficiently manipulate GPCR gene expression in mammalian cell lines and should facilitate studies on GPCR subcellular localization and internalization.

This protocol describes confocal microscopy detection of G protein-coupled receptor (GPCR) internalization in mammalian cells. It includes the basic cell culture, transfection, and confocal microscopy procedure and provides an efficient and easily interpretable method to detect the subcellular localization and internalization of fusion-expressed GPCR.

공 초점 현미경으로 리간드 - 활성화 된 G 단백질 결합 수용체 내재화 검출

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

Dictyostelium discoideum 응답 심각한 기계적인 자극의 평가

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...