이 작품은 국립 과학 재단 (NSF)에 의해 지원 되었다 no를 부여. IOS 1354956입니다. 우리 닥터 앤드류 D. 워렌 (나비목 및 생물 다양성, 플로리다 자연사 박물관의, 프로 리 다의 대학에 대 한 맥과 어 센터) 나비 이미지를 사용 하는 허가 대 한 감사 합니다.

1. 곤충 종, 솔루션 및 먹이 역 설치의 준비
참고: 양배추 나비 (배 rapae L., 흰나비과) 선택 됩니다 대표 나비목의 종족으로 그들은 액체 이해 능력과 놓이고 형태학22,23의 이전 연구에서 사용 된 때문에. 집 파리 (파리 자리 부채 L., Muscidae)과 파란색 병 파리 (Calliphora vomitoria L., Calliphoridae) 때문에 그들은 종종 다공성 표면13에 먹이 관찰 하는 데 사용 됩니다.
<ol>
	<li>23 ° C와 18 L: 6D photoperiod 순서 P. rapae pupate 환경 챔버를 성인으로 등장 하기 전 까지는 인공에 그들 ( 재료의 표참조) 다이어트는 곤충 공급 업체 및 후방에서 애벌레로 설정 합니다. M주문입니다. 부채 및 pupae로 C. vomitoria 및 P. rapae으로 같은 환경 조건에서 후면. 그들은 먹이 실험 전에 번데기에서 등장 하는 후 성인 나비와 파리를 먹이 하지 마십시오.</li>
	<li>20% 자당 해결책 (제어) 및 유체 통풍 관에 대 한 테스트 20% 자당 나노 솔루션을 준비 합니다. 형광 자기 나노 입자를 추가 하 여 나노 솔루션을 준비 (철 산화물 polyacrylic 산 코팅, 약 20 nm 직경에서) 20% 자당 솔루션 (1 mg/mL의 dH2O 나노 20% 자당과24 솔루션, 1:1)입니다. 버퍼링 하는 인산 염 (PBS)의 1 배 솔루션 준비 (10 x 1 x dH2O, pH 7.4에에서 희석), 해 사용 되는.</li>
	<li>클램프와 별도 먹이 단계 (플랫 플랫폼) 수동 조작으로 구성 된 먹이 스테이션을 설정 (그림 1). 먹이 무대에 오목 슬라이드를 놓고 숨 구멍 크기 직경 1, 5, 8, 11, 20, 30, 41, 또는 60 µ m 및 멤브레인 필터의 기 공 크기 직경 나일론 그물 필터 또는 먹이 실험에 대 한 인근 10 µ m.</li>
</ol>
2. 먹이 프로토콜
<ol>
	<li>곤충의 몸, 다리, 및 날개 접힌된 휴지로 바꿈. 머리와 mouthparts 노출 되도록 곤충을 놓습니다. 곤충 먹이 단계 (그림 1) 위에 중단 되도록 조작자에 클램프에 의해 함께 개최 됩니다 두 현미경 슬라이드 사이 곤충의 날개를 놓습니다.</li>
	<li>오목 슬라이드의 중심에 20% 자당 해결책 또는 20% 자당 나노 솔루션은 micropipette 30 µ L 물방울을 관리 합니다. 되도록 필터 종이의 중심 나노 솔루션의 물방울과 오목 슬라이드에 특정 기 공 크기의 단일 필터 종이 놓습니다. 드롭릿에 필터 종이 사이의 접촉 확산 따라 필터, 숨 구멍 (그림 1)를 작성 하는 솔루션에 발생 합니다. 참고: 필터 종이 곤충 피드 필터 종이 위에 나노 입자의 잠재적인 존재를 최소화 하기 위해, 다른 방법 보다는 오히려 작은 물방울, 위에 배치 됩니다.</li>
	<li>mouthparts의 원심 영역 으로만 먹이 단계 (그림 1)에 젖은 필터 종이 연락할 수 있도록 조작자와 곤충을 놓습니다. 곤충 핀을 사용 하 여 확장 필터 종이에 mouthparts 45에 대 한 피드에 곤충을 허용을 s.</li>
	<li>필터 종이의 표면에 있을 수 있는 액체 영화에 먹이 곤충의 기회를 최소화 하기 위해 위치는 mouthparts를 (즉슬라이드의 평평한 부분에 닿아 필터 종이의 부분 접촉. 슬라이드의 오목한 부분 위에 직접). 곤충 먹이에 대 한 관심을 표현 하지 않습니다, 경우는 mouthparts 먹이 시간 동안 곤충 핀 필터 종이에 개최 수 있습니다.</li>
</ol>
3입니다. 해
<ol>
	<li>곤충의 몸을 커버 하는 충분 한 솔루션 되도록 PBS 솔루션을 50 m m 시계 유리에 놓습니다. 장소는 stereoscope 옆 장비 (봄 마이크로 위, 곤충 핀, 괜 찮 포인트 집게를 해 부 해 부)을 해 부 위치와 stereoscope 곤충 아래 시계 유리.</li>
	<li>수 유, 후 티슈 페이퍼에서 곤충을 제거 하 고 날개 폐쇄와 그것을 잡고. 봄 마이크로 해가 위로 머리, 다리, 및 곤충의 날개를 제거 하 고 시계 유리 (그림 2)에서 PBS 솔루션에 곤충을 놓습니다.</li>
	<li>필요한 경우, anesthetize 해 전에 곤충. 집게를 사용 하 여 복 부의 원심 영역 근처 표 피에 의해 곤충을 잡아. 지배적인 손을 가진 봄 마이크로 해 부가 위를 사용 하 여 복 부, 흉부에이 때까지 후부 끝에서 시작의 측면을 따라 앞쪽으로 표 피를 잘라. 표 피만 잘라 고 (그림 2) 손상 곤충 내부 영양 운하는 특별 한 주의 분리 했습니다.</li>
	<li>복 부 (그림 2) 내부 영양 운하를 공개 하 여 해가 위 표 피의 추가 컷을 확인 합니다. 곤충 핀의 도움으로 복 부 표 피, 뚱뚱한 몸, 및 다른 구조를 제거 하 고 후속 처리, 시계 유리에는 흉부 및 영양 운하를 떠나 시계 유리 밖에 서 그들을 재배치. 주: 해 부는 흉부와 복 부의 시점 근처 골목 같은 구조 (영양 운하 확장) 자르기, 발표할 예정 이다.</li>
	<li>자르기는 노출 되지 않습니다 경우 작물은 계시 될 때까지 위와 흉부에 추가 incisions 확인 합니다. 일단 자르기 표시, 시계 유리 (그림 2)에서 작물 영양 운하만 떠나는 곤충의 나머지 부분을 버려야 됩니다. 참고: lepidopteran 작물은 거의 투명 하 고 셀로판 같은 경우 하지 체액으로 가득 이며 확장을 인식 하기 어려울 수 있습니다 자연, 또는 경우 그것은 해 부 동안에.</li>
	<li>Coverslip (24 x 24 mm)에 자르기를 좋은 포인트 집게 해 부를 사용 하 여 후속 이미징 (그림 2).</li>
</ol>
4입니다. 섭취 한 나노 입자의 결정
<ol>
	<li>위치 좋은 포인트 해 집게를 사용 하 여에서 coverslip에 자르기 자르기 파열을 방지 하기 위해 신경. 20 배 확대에 이미징 거꾸로 confocal 현미경에 CY3 채널 (또는 단계 대조)를 사용 합니다. 밖으로 건조에서 그것을 방지 하기 위해 절 개 후 즉시 자르기 이미지.</li>
	<li>현미경의 운영 단계의 제어에 손에 바 (41.3 m m 길이 직경에서 8 m m) 자석 저 어를 개최.</li></ol>파 자르기 (자르기에서 약 10 m m 거리) 근처 앞 뒤로 자석 볶음 바 각각 앞뒤로 모션 발생 약 1 초 (그림 2).
	<li>마그네틱 볶음 바 흔들었다는 나노 입자에 대 한 자르기 있는지 검사 합니다. 천천히 앞뒤로 거의 투명 한 작물 안에 나노 입자 운동에 대 한 현미경의 눈 렌즈를 통해 찾고 있는 동안 운영 단계를 이동 합니다. 나노 입자는 작물에, 긍정적인 수 유를 나타내는 그들은 응답 합니다 (그림 2) 바 저 어는 자기와 함께 한 마음으로 파.</li>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeding+mechanisms+of+adult+Lepidoptera:+Structure,+function,+and+evolution+of+the+mouthparts.">Feeding mechanisms of adult Lepidoptera: Structure, function, and evolution of the mouthparts.</a> Ann Rev Entomol. 55, 307-327 (2010).</li><li>Tsai, C. -. C., Monaenkova, D., Beard, C. E., Adler, P. H., Kornev, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paradox+of+the+drinking-straw+model+of+the+butterfly+proboscis.">Paradox of the drinking-straw model of the butterfly proboscis.</a> J Exp Biol. 217, 2130-2138 (2014).</li><li>Bauder, J. A. S., Handschuh, S., Metscher, B. D., Krenn, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+morphology+of+the+feeding+apparatus+and+evolution+of+proboscis+length+in+metalmark+butterflies+(Lepidoptera:+Riodinidae).">Functional morphology of the feeding apparatus and evolution of proboscis length in metalmark butterflies (Lepidoptera: Riodinidae).</a> Biol J Linn Soc. 110, 291-304 (2013).</li></ol>

저자는 공개 없다.

곤충 진드기 기능 역사적으로 심한 형태학의 학문에서 유추 (예., 마시는 밀 짚25,26lepidopteran 코 기능 관련); 그러나, 실험적 증거를 통합 하는 최근 연구 곤충 mouthparts 및 구조-기능 관계2,,1213 의 복잡성에 대 한 우리의 이해에 패러다임의 변화 결과 , 22</sup...

많은 곤충 그룹 있다 mouthparts (proboscises) 액체에 먹이 대 한 적응, 꿀, 과일, 썩 어 등 수액 흐름 (예: Diptera1, 나비목2, Hymenoptera3), xylem (Hemiptera4), 눈물 (나비목 5)과 혈액 (Phthiraptera6, Siphonaptera7, Diptera7, Hemiptera8, 나비목9). 곤충의 체액에 피드 기능은 생태계 건강 (예: 수 분10), 질병 전송4,11, biodiversification2,12및 연구와 관련 수렴 진화13의. 다양 한 음식 소스에 불구 하 고 일부 액체 먹이 곤충 가운데 테마 마이크로 또는 나노 크기의 방울, 액체 필름 또는 다공성 표면에 국한 될 수 있는 액체의 작은 금액을 확보 하는 기능입니다.
액체 먹이 곤충 (20% 이상 모든 동물 종14,15)과 다양 한 음식 소스에 피드를 자신의 능력의 광범위 한 다양성을 감안할 때 그들의 먹이 이해 행동 및 액체 흡수 메커니즘은 많은 분야에서 중요 한. 곤충 진드기 기능, 예를 들어, 생체 모방 기술, 예를 들면, 소량 고용 된 것과 유사한 방법을 사용 하 여 체액의 수집 등의 작업을 수행할 수 있는 미세 소자의 개발에 역할을 담당해 왔습니다. 곤충16 그러나 유체 통풍 관 기계 장치,,의 연구에 근본적인 문제 뿐만 아니라 어떻게 곤충 체액에 피드 결정 이지만 메커니즘을 지 원하는 실험적인 증거를 획득. 전적으로 동작을 사용 하 여 (예: 코12,17프로 빙)로 먹이 대 한 지표로 충분 한 체액의 성공적인 이해를 확인 하지 않습니다 없으며 경로 결정 하는 방법 때문에 그 체액 여행 곤충을 통과. 또한, 소량 액체 더 나은 실험 수행 자연 먹이 시나리오를 어디 체액 제한 리소스2,12나타냅니다.
위상 대비 영상 어떻게 나비 다공성 표면12에서 체액의 작은 금액에 대 한 피드를 평가 하기 위해 바둑의 나비 (별 plexippus L.)와 함께 사용 되었다 엑스레이 모나 크 나비 코 따라 cuticular 계획 (legulae 등) 사이의 공간을 통해 모 세관 작업을 사용 하 여 음식 운하에 작은 구멍에 국한 하는 체액을가지고. 들어오는 액체 음식 운하 벽에 성장 하 고 고원 불안정12,18, 다음 머리에 빠는 펌프의 행동에 의해 나비의 용기에 운송 하 여 액체 다리에 붕괴에 영화를 형성 합니다. 기술은 쉽게 사용할 수 있는 x 선 위상 대비 영상 곤충12,19,,2021내부 유체 흐름을 시각화 하는 최적의 도구 이지만, 더 편리 하 고 메서드는 글귀 체액을 곤충의 능력의 빠른 평가 위해 필요 하 고 그들을 섭취.
D. plexippus 에 대 한 먹이 메커니즘 다른 나비목 및 파리 (Diptera) (다공성 표면에서 액체에 피드 두 그룹), 경우 확인 하려면 Lehnert 외. 13 여기에서 보고 되는 다공성 표면에서 작은 양의 액체에 먹이 곤충의 능력을 평가 하기 위한 기술 적용. 접촉을 사용 하는 연구 및 다공성 표면에 대 한 여기에 설명 된 프로토콜 이지만, 주소 풀 먹이 메커니즘 같은 다른 연구 방법론을 변경할 수 있습니다. 또한, 응용 프로그램 마이크로 및 bioinspired 기술을 포함 하 여 다른 필드를 확장 합니다.

<table><tbody><tr><td>20% sucrose solution</td><td>Domino Sugar</td><td></td><td>Sugar needed to produce the sucrose solution with dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Sigma-Aldrich</td><td>P5493</td><td>10X concentration diluted to 1X in dH&lt;sub&gt;2&lt;/sub&gt;O for insect dissections</td></tr><tr><td>Single depression concave slide</td><td>AmScope</td><td>BS-C6</td><td>Slide is necessary for feeding stage setup</td></tr><tr><td>Filter paper</td><td>EMD Millipore</td><td>NY6004700 (60 &amp;micro;m)</td><td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td></tr><tr><td>Filter paper</td><td>EMD Millipore</td><td>NY4104700 (41 &amp;micro;m)</td><td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td></tr><tr><td>Filter paper</td><td>EMD Millipore</td><td>NY3004700 (30 &amp;micro;m)</td><td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td></tr><tr><td>Filter paper</td><td>EMD Millipore</td><td>NY2004700 (20 &amp;micro;m)</td><td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td></tr><tr><td>Filter paper</td><td>EMD Millipore</td><td>NY1104700 (11 &amp;micro;m)</td><td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td></tr><tr><td>Filter paper</td><td>EMD Millipore</td><td>TCTP04700 (10 &amp;micro;m)</td><td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td></tr><tr><td>Filter paper</td><td>EMD Millipore</td><td>TETP04700 (8 &amp;micro;m)</td><td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td></tr><tr><td>Filter paper</td><td>EMD Millipore</td><td>TMTP04700 (5 &amp;micro;m)</td><td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td></tr><tr><td>Filter paper</td><td>EMD Millipore</td><td>RTTP04700 (1 &amp;micro;m)</td><td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td></tr><tr><td>Iris microdissecting scissors</td><td>Carolina Biological Supply Company</td><td>623555</td><td>Scissors used for dissections</td></tr><tr><td>Insect pins (#1)</td><td>Bioquip Products</td><td>1208B1</td><td>Pins used during dissections and feeding trials</td></tr><tr><td>Extra-fine point dissecting forceps</td><td>Carolina Biological Supply Company</td><td>624684</td><td>Dissecting equipment</td></tr><tr><td>Leica M205 C Stereoscope</td><td>Leica Microsystems</td><td>M205 C</td><td>Stereoscope used for dissections</td></tr><tr><td>Inverted confocal microscope</td><td>Olympus</td><td>IX81</td><td>Fluorescent microscope used to detect magnetic nanoparticles</td></tr><tr><td>Fisherbrand PTFE Disposable Stir Bar</td><td>Fisherscientific</td><td>S68067</td><td>Magnet used to detect nanoparticles</td></tr><tr><td>Kimtech Science Kimwipes</td><td>Kimberly-Clark Professional</td><td>34155</td><td>Tissues used to secure insects during feeding trials</td></tr><tr><td>House fly (Musca domestica) pupae</td><td>Mantisplace.com</td><td></td><td>insects for experiments</td></tr><tr><td>Blue bottle fly (Calliphora vomitoria) pupae</td><td>Mantisplace.com</td><td></td><td>insects for experiments</td></tr><tr><td>Cabbage butterfly (Pieris rapae) larvae</td><td>Carolina Biological Supply Company</td><td>144102</td><td>insects for experiments</td></tr><tr><td>Finnpipette F1&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>4641080N</td><td>micropipette for dispensing liquids</td></tr><tr><td>Finntip 250 pipette tips</td><td>ThermoFisher Scientific</td><td>9400250</td><td>micropipette tips</td></tr><tr><td>Microscope Glass cover slides (=coverslips) (24 x 24 mm)</td><td>AmScope</td><td>CS-S24-100</td><td>coverslips for viewing the insect&#039;s crop on confocal microscope</td></tr></tbody></table>

the ingestion of fluorescent, magnetic nanoparticles for determining fluid-uptake abilities in insects

액체 먹이 곤충 섭취 다양 한 환경에 풀, 필름로 또는 작은 구멍에 액체. 액체 수집의 연구 필요 평가 놓이고 구조와 함수 관계; 그러나, 액체 흡수 메커니즘 구조 건축, 실험적인 증거와 때때로 동반된의 관측에서 역사적으로 유추 됩니다. 여기, 우리 보고 나비 (나비목)와 비행 거리 (Diptera) 액체의 소량을 사용 하 여 액체 이해 능력을 평가 하기 위한 새로운 방법. 곤충은 20% 자당 해결책 특정 기 공 크기의 필터 논문에서 형광, 자성 나노 입자와 혼합으로 먹인 다. 자르기 (유체를 저장 하는 데 사용 되는 내부 구조)는 곤충에서 제거 하 고는 confocal 현미경에 배치. 자석이 곤충 체액을 섭취 수 있습니다 나타내는 나노 입자의 존재를 확인 하는 작물에 의해 손을 흔들었다. 이 방법론은 광범위 한 먹이 하는 메커니즘 (모 세관 작용 및 액체 다리 형성)은 잠재적으로 공유할 나비목과 Diptera 때 다공성 표면에서 먹이 공개 하는 데 사용 됩니다. 또한,이 방법은 먹이 액체 먹이 곤충, 질병 전송 및 biomimetics, 나노 또는 마이크로 크기의 회로 포함 하는 잠재적으로 다른 연구에서 그 중요 한을 포함 하 여 다양 한 가운데 메커니즘의 연구에 대 한 사용할 수 있습니다 어디 액체 수송 필요 확인을 합니다.

액체 먹이 곤충 가운데 액체 이해 능력에 패턴의 연구 먹이 발생 하는 시기의 결정을 필요 합니다. 여기에 설명 된 프로토콜은 나비목 및 Diptera13중 제한 기 공 크기 가설을 테스트 하는 데 사용 됩니다. 제한 기 공 크기 가설 기 공 크기 직경12먹이 도관 직경 보다 작으면 액체 채워진 구멍에서 액체 먹이 곤충 먹이 수 없는 상태. 모 공에?...

Watch this Scientific Journal Video about The Ingestion of Fluorescent, Magnetic Nanoparticles for Determining Fluid-uptake Abilities in Insects at JoVE.com

The Ingestion of Fluorescent, Magnetic Nanoparticles for Determining Fluid-uptake Abilities in Insects

액체 먹이 곤충 다공성 표면에서 액체의 분 양 취득 하는 기능이 있다. 이 프로토콜에는 직접 먹이 솔루션을 사용 하 여 형광, 자성 나노 입자와 다공성 표면에서 액체를 섭취 하는 곤충에 대 한 능력을 결정 하는 방법을 설명 합니다.

형광, 섭취 곤충에 액체 이해 능력을 결정 하기 위한 자성 나노 입자

Fluid-feeding insects ingest a variety of liquids, which are present in the environment as pools, films, or confined to small pores. Studies of liquid ...

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6.3K 조회수.  Kent State University at Stark. 액체 먹이 곤충 다공성 표면에서 액체의 분 양 취득 하는 기능이 있다. 이 프로토콜에는 직접 먹이 솔루션을 사용 하 여 형광, 자성 나노 입자와 다공성 표면에서 액체를 섭취 하는 곤충에 대 한 능력을 결정 하는 방법을 설명 합니다.

비디오: The Ingestion of Fluorescent, Magnetic Nanoparticles for Determining Fluid-uptake Abilities in Insects

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion fluxes at the cellular level are required for initiating action potentials in excitable cells.2 Sodium regulation plays an important role in both of these cases; however, no method currently exists for continuously monitoring sodium levels in vivo 3 and intracellular sodium probes 4 do not provide similar detailed results as calcium probes. In an effort to fill both of these voids, fluorescent nanosensors have been developed that can monitor sodium concentrations in vitro and in vivo.5,6 These sensors are based on ion-selective optode technology and consist of plasticized polymeric particles in which sodium specific recognition elements, pH-sensitive fluorophores, and additives are embedded.7-9 Mechanistically, the sodium recognition element extracts sodium into the sensor. 10 This extraction causes the pH-sensitive fluorophore to release a hydrogen ion to maintain charge neutrality within the sensor which causes a change in fluorescence. The sodium sensors are reversible and selective for sodium over potassium even at high intracellular concentrations.6 They are approximately 120 nm in diameter and are coated with polyethylene glycol to impart biocompatibility. Using microinjection techniques, the sensors can be delivered into the cytoplasm of cells where they have been shown to monitor the temporal and spatial sodium dynamics of beating cardiac myocytes.11 Additionally, they have also tracked real-time changes in sodium concentrations in vivo when injected subcutaneously into mice.3 Herein, we explain in detail and demonstrate the methodology for fabricating fluorescent sodium nanosensors and briefly demonstrate the biological applications our lab uses the nanosensors for: the microinjection of the sensors into cells; and the subcutaneous injection of the sensors into mice.

Fluorescent nanoparticles produced in our lab are used for imaging ion concentrations and ion fluxes in biological systems such as cells during signaling and interstitial fluid during physiological homeostasis.

생물 학적 시스템에서 이온 농도의 측정을위한 형광 Nanoparticles

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion ...

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생체공학

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

멀티 스펙트럼 이미징 유동세포계측법 의해 Nanoparticles과 박테리아의 세포 내면화 분석

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. The observation of Tribolium embryos with light sheet-based fluorescence microscopy has multiple advantages over conventional widefield and confocal fluorescence microscopy. Due to the unique properties of a light sheet-based microscope, three dimensional images of living specimens can be recorded with high signal-to-noise ratios and significantly reduced photo-bleaching as well as photo-toxicity along multiple directions over periods that last several days. With more than four years of methodological development and a continuous increase of data, the time seems appropriate to establish standard operating procedures for the usage of light sheet technology in the Tribolium community as well as in the insect community at large. This protocol describes three mounting techniques suitable for different purposes, presents two novel custom-made transgenic Tribolium lines appropriate for long-term live imaging, suggests five fluorescent dyes to label intracellular structures of fixed embryos and provides information on data post-processing for the timely evaluation of the recorded data. Representative results concentrate on long-term live imaging, optical sectioning and the observation of the same embryo along multiple directions. The respective datasets are provided as a downloadable resource. Finally, the protocol discusses quality controls for live imaging assays, current limitations and the applicability of the outlined procedures to other insect species.
This protocol is primarily intended for developmental biologists who seek imaging solutions that outperform standard laboratory equipment. It promotes the continuous attempt to close the gap between the technically orientated laboratories/communities, which develop and refine microscopy methodologically, and the life science laboratories/communities, which require 'plug-and-play' solutions to technical challenges. Furthermore, it supports an axiomatic approach that moves the biological questions into the center of attention.

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Imaging the morphogenesis of insect embryos with light sheet-based fluorescence microscopy has become state of the art. This protocol outlines and compares three mounting techniques appropriate for Tribolium castaneum embryos, introduces two novel custom-made transgenic lines well suited for live imaging, discusses essential quality controls and indicates current experimental limitations.

라이트 시트 기반의 형광 생활의 현미경 또는 고정 및 스테인드<em&gt; Tribolium castaneum</em&gt; 배아

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. ...

발생학

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, and issues commands to start or stop feeding. Deciphering the processes underlying the decision-making of timely and moderate feeding carries major implications in our understanding of physiological and psychological disorders related to feeding control. Simple, quantitative, and robust methods are required to measure the food ingestion of animals after experimental manipulation, such as forcibly increasing the activities of certain target neurons. Here, we introduced dye-labeling-based feeding assays to facilitate the neurogenetic study of feeding control in adult fruit flies. We review available feeding assays, and then describe our methods step-by-step from setup to analysis, which combine thermogenetic and optogenetic manipulation of neurons controlling feeding motivation with dye-labeled food intake assay. We also discuss the advantages and limitations of our methods, compared with other feeding assays, to help readers choose an appropriate assay.

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Quantitative food-intake assays with dyed food provide a robust and high-throughput means to evaluate feeding motivation. Combining the food consumption assay with thermogenetic and optogenetic screens is a powerful approach to investigate the neural circuits underlying appetite in adult Drosophila melanogaster.

양이 많은 음식 섭취 량 분석을 결합 하 고 강제로 초파리 에서 식욕을 공부 하는 뉴런을 활성화

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, ...

행동분석학

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

발광 생물 칼슘 표시 GFP - 쿼린을 기반으로 생체 기능성 뇌 영상의 접근 방식

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

신경과학

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and water-screening methods used currently. While conventional bacterial detection methods, such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA) can specifically detect pathogenic contaminants, they require extensive sample preparation and lengthy waiting periods. In addition, these practices demand sophisticated laboratory instruments and settings, and must be executed by trained professionals. Herein, a protocol is proposed for a simpler diagnostic technique that features the unique combination of magnetic and fluorescent parameters in a nanoparticle-based platform. The proposed multiparametric magneto-fluorescent nanosensors (MFnS) can detect E. coli O157:H7 contamination with as little as 1 colony-forming unit present in solution within less than 1 h. Furthermore, the ability of MFnS to remain highly functional in complex media such as milk and lake water has been verified. Additional specificity assays were also used to demonstrate the ability of MFnS to only detect the specific target bacteria, even in the presence of similar bacterial species. The pairing of magnetic and fluorescent modalities allows for the detection and quantification of pathogen contamination in a wide range of concentrations, exhibiting its high performance in both early- and late-stage contamination detection. The effectiveness, affordability, and portability of the MFnS make them an ideal candidate for point-of-care screening for bacterial contaminants in a wide range of settings, from aquatic reservoirs to commercially packaged foods.

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

The overall goal of this protocol is to synthesize functional nanosensors for the portable, cost-effective, and rapid detection of specifically targeted pathogenic bacteria through a combination of magnetic relaxation and fluorescence emission modalities.

E. 콜라이 O157:H7의 Foodborne 병원 체 검사를 사용 하 여 자기 형광 Nanosensor: 신속한 감지

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and ...

면역학 및 감염병학

Using Magnetometry to Monitor Cellular Incorporation and Subsequent Biodegradation of Chemically Synthetized Iron Oxide Nanoparticles

Magnetic nanoparticles, made of iron oxide, present a peculiar interest for a wide range of biomedical applications for which they are often internalized in cells and then left within. One challenge is to assess their fate in the intracellular environment with reliable and precise methodologies. Herein, we introduce the use of the vibrating sample magnetometer (VSM) to precisely quantify the integrity of magnetic nanoparticles within cells by measuring their magnetic moment. Stem cells are first labeled with two types of magnetic nanoparticles; the nanoparticles have the same core produced via a fast and efficient microwave-based nonaqueous sol gel synthesis and differ in their coating: the commonly used citric acid molecule is compared to polyacrylic acid. The formation of 3D cell-spheroids is then achieved via centrifugation and the magnetic moment of these spheroids is measured at different times with the VSM. The obtained moment is a direct fingerprint of the nanoparticles&#8217; integrity, with decreasing values indicative of a nanoparticle degradation. For both nanoparticles, the magnetic moment decreases over culture time revealing their biodegradation. A protective effect of the polyacrylic acid coating is also shown, when compared to citric acid.

Iron oxide nanoparticles are synthesized via a nonaqueous sol gel procedure and coated with anionic short molecules or polymer. The use of magnetometry for monitoring the incorporation and biotransformations of magnetic nanoparticles inside human stem cells is demonstrated using a vibrating sample magnetometer (VSM).

자기 측정을 사용하여 화학적으로 합성된 산화철 나노 입자의 세포 통합 및 후속 생분해를 모니터링합니다.

Magnetic nanoparticles, made of iron oxide, present a peculiar interest for a wide range of biomedical applications for which they are often internalized ...

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are the main branches of innate immunity, and many of the factors regulating these responses are evolutionarily conserved between invertebrates and mammals. Phagocytosis, the central component of cellular innate immunity, is carried out by specialized blood cells of the immune system. The fruit fly, Drosophila melanogaster, has emerged as a powerful genetic model to investigate the molecular mechanisms and physiological impacts of phagocytosis in whole animals. Here we demonstrate an injection-based in vivo phagocytosis assay to quantify the particle uptake and destruction by Drosophila blood cells, hemocytes. The procedure allows researchers to precisely control the particle concentration and dose, making it possible to obtain highly reproducible results in a short amount of time. The experiment is quantitative, easy to perform, and can be applied to screen for host factors that influence pathogen recognition, uptake, and clearance.

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

This protocol describes an in vivo phagocytosis assay in adult Drosophila melanogaster to quantify phagocyte recognition and clearance of microbial infections.

과일 파리, 초파리 melanogaster에 비보 먹어서 분석 결과 사용 하 여의 세포 면역 반응 평가

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are ...

A Magnetic Nanoparticle-Based Immunoassay Using a Microfluidic Device

Source: Hern&#225;ndez-Ortiz, J. A., et al. <a href="https://app.jove.com/v/63899">Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-Based Immunoassays.</a> J. Vis. Exp. (2022).
This video demonstrates a microfluidic system for a magnetic nanoparticle-based immunoassay. The detection system utilizes a microfluidic device incorporating a magnetic trap within its channels. Pre-formed immunocomplexes, comprised of antigen-coated magnetic nanoparticles bound to primary and peroxidase-conjugated secondary antibodies, are introduced into the channels. The immunocomplexes become captured in the porous magnetic trap upon applying an external magnetic field. Using a fluorogenic substrate emitting fluorescence upon catalysis by the peroxidases, the captured immunocomplexes are detected under a microscope.

미세유체 소자를 이용한 자성 나노입자 기반 면역분석법

1. Device preparation

	Fill the channels with distilled water using a syringe. Make sure there are no leaks or resistance to flow. Immerse the device in ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

Detecting Bacterial Contamination Using Magneto-fluorescent Nanosensors

In order to prepare solutions for reading in the magnetic relaxometry, 300 microliters of PBS is first pipetted into an eppendorf tube. Then, a sample of bacterial stock is added, followed by the addition of nanosensors. The solution is then transferred to a glass tube, and a piece of parafilm is placed on top in order to prevent evaporation.
The glass tube is then placed inside a larger NMR tube and inserted into the magnetic relaxometer. A baseline solution containing no bacteria and only nanosensor in PBS is used to get a baseline T2 reading as shown. Then, solutions containing various concentrations of bacteria are inserted into the magnetic relaxometer for analysis, and the changes in T2 values are caused by the binding between the nanosensors and the bacteria.
As shown, the presence of as few as one bacterial CFU can be detected within minutes using this modality. However, as the bacterial concentration increases, the MR readings are less quantifiable, which is why the use of fluorescence emission data is also tantamount to an accurate bacterial quantification. Before fluorescence data can be collected, the sample must first be centrifuged.
The solution is transferred from the glass tube to an eppendorf tube, and then centrifuged. This separates the bacteria and the nanosensors bound to it from free-floating nanosensors in solution. The supernatant is discarded and the bacterial pellet is resuspended in PBS. Finally, the sample may be analyzed via fluorescence emission. The strength of the emission will be relative to the amount of nanosensors remaining in solution, and therefore, also to the amount of bacteria present.

형광, 섭취 곤충에 액체 이해 능력을 결정 하기 위한 자성 나노 입자

요약

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