저자는 중국 국립 자연과학 재단(61822505; 11774101; 61627827; 81630046), 중국 광동성 과학 기술 기획 프로젝트(2015B02020233016), 광저우 의 과학기술 프로그램(20101년)의 재정 지원을 인정하고자 합니다.

모든 동물 실험은 중국 광저우 주 사우스 차이나 사범 대학의 기관 동물 관리 및 사용 위원회가 제공하는 지침에 따라 수행되었습니다.
1. 시스템 설정
<ol><li>광학 경로(도 1)<ol>
<li>시스템 레이저 소스로 532 nm 펄스 레이저를 사용합니다. 레이저의 반복 속도를 10kHz로 설정하고, 출력 에너지는 100%로 설정하고, 트리거 설정을 사용자 정의 프로그램을 사용하여 외부 트리거로 설정합니다.</li>
		<li>광섬유 커플러(FC1)를 통해 레이저 빔을 단일 모드 섬유(SMF)에 결합합니다. 2차원 동력 단계(모터, 최대 속도: 20mm/s)에서 광섬유 콜리메이터(FC2)를 사용하여 레이저 빔을 콜리메이트합니다.</li>
		<li>2축 갈바노미터 스캐너(갈바)를 사용하여 레이저 빔을 편향시하십시오. 이동 가능한 미러(M1)를 사용하여 빔을 반사합니다. 4× 목표 렌즈를 통해 빔에 초점을 맞춥니다(OL, 숫자 조리개: 0.1).</li>
		<li>XY 번역기 마운트(TM)를 사용하여 자체 제작 중공 초음파 트랜스듀서(UT, 중앙 주파수: 25MHz)를 수정합니다. 대역폭: 90% 이상; 중앙 구멍 : OL12의하단에 3mm) . 초음파 트랜스듀서의 중앙 구멍을 통해 집중 빔을 전달합니다.</li>
	</ol>
</li>
	<li>경로 스캔<ol>
<li>WIM 동안 필드 프로그래밍 가능한 게이트 어레이(FPGA 2)를 사용하여 Galva를 잠급니다. 사용자 정의 프로그램을 통해 적절한 검색 범위 및 스캔 속도를 설정합니다.</li>
		<li>RIM 동안 필드 프로그래밍 가능한 게이트 어레이(FPGA 1)를 사용하여 모터를 잠급니다. FPGA 2를 사용하여 스캔 빈도 및 스캔 포인트 수를 설정합니다. 사용자 정의 프로그램을 사용하여 시작 및 스캔 중지를 제어합니다.</li>
	</ol>
</li>
	<li>데이터 수집<ol>
<li>50dB 증폭기(AMP)를 사용하여 PA 신호를 증폭시합니다. DAQ(데이터 수집 카드)로 신호를 디지털화합니다. FPGA 1 또는 FPGA 2를 통해 트리거 신호를 가져옵니다.</li>
		<li>그래픽 처리 장치(GPU)를 사용하여 데이터를 처리하고 이미지를병렬로 표시합니다(13)</li>
	</ol>
</li>
	<li>CCD 이미징 시스템<ol>
<li>링 모양의 흰색 LED(색상 온도: 6500 K; 점도: 40000 럭스; 직경: 7.5cm) 조명 소스로. M1을 제거하고 고정 거울(M2)을 사용하여 빛을 반사합니다.</li>
		<li>PA 이미징 시스템에서 CCD 카메라(630만 화소)를 사용하여 이미지를 기록합니다. 디스플레이 소프트웨어로 이미지를 표시합니다.</li>
	</ol>
</li>
</ol>2. 시스템 정렬
<ol><li>물 탱크 (10cm × 10cm × 4.4 cm; 하단 창: 3 cm × 3 cm)를 선택합니다. 폴리에틸렌 멤브레인(멤브레인 두께: 10 μm)을 사용하여 전체 물 탱크를 덮습니다. 충분한 초순수물을 추가합니다.</li>
	<li>작업 단계에 물 탱크를 놓습니다.</li>
	<li>레이저 스위치를 켭니다. 레이저 제어 프로그램을 선택합니다. 5 분 동안 예열. 펌핑 스위치의 "ON" 버튼을 누릅니다. 레이저 매개 변수를 단계 당 1.1.1로 설정합니다. 레이저의 배플을 엽니다.</li>
	<li>A라인 수집 프로그램을 선택합니다. "시작" 버튼을 눌러 단일 점 신호를 캡처하고 현재 A라인 신호의 진폭과 스펙트럼을 표시합니다.</li>
	<li>물 탱크의 바닥에 블레이드를 놓습니다. 어쿠스틱 커플링을 위해 수조에 UT의 하단 부분을 담급하십시오. UT의 하단 부분에 거품을 피하십시오.</li>
	<li>갈바의 위치를 조정하고, UT와 OL 간에 XY 번역기를 조정하여 진동 신호를 피하고, 이것이 공초점인지 확인합니다.</li>
	<li>작업 단계의 높이를 조정하여 신호의 진폭을 최대화하고 초점 위치를 결정합니다.</li>
</ol>3. 동물 실험
<ol><li>20\u201230 g의 체중이 있는 5\u20126 주 된 BALB/c 마우스를 사용하십시오.</li>
	<li>실험 전에 우레탄(1g/kg)을 사용하여 동물을 마취시화한다.</li>
	<li>WIM과 RIM 간의 전환을 수행합니다.<ol>
<li>평면 초음파 트랜스듀서를 사용합니다. 트리머와 탈모 크림을 사용하여 마우스 뒷면의 모피를 면도합니다. 마우스를 홀더(8cm × 2.8cm × 2cm)에 놓습니다.</li>
		<li>화상 진찰 영역이 초음파 젤을 사용하여 폴리에틸렌 멤브레인과 접촉하도록 허용하십시오. 접촉 부분에서 거품을 피하십시오.</li>
		<li>음향 커플링을 위해 작업 단계에 홀더를 배치합니다. 2.3\u20122.4 단계를 수행하여 레이저를 시작하고 A라인 신호를 수집합니다. 2.6\u20122.7 단계를 따라 정렬합니다. 정렬 후 컬렉션을 종료하려면 "중지"를 누릅니다.</li>
		<li>WIM 프로그램을 선택합니다. 새로 만든 폴더의 이름을 지정합니다. "스캐닝 속도" 탭의 스캔 매개변수를 20mm/s로 설정하고, "스캐닝 영역" 탭의 "20mm*20mm", "단계" 탭에서 "20"으로 설정합니다. 수집 버튼을 클릭하여 스캔을 시작합니다.</li>
		<li>중지 버튼을 클릭하여 획득 후 스캔을 종료합니다. 0으로 돌아가기를 클릭하여 모터를 0으로 가져옵니다. 레이저 배플을 닫습니다. 트리거 설정을 내부 트리거로 설정합니다. 스위치를 펌핑하기 위해 OFF 버튼을 누릅니다.</li>
		<li>WIM 트리거를 RIM 트리거로 교체하고 외부 레이저 트리거에 연결합니다. 스위치를 펌핑하기 위해 ON 버튼을 누릅니다. 트리거 설정을 외부 트리거로 설정합니다. 종료 버튼을 클릭하여 WIM 프로그램을 종료합니다.</li>
		<li>2.4 단계를 사용하여 A라인 신호를 수집합니다. 레이저 배플을 엽니다. 2.6\u20122.7 단계를 따라 정렬합니다. 정렬 후 컬렉션을 종료하려면 중지를 누릅니다.</li>
		<li>RIM 프로그램을 선택합니다. 새로 만든 폴더의 이름을 지정합니다. 수집 버튼을 클릭하여 스캔을 시작합니다.</li>
		<li>수집을 완료한 후 스캐닝을 종료하려면 중지 버튼을 클릭합니다. EXIT 버튼을 클릭하여 RIM 프로그램을 종료합니다.</li>
		<li>화상 진찰의 결론에 자궁 경관 탈구를 사용하여 동물을 안락사시.</li>
	</ol></li>
	<li>혈관 시각화의 WIM을 수행합니다.<ol>
<li>집중 초음파 트랜스듀서를 사용하소서(중앙 주파수: 25MHz; 대역폭: 90% 이상; 초점 거리 : 8mm). 마우스 귀 또는 두피의 머리카락을 제거합니다.<ol>
<li>메스를 사용하여 마우스의 두개골 측두엽의 측면(두개골에 대한 깊이)의 측면에 작은 절개를 합니다. 안과 가위를 사용하여 이 절개부터 시작합니다. 두개골의 바깥쪽 주위에 두피를 자른다. 출혈 점을 압축하여 출혈을 멈춥춥시다. 정상적인 식염수로 상처를 씻으시다. 마우스를 홀더에 놓습니다.</li>
		</ol>
</li>
		<li>화상 진찰 영역이 초음파 젤을 사용하여 폴리에틸렌 멤브레인과 접촉하도록 허용하십시오. 접촉부위(보충도 1)에서거품을 피하십시오.</li>
		<li>음향 커플링을 위해 작업 단계에 홀더를 배치합니다. 2.3\u20122.4 단계를 사용하여 레이저를 열고 A 라인 신호를 수집합니다. 2.6\u20122.7 단계를 사용하여 정렬합니다. 정렬 후 컬렉션을 종료하려면 중지를 누릅니다.</li>
		<li>WIM 프로그램을 선택합니다. 새로 만든 폴더의 이름을 지정합니다. "스캐닝 속도" 탭에서 스캐닝 매개변수를 "10mm/s", "스캐닝 영역" 탭 아래에 "10mm*10mm", "단계" 탭에서 "10"으로 설정합니다. 수집 버튼을 클릭하여 스캔을 시작합니다.</li>
		<li>수집을 완료한 후 스캐닝을 종료하려면 중지 버튼을 클릭합니다. 0으로 돌아가기를 클릭하여 모터가 0으로 돌아오도록 합니다. 종료 버튼을 클릭하여 WIM 프로그램을 종료합니다.</li>
		<li>동물이 마취 하에 있는 동안 시술의 결론에 동물을 안락사.</li>
	</ol></li>
	<li>작은 동물의 동적 모니터링을 위해 RIM을 수행합니다.<ol>
<li>마우스 복부의 머리카락을 면도합니다. 마우스를 홀더에 놓습니다.</li>
		<li>화상 진찰 영역이 초음파 젤을 사용하여 폴리에틸렌 멤브레인과 접촉하도록 허용하십시오. 접촉 영역에서 거품을 피하십시오.</li>
		<li>음향 커플링을 위해 작업 단계에 홀더를 배치합니다. 단계 2.3\u20122.4를 수행하여 레이저를 시작하고 A라인 신호를 수집합니다. 2.6\u20122.7 단계를 수행하여 정렬합니다. 정렬 후 컬렉션을 종료하려면 중지를 누릅니다.</li>
		<li>RIM 프로그램을 선택합니다. 새로 만든 폴더의 이름을 지정합니다. 수집 버튼을 클릭하여 스캔을 시작합니다.</li>
		<li>수집을 완료한 후 스캐닝을 종료하려면 중지 버튼을 클릭합니다. EXIT 버튼을 클릭하여 RIM 프로그램을 종료합니다.</li>
	</ol></li>
	<li>RIM 데이터를 사용하여 사용자 정의 프로그램에 의한 깊이 방향을 따라 최대 진폭 투영(MAP)을 재구성합니다. 동물의 동적 변화를 관찰한다.</li>
</ol>

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W., Lee, E. S., Kim, S. H., Lee, 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=Real-time+GPU-accelerated+processing+and+volumetric+display+for+wide-field+laser-scanning+optical-resolution+photoacoustic+microscopy.">Real-time GPU-accelerated processing and volumetric display for wide-field laser-scanning optical-resolution photoacoustic microscopy.</a> Biomedical Optics Express. 6 (12), 4650-4660 (2015).</li></ol>

모든 동물 실험은 기관 동물 관리 및 사용 위원회의 승인 된 지침 및 규정에 따라 수행되었습니다. 저자는 원고에 관련 재정적 이익이 없으며 공개 할 다른 잠재적 이해 상충이 없습니다.

여기서 우리는 혈관의 구조와 혈액의 관련 동적 변화를 포착하기 위해 설계 및 개발된 비침습적 혈관 시각화를 위한 이중 래스터 스캐닝 광음향 소형 동물 심화를 제시했습니다. DRS-PAI의 장점은 WIM과 RIM을 하나의 시스템으로 통합하여 작은 동물의 혈관 동적 및 혈관 네트워크 구조를 보다 쉽게 연구할 수 있다는 것입니다. 이 시스템은 고해상도 광야 혈관 시각화 및 실시간 혈액 역학을 제공할 수 ...

기본 생물 의학 분야에서, 작은 동물은 인간의 생리 적 기능을 시뮬레이션 할 수 있습니다. 따라서, 작은 동물 화상 진찰은 인간 동종 질병의 연구를 지도하고 효과적인 처리를 찾는 데 중요한 역할을한다 1. 광음향 영상(PAI)은 광학 영상 및 초음파 이미징2의장점을 결합한 비침습적 이미징 기술이다. 광음향 현미경 검사법(PAM)은 작은 동물의 기초 연구를 위한 귀중한 이미징방법이다 3. PAM은 광학 여기 및 초음파 검출4를기반으로 고해상도, 심층 침투, 고특이성 및 고대비 이미지를 쉽게 얻을 수 있습니다.
특정 파장을 가진 펄스 레이저는 조직의 내인성 크로모포러스에 의해 흡수됩니다. 이어서, 조직의 온도가 상승하여 사진 유발 초음파의 생산이 초래됩니다. 초음파는 초음파 트랜스듀서에 의해 검출 될 수있다. 신호 획득 및 이미지 재구성 후 흡수제의 공간 분포를 얻을 수 있다5. 한편으로는, 전체 기관 혈관 네트워크의 시각화는 넓은 시야를 요구합니다. 넓은 필드 스캐닝 과정은 일반적으로 고해상도6,7,8을보장하는 데 오랜 시간이 걸립니다. 다른 한편으로는, 작은 동물의 혈역학 활동을 관찰하는 것은 빠른 실시간 화상 진찰을 요구합니다. 실시간 이미징은 작은 동물의 생명 징후를 실시간으로9,10,11로연구하는 데 유익합니다. 실시간 이미징의 시야는 일반적으로 높은 업데이트 속도를 보장하기에 충분히 작습니다. 따라서, 종종 넓은 시야와 실시간 이미징을 달성하는 것 사이에는 절충이 있습니다. 이전에는 광시야 이미징 또는 실시간 이미징에 각각 2개의 서로 다른 시스템이 사용되었습니다.
이 작품은 2차원 전동 번역 단계와 2축 갈바노미터 스캐너를 기반으로 한 실시간 이미징을 기반으로 하는 와이드 필드 이미징을 통합한 이중 래스터 스캐닝 광음향 이미저(DRS-PAI)를 보고합니다. 광시야 이미징 모드(WIM)는 혈관 형태를 보여주기 위해 수행됩니다. 실시간 이미징 모드(RIM)의 경우 현재 두 가지 기능이 있습니다. 먼저 RIM은 실시간 B 스캔 이미지를 제공할 수 있습니다. 깊이 방향을 따라 혈관의 변위를 측정함으로써 호흡 또는 펄스의 특성이 드러낼 수 있습니다. 둘째, RIM은 WIM 이미지의 특정 영역을 정량적으로 측정할 수 있습니다. 로컬 WIM 지역의 유사한 이미지를 제공함으로써 로컬 변경 내용을 정확하게 밝힐 수 있습니다. 이 시스템은 혈관 시각화의 넓은 현장 이미징과 로컬 동적의 실시간 이미징 사이의 유연한 전환을 설계합니다. 이 시스템은 작은 동물 화상 진찰에 대한 필요가 있는 기본적인 생물 의학 연구에서 바람직합니다.

<table>
	<tbody>
		<tr>
			<td>12 bit multi-purpose digitizer</td>
			<td>Spectrum</td>
			<td>M3i.3221</td>
			<td>Data acquisition card</td>
		</tr>
		<tr>
			<td>A-line collected program</td>
			<td>National Instrument</td>
			<td>LabVIEW</td>
			<td>User-defined program</td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>RF Bay</td>
			<td>LNA-650</td>
			<td>Amplifier</td>
		</tr>
		<tr>
			<td>Depilatory Cream</td>
			<td>Veet</td>
			<td>33-II</td>
			<td>Animal depilatory</td>
		</tr>
		<tr>
			<td>Fiberport Coupler</td>
			<td>Thorlab</td>
			<td>PAF-X-7-A</td>
			<td>Fiber Coupler</td>
		</tr>
		<tr>
			<td>Field Programmable Gate Array</td>
			<td>Altera</td>
			<td>Cyclone IV</td>
			<td>Trigger Control</td>
		</tr>
		<tr>
			<td>Fixed Focus Collomation Packages</td>
			<td>Thorlabs</td>
			<td>F240FC-532</td>
			<td>Fiber Collimator</td>
		</tr>
		<tr>
			<td>Foused ultrasonic transducer</td>
			<td>Self-made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Graphics Processing Unit</td>
			<td>NVIDIA</td>
			<td>GeForce GTX 1060</td>
			<td>Processing data</td>
		</tr>
		<tr>
			<td>Holder</td>
			<td>Self-made</td>
			<td></td>
			<td>Animal fixation</td>
		</tr>
		<tr>
			<td>Laser control program</td>
			<td>National Instrument</td>
			<td>LabVIEW</td>
			<td>User-defined program</td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>Guangdong Medical Laboratory Animal Center</td>
			<td>BALB/c</td>
			<td>Animal Model</td>
		</tr>
		<tr>
			<td>Microscope camera</td>
			<td>Mshot</td>
			<td>MS60</td>
			<td>CCD camera</td>
		</tr>
		<tr>
			<td>Microscope Objective</td>
			<td>Daheng Optics</td>
			<td>GCO-2111</td>
			<td>Objective Lens</td>
		</tr>
		<tr>
			<td>Mirror</td>
			<td>Daheng Optics</td>
			<td>GCC-1011</td>
			<td>Moveable/Fixed Mirror</td>
		</tr>
		<tr>
			<td>Moving Magnet Capacitive Detector Galvanometer Scanner</td>
			<td>Century Sunny</td>
			<td>S8107</td>
			<td>real-time scanner</td>
		</tr>
		<tr>
			<td>Mshot image analysis system</td>
			<td>Mshot</td>
			<td></td>
			<td>Display software</td>
		</tr>
		<tr>
			<td>Normal Saline</td>
			<td>CR DOUBLE-CRANE</td>
			<td>H34023609</td>
			<td>Normal Saline</td>
		</tr>
		<tr>
			<td>Ophthalmic Scissors</td>
			<td>SUJIE</td>
			<td></td>
			<td>Scalp Remove</td>
		</tr>
		<tr>
			<td>Planar ultrasonic transducer</td>
			<td>Self-made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Wrap</td>
			<td>HJSJLSL</td>
			<td></td>
			<td>Polyethylene Membrane</td>
		</tr>
		<tr>
			<td>Program Control Software</td>
			<td>National Instrument</td>
			<td>LabVIEW</td>
			<td>User-defined Program</td>
		</tr>
		<tr>
			<td>Pulsed Q-swithched Laser</td>
			<td>Laser-export</td>
			<td>DTL-314QT</td>
			<td>532-nm pulse Laser</td>
		</tr>
		<tr>
			<td>Real-time imaging program</td>
			<td>National Instrument</td>
			<td>LabVIEW</td>
			<td>User-defined program</td>
		</tr>
		<tr>
			<td>Ring-shaped white LED</td>
			<td>Self-made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Shaver</td>
			<td>Codos</td>
			<td>CP-9200</td>
			<td>Animal Shaver</td>
		</tr>
		<tr>
			<td>Single-Mode Fibers</td>
			<td>Nufern</td>
			<td>460-HP</td>
			<td>Single-mode fiber</td>
		</tr>
		<tr>
			<td>Surgical Blade</td>
			<td>SUJIE</td>
			<td>11</td>
			<td>Blade</td>
		</tr>
		<tr>
			<td>Surgical Scalpel</td>
			<td>SUJIE</td>
			<td>7</td>
			<td>Scalp Remove</td>
		</tr>
		<tr>
			<td>Translation Stage</td>
			<td>Jiancheng Optics</td>
			<td>LS2-25T</td>
			<td>wide-field scanning stage</td>
		</tr>
		<tr>
			<td>Ultrasonic Transducer</td>
			<td>Self-made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound gel</td>
			<td>GUANGGONG PAI</td>
			<td>ZC4252418</td>
			<td>Acoustic Coupling</td>
		</tr>
		<tr>
			<td>Urethane</td>
			<td>Tokyo Chemical Industry</td>
			<td>C0028</td>
			<td>Animal Anestheized</td>
		</tr>
		<tr>
			<td>Water tank</td>
			<td>Self-made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wide-field imaging program</td>
			<td>National Instrument</td>
			<td>LabVIEW</td>
			<td>User-defined program</td>
		</tr>
		<tr>
			<td>XY Translator Mount</td>
			<td>Self-made</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

dual raster-scanning photoacoustic small-animal imager for vascular visualization

작은 동물에 혈관 네트워크의 화상 진찰은 기본적인 생물 의학 연구에서 중요한 역할을 했습니다. 광음향 이미징 기술은 작은 동물의 이미지학에 적용할 수 있는 큰 잠재력을 가지고 있습니다. 작은 동물의 넓은 분야 광음향 이미징은 높은 현면 해상도, 깊은 침투 및 여러 대조를 가진 이미지를 제공 할 수 있습니다. 또한, 실시간 광음향 영상 시스템은 작은 동물 생리적 특징의 동적 모니터링을 연구하는 데 사용될 수 있는 작은 동물 혈관의 혈역학적 활동을 관찰하는 것이 바람직하다. 여기에, 이중 래스터 스캐닝 광음향 이미저를 제시, 전환 이중 모드 이미징 기능을 특징으로. 넓은 시야 이미징은 2차원 전동 번역 단계에 의해 구동되며 실시간 이미징은 갈바노미터로 실현됩니다. 다양한 매개 변수 및 이미징 모드를 설정하여 작은 동물 혈관 네트워크의 생체 내 시각화를 수행할 수 있습니다. 실시간 이미징은 약물 유도 등의 펄스 변화 및 혈류 변화를 관찰하는 데 사용할 수 있습니다. 넓은 필드 화상 진찰은 종양 혈관의 성장 변경을 추적하기 위하여 이용될 수 있습니다. 이들은 기본적인 생물 의학 연구의 각종 분야에서 채택되기 쉽습니다.

DRS-PAI의 회로도는 도 1에표시됩니다. 이 시스템은 RIM을 통해 WIM 간에 유연하고 반복 가능한 전환을 가능하게 합니다. 획득한 PA 신호는 PA B-스캔 및 MAP 이미지를 생성하기 위해 신속하게 처리됩니다. CCD 카메라는 샘플 사진을 제공할 수 있습니다.
DRS-PAI의 모든 구성 요소는 이미저 설정(그림2)에통합및 조립되어 조립 및 조작이 용이?...

Watch this Scientific Journal Video about Dual Raster-Scanning Photoacoustic Small-Animal Imager for Vascular Visualization at JoVE.com

Dual Raster-Scanning Photoacoustic Small-Animal Imager for Vascular Visualization

이중 래스터 스캐닝 광음향 이미저를 설계하여 와이드 필드 이미징과 실시간 이미징을 통합했습니다.

혈관 시각화를 위한 이중 래스터 스캐닝 광음향 소형 동물 이미지

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great ...

dual-raster-scanning-photoacoustic-small-animal-imager-for-vascular

Research

JoVE Journal

Bioengineering

4.0K 조회수.  South China Normal University. 이중 래스터 스캐닝 광음향 이미저를 설계하여 와이드 필드 이미징과 실시간 이미징을 통합했습니다.

비디오: Dual Raster-Scanning Photoacoustic Small-Animal Imager for Vascular Visualization

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

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors1,2,3. CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patient&prime;s response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)4,5. This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid6,7. PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.

We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic disease.

Photoacoustic Flowmetry를 사용하여 순환 흑색증 전지의 탐지 및 격리

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

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 ...

Photoacoustic Cystography

Conventional pediatric cystography, which is based on diagnostic X-ray using a radio-opaque dye, suffers from the use of harmful ionizing radiation. The risk of bladder cancers in children due to radiation exposure is more significant than many other cancers. Here we demonstrate the feasibility of nonionizing and noninvasive photoacoustic (PA) imaging of urinary bladders, referred to as photoacoustic cystography (PAC), using near-infrared (NIR) optical absorbents (i.e. methylene blue, plasmonic gold nanostructures, or single walled carbon nanotubes) as an optical-turbid tracer. We have successfully imaged a rat bladder filled with the optical absorbing agents using a dark-field confocal PAC system. After transurethral injection of the contrast agents, the rat's bladders were photoacoustically visualized by achieving significant PA signal enhancement. The accumulation was validated by spectroscopic PA imaging. Further, by using only a laser pulse energy of less than 1 mJ/cm2 (1/20 of the safety limit), our current imaging system could map the methylene-blue-filled-rat-bladder at the depth of beyond 1 cm in biological tissues in vivo. Both in vivo and ex vivo PA imaging results validate that the contrast agents were naturally excreted via urination. Thus, there is no concern regarding long-term toxic agent accumulation, which will facilitate clinical translation.

Photoacoustic cystography (PAC) has a great potential to map urinary bladders, a radiation sensitive internal organ in pediatric patients, without using any ionizing radiation or toxic contrast agent. Here we demonstrate the use of PAC for mapping urinary bladders with an injection of optical-opaque tracers in rats in vivo.

광 음향 Cystography

Conventional pediatric cystography, which is based on diagnostic X-ray using a radio-opaque dye, suffers from the use of harmful ionizing radiation. The ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

매트릭스 수정에 의해 유도 된 세포 접착 및 탈 부착의 실시간 변화를 측정 셀 기판 임피던스와 라이브 세포 이미징을 사용하여

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

혈관 조직 재생을위한 엔지니어링 3D Cellularized 콜라겐 젤

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

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 ...

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

In vivo small-animal imaging has an important role to play in preclinical studies. Photoacoustic tomography (PAT) is an emerging hybrid imaging modality that shows great potential for both preclinical and clinical applications. Conventional optical parametric oscillator-based PAT (OPO-PAT) systems are bulky and expensive and cannot provide high-speed imaging. Recently, pulsed-laser diodes (PLDs) have been successfully demonstrated as an alternative excitation source for PAT. Pulsed-laser diode PAT (PLD-PAT) has been successfully demonstrated for high-speed imaging on photoacoustic phantoms and biological tissues. This work provides a visualized experimental protocol for in vivo brain imaging using PLD-PAT. The protocol includes the compact PLD-PAT system configuration and its description, animal preparation for brain imaging, and a typical experimental procedure for 2D cross-sectional rat brain imaging. The PLD-PAT system is compact and cost-effective and can provide high-speed, high-quality imaging. Brain images collected in vivo at various scan speeds are presented.

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

A compact pulsed-laser diode-based photoacoustic tomography (PLD-PAT) system for high-speed in vivo brain imaging in small animals is demonstrated.

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 ...

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

듀얼에 대 한 생체 모방 지하실 멤브레인으로 ultrathin Porated 탄성 Hydrogels 세포 배양

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...

혈관 시각화를 위한 이중 래스터 스캐닝 광음향 소형 동물 이미지

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