이 연구는 싱가포르 교육부 (ARC2 / 15 : M4020238)와 싱가포르 보건 의료 연구 협의회 (NMRC / OFIRG / 0005 / 2016 : M4062012)가 후원하는 Tier 2 보조금으로 지원됩니다. 저자는 Machine Shop의 도움으로 Chow Wai Hoong Bobby에게 감사드립니다.

모든 동물 실험은 싱가포르 남양 기술 대학교 (Animal Protocol Number ARF-SBS / NIE-A0263)의 기관 동물 관리 및 사용위원회의 승인을받은 지침 및 규정에 따라 수행되었습니다. 1. 시스템 설명 <ol><li> 그림 1a 와 같이 원형 스캐너 안에 PLD를 장착하십시오. PLD를 레이저 드라이버 장치 (LDU)에 연결하십시오. 참고 : PLD는 최대 펄스 에너지가 ~ 1.42 mJ이고 반복 속도가 최대 7 kHz 인 ~ 803 nm의 파장에서 ~ 136 ns 펄스를 제공합니다. 레이저 구동 유닛 (LDU)은 온도 제어기, 가변 전원, 전원 (12V) 및 함수 발생기를 포함한다. 재료 표를 참조하십시오. 가변 파워 서플라이는 레이저 파워를 제어하는데 사용되며, 함수 발생기는 PLD의 반복률을 변경하는 데 사용됩니다. </li><li> PLD 레이저를 켭니다. PLD 반복률을 &quot;7,000&quot;으로 설정합니다. Hz를 LDU의 함수 발생기를 사용하여 계산합니다. 가변 전원 장치의 전압을 &quot;3.1&quot;V로 설정하여 펄스 에너지를 1.42mJ로 증가시킵니다. </li><li> 그림 1a 와 같이 광 확산기 (OD)를 PLD 출구 창 앞에 설치하여 출력 빔을 균일하게 만듭니다. 참고 : 미세 입자 ( 예 : 1,500- 그릿 광택)가있는 디퓨저를 사용하십시오. </li><li> UST 홀더에 초점을 맞춘 UST를 그림 1a 와 같이 스캐닝 영역의 중심을 향하도록 장착합니다. 참고 : UST의 중심 주파수는 2.25MHz이고 초점 길이는 1.9 인치입니다. </li><li> 그림 1a 와 같이 초음파 감지기를 아크릴 탱크 안에 놓습니다 . UST가 완전히 잠기도록 탱크를 물로 채 웁니다. 참고 : 수중 매체는 뇌 (샘플)의 광 음향 신호를 UST에 연결하는 데 사용됩니다. 아크릴 물 탱크 (WT, 재료 표 참조)는 맞춤형입니다작은 동물 이미징을 위해 설계되었습니다. 물 탱크 설계도는 그림 1b에 나와 있습니다. </li><li> 펄서 / 리시버 장치 (PRU; 재료 표 참조)를 사용하여 샘플에서 PA 신호를 확인하십시오. 참고 :이 신호는 100MS / s 샘플링 속도로 12 비트 DAQ 카드 ( 재료 표 참조)로 디지털화되어 컴퓨터에 저장되었습니다. </li></ol> 2. 쥐 뇌 이미징을위한 동물 준비 참고 : 위에서 설명한 PLD - PAT 시스템은 작은 동물의 두뇌 이미징을 위해 시연되었습니다. 이 실험을 위해 건강한 암컷 쥐들 ( Table of Materials 참조)이 사용되었습니다. <ol><li> 동물에게 케타민 2 mL, 자일 라진 1 mL 및 식염수 1 mL (0.2 mL / 100 g 용량)의 칵테일을 복강 내 주사하여 마취시킨다. </li><li> 머리 깎기를 사용하여 동물의 두피에있는 모피를 제거하십시오. 면도 크림에 부드럽게 제모 크림을 바릅니다.모피의 더 고갈을위한 영역. <ol><li> 면봉을 사용하여 4-5 분 후에 적용된 크림을 제거하십시오. </li><li> 마취 및 레이저 조명으로 인한 건조를 방지하기 위해 동물의 눈에 인공 눈물 연고를 바르십시오. </li></ol></li><li> 실습용 잭에 호흡 마스크 ( Table of Materials 참조)가 장착 된 주문 제작 동물 홀더 ( 재료 표 참조)를 장착하십시오. </li><li> 동물을 놓기 쉬운 위치에 놓습니다. 이미징 중 동물의 움직임을 피하기 위해 외과 용 테이프를 사용하여 홀더에 고정시킵니다. </li><li> 호흡 마스크가 쥐의 코와 입을 덮어 흡입 마취를 전달하는지 확인하십시오. </li></ol> 3. 생쥐 생쥐 뇌 이미징 <ol><li> 호흡 마스크를 마취 기계에 연결하십시오. 마취 기계를 켜고 0.75 % 이소 플루 란으로 산소 1.0 L / min을 전달하도록 설정하십시오. <ol><li> 맥박 산소 농도계를 클램핑하십시오.동물의 생리적 상태를 모니터하기 위해 꼬리를 잡아 당긴다. </li></ol></li><li> 쥐의 두피에 무색의 초음파 젤 층을 바르십시오. 랩 - 잭 위치를 스캐너 중앙으로 조정하십시오. 호흡 마스크는 이미징 창에 맞게 사용자 정의됩니다. 시중에서 구할 수있는 코 콘 10 %를 자르고 장갑 조각에 연결하십시오. <ol><li> 이미징 비행기가 UST의 초점에 있도록 실험실 잭의 높이를 수동으로 조정하십시오. </li></ol></li><li> 필요에 따라 데이터 수집 소프트웨어의 매개 변수를 설정하십시오 (재료 표 참조). 데이터 수집 소프트웨어 프로그램을 실행하여 수집 ( 즉, 이미징)을 시작하십시오. 참고 :이 프로그램은 UST를 회전하고 A 라인 PA 신호를 수집하는 데 사용됩니다. 수집 된 A- 라인은 컴퓨터에 저장됩니다. </li><li> 전체 이미징 기간 동안 동물을 관찰하고 이미징이 완료된 후 PAT 재건을 진행하십시오. </li><li> 데이터 수집이 끝나면,재구성 소프트웨어 프로그램을 사용하여 A- 라인에서 횡단면 뇌 영상을 재구성하십시오. </li><li> 마취 시스템을 끄고 동물을 무대에서 꺼내서 새장으로 돌려 놓고 의식을 회복 할 때까지 모니터하십시오. 참고 : 예를 들어, UST가 5 초 동안 회전하면 PLD는 35,000 (= 5 x 7,000) 펄스를 제공하고 UST는 35,000 개의 A 라인을 수집합니다. 35,000 개의 A 라인은 평균 70 개 이상의 신호를 평균화하여 (평균 A 라인 = 35,000 / 70 = 500) 평균 500 개로 축소됩니다. 그림 1c 는 레이저 펄스 및 A 라인 수집의 조명을 보여줍니다. 지연 - 합 역 투영 알고리즘을 기반으로 한 재구성 프로그램을 사용해야합니다. </li></ol><img alt="그림 1" class="xfigimg" src="/files/ftp_upload/55811/55811fig1.jpg" /> 그림 1 : PLD-PAT 시스템의 회로도 ( a ) PLD-PAT의 개략도. PLD : 펄스 레이저 다이오드, CSP : circuAM : 마취기, M : 모터, MPU : 모터 풀리 장치, LDU : 레이저 드라이버 장치, PRU : 펄서 / 리시버 장치, UST : 초음파 변환기, WT : 물 탱크, PF : 고분자 필름 및 DAQ : 데이터 수집 카드. ( b ) 생체 내 소 동물 뇌 영상을위한 수조의 평면도, 평면도 (1) ​​및 단면도 (2). A : 미터 나사, B : 아크릴 환형 판, C : 실리콘 &quot;O&quot;링, D : 100 μm 두께, 투명 폴리에틸렌 커버. 탱크는 바닥에 직경 9 cm의 구멍이 있었고, 초음파 및 광학적으로 투명한 100 μm 두께의 폴리에틸렌 막으로 봉인되었습니다. ( c ) PLD 및 A- 라인으로부터의 레이저 펄스의 조명의 도식. 5 초 연속 스캔 시간. <a href="http://ecsource.jove.com/files/ftp_upload/55811/55811fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

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S., Beard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulsed+near-infrared+laser+diode+excitation+system+for+biomedical+photoacoustic+imaging.">Pulsed near-infrared laser diode excitation system for biomedical photoacoustic imaging.</a> Opt Lett. 31 (23), 3462-3464 (2006).</li><li>Kolkman, R. G. M., Steenbergen, W., van Leeuwen, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+photoacoustic+imaging+of+blood+vessels+with+a+pulsed+laser+diode.">In vivo photoacoustic imaging of blood vessels with a pulsed laser diode.</a> Lasers Med Sci. 21 (3), 134-139 (2006).</li><li>Daoudi, K., 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=Handheld+probe+integrating+laser+diode+and+ultrasound+transducer+array+for+ultrasound/photoacoustic+dual+modality+imaging.">Handheld probe integrating laser diode and ultrasound transducer array for ultrasound/photoacoustic dual modality imaging.</a> Opt Express. 22 (21), 26365-26374 (2014).</li><li>Upputuri, P. K., Pramanik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulsed+laser+diode+based+optoacoustic+imaging+of+biological+tissues.">Pulsed laser diode based optoacoustic imaging of biological tissues.</a> Biomed Phys Eng Express. 1 (4), 045010-045017 (2015).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> American National Standard for Safe Use of Lasers ANSI Z136.1-2000. , (2000).</li></ol>

저자는 원고에 관련있는 금전적 이해 관계가없고 공개 할 다른 잠재적 인 이해 상충은 없다.

이 작품은 쥐 PLD - PAT 시스템을 사용하여 생체 내 뇌 영상을 수행하기위한 프로토콜을 제공합니다. 이 프로토콜에는 쥐의 뇌 영상에 대한 일러스트레이션뿐만 아니라 이미징 시스템과 그 정렬에 대한 자세한 설명이 포함되어 있습니다. 기존의 OPO 기반 PAT 시스템은 값 비싸고 부피가 크며 5-10 분 내에 하나의 단면 이미지를 제공 할 수 있습니다. PLD-PAT 시스템은 콤팩트하고 휴대 가능하며 저?...

<html> Photoacoustic tomography (PAT)는 임상 및 전임상 연구 1 , 2 , 3 , 4 , 5 에서 많은 응용 분야가있는 하이브리드 이미징 모달입니다. PAT에서 나노초 레이저 펄스는 생물학적 조직을 조사합니다. 조직 발색단에 의한 입사광의 흡수는 국부적 인 온도 상승을 일으키고, 이는 음파의 형태로 방출되는 압력 파를 산출합니다. 초음파 검출기는 샘플 주변의 다양한 위치에서 광 음향 신호를 수집합니다. 광 음향 (PA) 신호는 광섬유 이미지를 생성하기 위해 다양한 알고리즘 (예 : 지연 - 합 알고리즘) 6 을 사용하여 재구성됩니다. 이 하이브리드 이미징 모달리티는 고해상도, 심 조직 이미징 및 높은 광학 흡수 대비를 제공합니다 .class = &quot;xref&quot;&gt; 8. 최근에는 더 긴 파장 (~ 1,064 nm)과 인산염 프탈로시아닌 (phosphorus phthalocyanine)이라는 외인성 조영제를 사용하여 닭 가슴 조직에서 ~ 12 cm 이미징 깊이 9 가 달성되었습니다. 이 깊이 감도는 공 촛점 형광 현미경, 2 광자 형광 현미경, 10 광 결맞음 단층 촬영 등과 같은 다른 광학 방법의 깊이 감도보다 훨씬 높습니다 . 하나 이상의 파장을 사용하여 PAT는 장기의 구조적 및 기능적 변화를 나타낼 수 있습니다 . 많은 인간의 질병에 대해, 작은 동물 모델은 잘 확립되어 있습니다 12 , 13 , 14 , 15 . 작은 동물의 이미징을 위해 몇 가지 양상이 입증되었습니다. 이러한 모든 접근법 중 PA 이미징은 앞서 언급 한 장점으로 인해 빠르게 주목을 받았습니다. 아빠T는 작은 동물 4 , 16 , 17 , 18 의 조직과 기관 ( 즉, 심장, 폐, 간, 눈, 비장, 뇌, 피부, 척수, 신장 등 )에서 혈관을 영상화 할 수있는 가능성을 보여주었습니다. PAT는 소 동물 뇌 영상을위한 잘 확립 된 양식입니다. PA 파는 발색단에 의한 광 흡수로 인해 생성되므로 다중 파장 PAT는 총 헤모글로빈 농도 (HbT)와 산소 포화도 (SO 2 ) 19 , 20 , 21 , 22 의 매핑을 허용합니다. 뇌의 신경 혈관 이미징은 외인성 조영제 12 , 23 , 24 의 도움으로 이루어졌습니다. PA 양식은 뇌 건강에 대한 더 나은 이해를 돕습니다.분자 및 유전 수준에서 정보를 제공합니다. 작은 동물 이미징의 경우, Nd : YAG / OPO 레이저가 PAT 여기 소스로 널리 사용됩니다. 이 레이저는 ~ 10-Hz 반복 속도로 약 5 ns의 근적외선 펄스 (에너지가 OPO 출력 창에서 ~ 100 mJ)를 전달합니다. 이러한 레이저가 장착 된 PA 시스템은 값이 비싸고 부피가 크며 레이저 소스의 반복률이 낮기 때문에 단일 요소 초음파 변환기 (UST)로 저속 이미징이 가능합니다. 이러한 PA 시스템의 일반적인 A- 라인 획득 시간은 단면 25 당 ~ 5 분입니다. 그러한 긴 측정 시간을 가진 영상 시스템은 전신 영상, 시간 - 해상도 기능 영상 등 을위한 생리적 매개 변수를 제어하기가 어렵 기 때문에 작은 동물 영상에 이상적이지 않습니다 . 다중 단일 소자 UST를 채택함으로써, 어레이 기반의 UST 또는 높은 반복 속도의 레이저를 사용하면 PA의 이미징 속도를 높일 수 있습니다시스템. 샘플 주위의 모든 PA 신호를 수집하기 위해 하나의 단일 요소 UST 만 사용하면 시스템의 이미징 속도가 제한됩니다. 고속 또는 고감도의 이미징 기술을 위해 원형 또는 반원형 형상으로 배열 된 여러 개의 단일 요소 UST가 시연됩니다. 선형, 반원형, 원형 ​​및 부피 측정 어레이와 같은 어레이 기반 UST 26 이 실시간 이미징에 성공적으로 사용되었습니다 1 . 이러한 어레이 기반 UST는 이미징 속도를 높이고 측정 감도를 낮추지 만 비싸다. 그러나 어레이 기반 UST를 사용하는 PA 시스템의 이미징 속도는 여전히 레이저의 반복 속도에 의해 제한됩니다. 펄스 레이저 기술은 높은 반복 속도의 펄스 레이저 다이오드 (PLD)를 만들기 위해 발전했습니다. 7,000 frames / s 임상용 초음파 플랫폼을 사용하는 PLD로 B- 스캔 광 음향 이미징이 시연되었습니다. 이러한 PLD는 th의 영상 속도를 향상시킬 수있다.e PAT 시스템, 심지어 단일 요소 UST 순환 스캐닝 형상. 단일 요소 UST는 어레이 기반 UST와 달리 저렴하고 민감합니다. 지난 10 년 동안, PA 영상의 자극 원으로서 고 반복률 PLD의 사용에 관한 연구는 거의보고되지 않았다. 섬유 기반의 근적외선 PLD가 팬텀 28의 PA 이미징에 대해 입증되었습니다. 인간의 피부 아래 ~ 1mm 깊이에서 혈관을 생체 내 이미징 한 것은 저에너지 PLD 29를 사용하여 입증되었습니다. PLD 기반의 광학 해상도 photoacoustic microscope (ORPAM)이보고되었습니다. PLD를 사용하여 0.43 Hz의 프레임 속도에서 ~ 1.5 cm 깊이 이미징이 입증되었습니다 30 . 아주 최근에, PLD-PAT 시스템은 생물학적 조직에서 약 3 초 및 약 2 cm 이미징 깊이에서 이미지를 제공하는 것으로보고되었다. 이 연구는 그러한 저가의 소형 시스템이 높은 쿼아를 제공 할 수 있음을 입증했다.lity 이미지, 고속에서도. PLD-PAT 시스템은 높은 프레임 속도 (7,000fps) 광 음향 이미징, 표면 혈관 이미징, 손가락 관절 영상, 2cm 깊이 조직 이미징, 소형 동물 뇌 영상 등을 위해 사용될 수 있습니다 . 단일 파장 및 PLD로부터의 낮은 펄스 에너지 펄스는 멀티 스펙트럼 및 심 조직 이미징으로의 적용을 제한합니다. 실험은 전임상 적용에 사용 된 것과 동일한 PLD-PAT 시스템을 사용하여 작은 동물에 대해 수행되었습니다. 이 연구의 목적은 작은 동물의 생체 내 2D 횡단 뇌 이미징을위한 PLD-PAT 시스템의 시각화 된 실험 데모를 제공하는 것입니다.

<table border="0" cellpadding="0" cellspacing="0" width="1212">
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	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:243px;">Pulsed laser diode</td>
			<td style="width:195px;">Quantel, France</td>
			<td style="width:327px;">QD-Q1910-SA-TEC</td>
			<td style="width:449px;">It is the excitation laser source with specifications 803 nm, 1.4mJ per pulse, 136 ns pulse, 7kHz maximum, dimentions : 11.0 x 6.0 x 3.6 cm, weight: ~150 gm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Stepper motor with gearbox</td>
			<td style="width:195px;">LIN Engineering (Servo Dynamics)</td>
			<td style="width:327px;">Motor: CO-5718-01P, Gearbox: DPL64/1, I = 10 for NEMA 23; power supply PW100-48</td>
			<td style="width:449px;">To move the detector holder in a circular geometry. Torque: 2.08 N-m, Rotor inertia: 2.6 kg-cm2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Ultrasonic pulser/receiver</td>
			<td>Olympus</td>
			<td>5072PR</td>
			<td style="width:449px;">To receive, filter and ampligy the PA signal from UST. Its bandwidth is 35MHz, and gain is &#177;59 dB.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Ultrasound Transducer</td>
			<td>Olympus</td>
			<td>V306-SU-NK-CF1.9IN/Q4200069</td>
			<td style="width:449px;">Ultrasonic sensors used for photoacoustic detection. Central freqency 2.25 MHz, 0.5 in, Cylindrical focus 1.9 inch</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">PCIe DAQ (Data acquisition) Card</td>
			<td>GaGe</td>
			<td>CSE4227/ A6000610/B0E00610</td>
			<td style="width:449px;">12 bit, 100 Ms/s, 2 channels, 1 Gs on board memory, PCIe x16 interface</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Rats</td>
			<td>In Vivos Pte Ltd, Singapore</td>
			<td style="width:327px;">NTac:SD, Sprague Dawley / SD</td>
			<td>Female, weight 100&#177;10g</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Acrylic water tank </td>
			<td style="width:195px;">NTU workshop</td>
			<td style="width:327px;">Custom-made</td>
			<td style="width:449px;">It contains the water that acts as an acoustic coupling medium between brain and detector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Circular Scanner </td>
			<td style="width:195px;">NTU workshop</td>
			<td style="width:327px;">Custom-made</td>
			<td>Scanner is made out of Alluminum </td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Anesthetic Machine</td>
			<td>medical plus pte ltd</td>
			<td style="width:327px;">Non-Rebreathing Anaesthesia machine with oxygen concentrator.</td>
			<td>Supplies oxygen and isoflurane to animal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Pulse Oxymeter portable</td>
			<td>Medtronic</td>
			<td style="width:327px;">PM10N with veterinary sensor</td>
			<td>Monitors the pulse oxymetry of the animal</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Ultrasound gel</td>
			<td style="width:195px;">Progress/parker acquasonic gel</td>
			<td>PA-GEL-CLEA-5000</td>
			<td>Clear ultrasound gel</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:243px;">Data acqusison software</td>
			<td style="width:195px;">National Instruments Corporation,Austin,TX,USA)</td>
			<td style="width:327px;">NI LabVIEW 2015 SP1</td>
			<td style="width:449px;">LabVIEW based program was developed in our laboratory for controlling the stepper motor and acquring the PA singnals from the detector</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Data processing software</td>
			<td style="width:195px;">Matlab (Mathworks, Natick, MA, USA)</td>
			<td style="width:327px;">Matlab R2012b</td>
			<td style="width:449px;">Matlab code for reconstruction of PA images was developed in our lab</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Temperature controller </td>
			<td>LaridTech, MO,USA</td>
			<td>MTTC1410</td>
			<td>It will constantly control temperature of the PLD </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">12 V power supply </td>
			<td>Voltcraft </td>
			<td style="width:327px;">PPS-11810</td>
			<td>To supply operating voltage for PLD</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Variable power supply </td>
			<td>BASETech</td>
			<td>BT-153</td>
			<td>To change the laser output power</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Funtion generator </td>
			<td style="width:195px;">Funktionsgenerator</td>
			<td>FG250D</td>
			<td style="width:449px;">To change the repetetion rate of the PLD. It will provide TTL signal to synchronize the DAQ with the laser excitation.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Animal distributor</td>
			<td style="width:195px;">In Vivos Pte Ltd, Singapore</td>
			<td style="width:327px;"></td>
			<td>Animal distributor that supplies small animals for research purpose.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Animal holder</td>
			<td style="width:195px;">NTU workshop</td>
			<td style="width:327px;">Custom-made</td>
			<td>Used for holding the animal on its abdomen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Breathing mask</td>
			<td style="width:195px;">NTU workshop</td>
			<td style="width:327px;">Custom-made</td>
			<td>Used along with animal holder to supply anesthesia mixture to the animal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Pentobarbital sodium</td>
			<td style="width:195px;">Valabarb</td>
			<td style="width:327px;"></td>
			<td>Used for euthanizing the animal after the expeirment.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Optical diffuser</td>
			<td style="width:195px;">Thorlabs</td>
			<td style="width:327px;">DG10-1500</td>
			<td>Used to to make the laser beam homogeneous</td>
		</tr>
	</tbody>
</table>

a high-performance compact photoacoustic tomography system for in vivo small-animal brain imaging

생체 내 작은 동물 영상은 전임상 연구에서 중요한 역할을합니다. Photoacoustic tomography (PAT)는 전임상 및 임상 적용 모두에 대한 큰 잠재력을 보여주는 신흥 하이브리드 이미징 양식입니다. 기존의 광 매개 오실레이터 기반 PAT (OPO-PAT) 시스템은 부피가 크고 값 비싸고 고속 이미징을 제공 할 수 없습니다. 최근 펄스 레이저 다이오드 (PLD)가 PAT의 대안적인 여기 소스로서 성공적으로 시연되었습니다. 펄스 레이저 다이오드 PAT (PLD-PAT)는 광 음향 팬텀 및 생물 조직에서의 고속 이미징에 성공적으로 시연되었습니다. 이 작품은 PLD - 팻을 사용하여 생체 내 뇌 이미징을위한 시각화 실험 프로토콜을 제공합니다. 이 프로토콜에는 소형 PLD-PAT 시스템 구성과 그 기술, 뇌 이미징을위한 동물 준비 및 2D 횡단면 쥐 뇌 영상을위한 전형적인 실험 절차가 포함됩니다. PLD-PAT 시스템은 콤팩트하고 비용은 저렴합니다.fective이며 고속, 고품질 이미지를 제공 할 수 있습니다. 생체 내 에서 다양한 스캔 속도로 수집 된 뇌 이미지가 제시됩니다.

이 섹션에서는 설명 된 PLD-PAT 시스템의 기능을 보여주는 생체 내 뇌 영상 결과를 보여줍니다. PLD-PAT 시스템의 고속 이미징 기능을 입증하기 위해 두 가지 건강한 쥐의 생체 내 뇌 영상을 수행했습니다. 그림 2 는 다양한 스캔 속도에서 암컷 쥐 (93g)의 뇌 이미지를 보여줍니다. 그림 2a 와 b는 두뇌 영역에서 두피를 제...

Watch this Scientific Journal Video about A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging at JoVE.com

A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging

소형 동물에서의 고속 생체 내 뇌 이미징을위한 소형 펄스 레이저 다이오드 기반 광 음향 단층 촬영 (PLD-PAT) 시스템이 시연되었습니다.

다음을위한 고성능 컴팩트 광 음향 단층 촬영 시스템<em&gt; In Vivo</em&gt; 소형 동물 뇌 영상

In vivo small-animal imaging has an important role to play in preclinical studies. Photoacoustic tomography (PAT) is an emerging hybrid imaging modality ...

a-high-performance-compact-photoacoustic-tomography-system-for-vivo

Research

JoVE Journal

Bioengineering

A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging

10.4K 조회수.  Nanyang Technological University.  소형 동물에서의 고속 생체 내 뇌 이미징을위한 소형 펄스 레이저 다이오드 기반 광 음향 단층 촬영 (PLD-PAT) 시스템이 시연되었습니다. 

비디오: A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

입체 광학 해상도 Photoacoustic 현미경

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

연구

생체공학

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

에 대한 바이러스 나노 입자<em&gt; 생체에</em&gt; 종양 이미징

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging technologies. Existing retinal imaging modalities, such as fundus photography1, confocal scanning laser ophthalmoscopy (cSLO)2, and optical coherence tomography (OCT)3, have significant contributions in monitoring disease onsets and progressions, and developing new therapeutic strategies. However, they predominantly rely on the back-reflected photons from the retina. As a consequence, the optical absorption properties of the retina, which are usually strongly associated with retinal pathophysiology status, are inaccessible by the traditional imaging technologies.
Photoacoustic ophthalmoscopy (PAOM) is an emerging retinal imaging modality that permits the detection of the optical absorption contrasts in the eye with a high sensitivity4-7 . In PAOM nanosecond laser pulses are delivered through the pupil and scanned across the posterior eye to induce photoacoustic (PA) signals, which are detected by an unfocused ultrasonic transducer attached to the eyelid. Because of the strong optical absorption of hemoglobin and melanin, PAOM is capable of non-invasively imaging the retinal and choroidal vasculatures, and the retinal pigment epithelium (RPE) melanin at high contrasts 6,7. More importantly, based on the well-developed spectroscopic photoacoustic imaging5,8 , PAOM has the potential to map the hemoglobin oxygen saturation in retinal vessels, which can be critical in studying the physiology and pathology of several blinding diseases 9 such as diabetic retinopathy and neovascular age-related macular degeneration.
Moreover, being the only existing optical-absorption-based ophthalmic imaging modality, PAOM can be integrated with well-established clinical ophthalmic imaging techniques to achieve more comprehensive anatomic and functional evaluations of the eye based on multiple optical contrasts6,10 . In this work, we integrate PAOM and spectral-domain OCT (SD-OCT) for simultaneously in vivo retinal imaging of rat, where both optical absorption and scattering properties of the retina are revealed. The system configuration, system alignment and imaging acquisition are presented.

Photoacoustic ophthalmology (PAOM), an optical-absorption-based imaging modality, provides the complementary evaluation of the retina to the currently available ophthalmic imaging technologies. We report the using of PAOM integrated with spectral-domain optical coherence tomography (SD-OCT) for simultaneous multimodal retinal imaging in rats.

통합 Photoacoustic Ophthalmoscopy 및 스펙트럼 도메인 광 결맞음 단층 촬영 장치

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging ...

Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

A detailed understanding of the complexity and relative variability within the human cardiac venous system is crucial for the development of cardiac devices that require access to these vessels. For example, cardiac venous anatomy is known to be one of the key limitations for the proper delivery of cardiac resynchronization therapy (CRT)1 Therefore, the development of a database of anatomical parameters for human cardiac venous systems can aid in the design of CRT delivery devices to overcome such a limitation. In this research project, the anatomical parameters were obtained from 3D reconstructions of the venous system using contrast-computed tomography (CT) imaging and modeling software (Materialise, Leuven, Belgium). The following parameters were assessed for each vein: arc length, tortuousity, branching angle, distance to the coronary sinus ostium, and vessel diameter.
	CRT is a potential treatment for patients with electromechanical dyssynchrony. Approximately 10-20% of heart failure patients may benefit from CRT2. Electromechanical dyssynchrony implies that parts of the myocardium activate and contract earlier or later than the normal conduction pathway of the heart. In CRT, dyssynchronous areas of the myocardium are treated with electrical stimulation. CRT pacing typically involves pacing leads that stimulate the right atrium (RA), right ventricle (RV), and left ventricle (LV) to produce more resynchronized rhythms. The LV lead is typically implanted within a cardiac vein, with the aim to overlay it within the site of latest myocardial activation. 
	We believe that the models obtained and the analyses thereof will promote the anatomical education for patients, students, clinicians, and medical device designers. The methodologies employed here can also be utilized to study other anatomical features of our human heart specimens, such as the coronary arteries. To further encourage the educational value of this research, we have shared the venous models on our free access website: <a href="http://www.vhlab.umn.edu/atlas" target="_blank">www.vhlab.umn.edu/atlas</a>.

The objective of this research is to recreate and then access the anatomy of the human cardiac venous system using 3D reconstructions generated from contrast-computed tomography scans.

콘트라스트 계산하기 관류 고정 시편의 단층 촬영을 이용하여 인간의 심장 정맥 시스템의 해부학 적 재건

A detailed  understanding of the complexity and relative variability within the human  cardiac venous system is crucial for the development of cardiac ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

소형 동물 모델에서의 동정맥 (AV) 루프는 혈관 신생과 혈관을 조성한 조직 공학을 공부하기

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the optical mean free path (~1 mm in skin) with high resolution. By combining optical absorption contrast with the high spatial resolution of ultrasound in a single modality, this technique can penetrate deep tissues. Photoacoustic microscopy systems can have either a low acoustic resolution and probe deeply or a high optical resolution and probe shallowly. It is challenging to achieve high spatial resolution and large depth penetration with a single system. This work presents an AR-OR-PAM system capable of both high-resolution imaging at shallow depths and low-resolution deep-tissue imaging of the same sample in vivo. A lateral resolution of 4 &#181;m with 1.4 mm imaging depth using optical focusing and a lateral resolution of 45 &#181;m with 7.8 mm imaging depth using acoustic focusing were successfully demonstrated using the combined system. Here,&#160;in vivo&#160;small-animal blood vasculature imaging is performed to demonstrate its biological imaging capability.

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Here a switchable acoustic resolution (AR) and optical resolution (OR) photoacoustic microscopy (AR-OR-PAM) system capable of both high resolution imaging at shallow depth and low resolution deep tissue imaging on the same sample in vivo is demonstrated.

전환 가능한 음향 및 광학 해상도 Photoacoustic Microscopy for<em&gt; In Vivo</em&gt; 소 동물 혈관 조영술

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the ...

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high resolutions in live animal models. This allows for repeated imaging of the same rodent over time. This feature not only reduces the total number of rodents required in an experimental design and thereby reduces the inter-subject variation that can arise, but also allows researchers to assess longitudinal or life-long responses to an intervention. To acquire high quality images that can be processed and analyzed to more accurately quantify outcomes of bone micro-architecture, users of in vivo &#181;CT scanners must properly anesthetize the rat, and position and restrain the hind limb. To do this, it is imperative that the rat be anesthetized to a level of complete relaxation, and that pedal reflexes are lost. These guidelines may be modified for each individual rat, as the rate of isoflurane metabolism can vary depending on strain and body size. Proper technique for in vivo &#181;CT image acquisition enables accurate and consistent measurement of bone micro-architecture within and across studies.

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

This paper instructs users of in vivo micro-computed tomography (&#181;CT) scanners how to anesthetize, correctly position and restrain the hind limb of a rat for minimal movement during high-resolution imaging of the tibia. The result is high quality images that can be processed to accurately quantify bone micro-architecture.

적절 한 위치와 뼈 마이크로 아키텍처를 사용 하 여 생체 내에서 마이크로-단층의 집중된 고해상도 이미징 쥐 뒷 다리의 구속

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high ...

Hand-held Clinical Photoacoustic Imaging System for Real-time Non-invasive Small Animal Imaging

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, we report a combined photoacoustic and clinical ultrasound imaging system by integrating an ultrasound probe with light delivery for small animal imaging. We demonstrate this by showing sentinel lymph node imaging in small animals along with minimally invasive real-time needle guidance. A clinical ultrasound platform with access to raw channel data allows the integration of photoacoustic imaging leading to a handheld real-time clinical photoacoustic imaging system. Methylene blue was used for sentinel lymph node imaging at 675 nm wavelength. Additionally, needle guidance with dual modal ultrasound and photoacoustic imaging was shown using the imaging system. Depth imaging of up to 1.5 cm was demonstrated with a 10 Hz laser at a photoacoustic imaging frame rate of 5 frames per second.

A clinical handheld photoacoustic imaging system will be demonstrated for real-time non-invasive small animal imaging.

휴대용 임상 Photoacoustic 실시간 비-침략 적 작은 동물 이미징을 위한 이미징 시스템

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

엄밀한 포함의 고정 및 스테인드, 전체, 마이크로 단층에 의해 섹션-무료 3D 조직학에 대 한 밀리미터 단위 표본

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

피부에 있는 유전으로 표를 붙인 기자 세포의 Vivo 2 색 2-광자 화상 진찰에서

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

다음을위한 고성능 컴팩트 광 음향 단층 촬영 시스템

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