Name: a high-performance compact photoacoustic tomography system for in vivo small-animal brain imaging
Uploaded: 2017-06-21T14:00:00.000+00:00
Duration: 5 min 32 s
Description: Watch this Scientific Journal Video about A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging at JoVE.com

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

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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

Computed Tomography-guided Time-domain Diffuse Fluorescence Tomography in Small Animals for Localization of Cancer Biomarkers

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light absorption by tissue chromophores (e.g., hemoglobin, water, lipids, melanin) typically limits optical signal propagation through thicknesses larger than a few millimeters2. Compared to other visible wavelengths, tissue absorption for red and near-infrared (near-IR) light absorption dramatically decreases and non-elastic scattering becomes the dominant light-tissue interaction mechanism. The relatively recent development of fluorescent agents that absorb and emit light in the near-IR range (600-1000 nm), has driven the development of imaging systems and light propagation models that can achieve whole body three-dimensional imaging in small animals3.
Despite great strides in this area, the ill-posed nature of diffuse fluorescence tomography remains a significant problem for the stability, contrast recovery and spatial resolution of image reconstruction techniques and the optimal approach to FMI in small animals has yet to be agreed on. The majority of research groups have invested in charge-coupled device (CCD)-based systems that provide abundant tissue-sampling but suboptimal sensitivity4-9, while our group and a few others10-13 have pursued systems based on very high sensitivity detectors, that at this time allow dense tissue sampling to be achieved only at the cost of low imaging throughput. Here we demonstrate the methodology for applying single-photon detection technology in a fluorescence tomography system to localize a cancerous brain lesion in a mouse model.
The fluorescence tomography (FT) system employed single photon counting using photomultiplier tubes (PMT) and information-rich time-domain light detection in a non-contact conformation11. This provides a simultaneous collection of transmitted excitation and emission light, and includes automatic fluorescence excitation exposure control14, laser referencing, and co-registration with a small animal computed tomography (microCT) system15. A nude mouse model was used for imaging. The animal was inoculated orthotopically with a human glioma cell line (U251) in the left cerebral hemisphere and imaged 2 weeks later. The tumor was made to fluoresce by injecting a fluorescent tracer, IRDye 800CW-EGF (LI-COR Biosciences, Lincoln, NE) targeted to epidermal growth factor receptor, a cell membrane protein known to be overexpressed in the U251 tumor line and many other cancers18. A second, untargeted fluorescent tracer, Alexa Fluor 647 (Life Technologies, Grand Island, NY) was also injected to account for non-receptor mediated effects on the uptake of the targeted tracers to provide a means of quantifying tracer binding and receptor availability/density27. A CT-guided, time-domain algorithm was used to reconstruct the location of both fluorescent tracers (i.e., the location of the tumor) in the mouse brain and their ability to localize the tumor was verified by contrast-enhanced magnetic resonance imaging.
Though demonstrated for fluorescence imaging in a glioma mouse model, the methodology presented in this video can be extended to different tumor models in various small animal models potentially up to the size of a rat17.

Diffuse fluorescence tomography offers a relatively low-cost and potentially high-throughout approach to preclinical in vivo tumor imaging. The methodology of optical data collection, calibration, and image reconstruction is presented for a computed tomography-guided non-contact time-domain system using fluorescent targeting of the tumor biomarker epidermal growth factor receptor in a mouse glioma model.

암 생체 현지화위한 작은 동물의 계산된 Tomography 주 도형 시간 도메인 미만성 형광 Tomography

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light ...

의학

Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously attaining anatomical, functional and molecular contrast in deep optically opaque tissues. While enormous potential has been recently demonstrated in the application of optoacoustics for small animal research, vast efforts have also been undertaken in translating this imaging technology into clinical practice. We present here a newly developed optoacoustic tomography approach capable of delivering high resolution and spectrally enriched volumetric images of tissue morphology and function in real time. A detailed description of the experimental protocol for operating with the imaging system in both hand-held and stationary modes is provided and showcased for different potential scenarios involving functional and molecular studies in murine models and humans. The possibility for real time visualization in three dimensions along with the versatile handheld design of the imaging probe make the newly developed approach unique among the pantheon of imaging modalities used in today&#8217;s preclinical research and clinical practice.

We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice.

범용 실시간으로 깊은 조직 인간의 혈관 조영술과 기능 전임상 연구를위한 3 차원 광 음향 이미징 프로브를 손 개최

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously ...

Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central nervous system of small animals. However, transdermal and transcranial neuroimaging is frequently affected by low sensitivity, image aberrations and loss of space resolution, requiring scalp or even skull removal before imaging. To overcome this challenge, a new protocol is presented to gain significant insights in brain hemodynamics by photoacoustic and high-frequency ultrasounds imaging with the animal skin and skull intact. The procedure relies on the passage of ultrasound (US) waves and laser directly through the fissures that are naturally present on the animal cranium. By juxtaposing the imaging transducer device exactly in correspondence to these selected areas where the skull has a reduced thickness or is totally absent, one can acquire high quality deep images and explore internal brain regions that are usually difficult to anatomically or functionally describe without an invasive approach. By applying this experimental procedure, significant data can be collected in both sonic and optoacoustic modalities, enabling to image the parenchymal and the vascular anatomy far below the head surface. Deep brain features such as parenchymal convolutions and fissures separating the lobes were clearly visible. Moreover, the configuration of large and small blood vessels was imaged at several millimeters of depth, and precise information were collected about blood fluxes, vascular stream velocities and the hemoglobin chemical state. This repertoire of data could be crucial in several research contests, ranging from brain vascular disease studies to experimental techniques involving the systemic administration of exogenous chemicals or other objects endowed with imaging contrast enhancement properties. In conclusion, thanks to the presented protocol, the US and PA techniques become an attractive noninvasive performance-competitive means for cortical and internal brain imaging, retaining a significant potential in many neurologic fields.

The present work describes a new protocol to perform non-invasive high-frequency ultrasound and photoacoustic based imaging on rat brain, to efficiently visualize deep subcortical regions and their vascular patterns by directing signals on skull foramina naturally present on animal cranium.

비 침습적 실질, 혈관 및 대사 고주파 초음파 및 광 음향 쥐 뇌 심부 이미징

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central ...

신경과학

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 ...

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, ...

Pulsed Laser Diode-Based Desktop Photoacoustic Tomography for Monitoring Wash-In and Wash-Out of Dye in Rat Cortical Vasculature

Photoacoustic (PA) tomography (PAT) imaging is an emerging biomedical imaging modality useful in various preclinical and clinical applications. Custom-made circular ring array-based transducers and conventional bulky Nd:YAG/OPO lasers inhibit translation of the PAT system to clinics. Ultra-compact pulsed laser diodes (PLDs) are currently being used as an alternative source of near-infrared excitation for PA imaging. High-speed dynamic in vivo imaging has been demonstrated using a compact PLD-based desktop PAT system (PLD-PAT). A visualized experimental protocol using the desktop PLD-PAT system is provided in this work for dynamic in vivo brain imaging. The protocol describes the desktop PLD-PAT system configuration, preparation of animal for brain vascular imaging, and procedure for dynamic visualization of indocyanine green (ICG) dye uptake and clearance process in rat cortical vasculature.&#160;

A compact pulsed laser diode-based desktop photoacoustic tomography (PLD-PAT) system is demonstrated for high-speed dynamic in vivo imaging of small animal cortical vasculature.

쥐 대뇌 피 질의 혈관 구조에서 염료의 세척 및 세척을 모니터링 하기 위한 펄스 레이저 다이오드 기반 데스크탑 광 음향 단층 촬영

Photoacoustic (PA) tomography (PAT) imaging is an emerging biomedical imaging modality useful in various preclinical and clinical applications. ...

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

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great potential for application in the imageology of small animals. The wide-field photoacoustic imaging of small animals can provide images with high spatiotemporal resolution, deep penetration, and multiple contrasts. Also, the real-time photoacoustic imaging system is desirable to observe the hemodynamic activities of small-animal vasculature, which can be used to research the dynamic monitoring of small-animal physiological features. Here, a dual-raster-scanning photoacoustic imager is presented, featuring a switchable double-mode imaging function. The wide-field imaging is driven by a two-dimensional motorized translation stage, while the real-time imaging is realized with galvanometers. By setting different parameters and imaging modes, in vivo visualization of small-animal vascular network can be performed. The real-time imaging can be used to observe pulse change and blood flow change of drug-induced, etc. The wide-field imaging can be used to track the growth change of tumor vasculature. These are easy to be adopted in various areas of basic biomedicine research.

A dual raster-scanning photoacoustic imager was designed, which integrated wide-field imaging and real-time imaging.

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 ...

A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in Rodents

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a physiological barrier preventing large molecules (&#62;500 Da) from entering the brain tissue. Microbubble-mediated focused ultrasound (FUS) can be used to induce a transient local BBB opening, allowing larger drugs to enter the brain parenchyma.
In addition to large-scale clinical devices for clinical translation, preclinical research for therapy response assessment of drug candidates requires dedicated small animal ultrasound setups for targeted BBB opening. Preferably, these systems allow high-throughput workflows with both high-spatial precision as well as integrated cavitation monitoring, while still being cost effective in both initial investment and running costs.
Here, we present a bioluminescence and X-ray guided stereotactic small animal FUS system that is based on commercially available components and fulfills the aforementioned requirements. A particular emphasis has been placed on a high degree of automation facilitating the challenges typically encountered in high-volume preclinical drug evaluation studies. Examples of these challenges are the need for standardization in order to ensure data reproducibility, reduce intra-group variability, reduce sample size and thus comply with ethical requirements and decrease unnecessary workload. The proposed BBB system has been validated in the scope of BBB opening facilitated drug delivery trials on patient-derived xenograft models of glioblastoma multiforme and diffuse midline glioma.

The blood-brain barrier (BBB) can be temporarily disrupted with microbubble-mediated focused ultrasound (FUS). Here, we describe a step-by-step protocol for high-throughput BBB opening in vivo using a modular FUS system accessible for non-ultrasound experts.

설치류의 혈액 뇌 장벽 개방을 위한 고처리량 이미지 유도 입체 신경 탐색 및 집중 초음파 시스템

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a ...

허혈성 뇌졸중의 비침습적 통합 PAUSAT 전뇌 영상

Integrated Photoacoustic, Ultrasound, and Angiographic Tomography (PAUSAT) for NonInvasive Whole-Brain Imaging of Ischemic Stroke

Presented here is an experimental ischemic stroke study using our newly developed noninvasive imaging system that integrates three acoustic-based imaging technologies: photoacoustic, ultrasound, and angiographic tomography (PAUSAT). Combining these three modalities helps acquire multi-spectral photoacoustic tomography (PAT) of the brain blood oxygenation, high-frequency ultrasound imaging of the brain tissue, and acoustic angiography of the cerebral blood perfusion. The multi-modal imaging platform allows the study of cerebral perfusion and oxygenation changes in the whole mouse brain after stroke. Two commonly used ischemic stroke models were evaluated: the permanent middle cerebral artery occlusion (pMCAO) model and the photothrombotic (PT) model. PAUSAT was used to image the same mouse brains before and after a stroke and quantitatively analyze both stroke models. This imaging system was able to clearly show the brain vascular changes after ischemic stroke, including significantly reduced blood perfusion and oxygenation in the stroke infarct region (ipsilateral) compared to the uninjured tissue (contralateral). The results were confirmed by both laser speckle contrast imaging and triphenyltetrazolium chloride (TTC) staining. Furthermore, stroke infarct volume in both stroke models was measured and validated by TTC staining as the ground truth. Through this study, we have demonstrated that PAUSAT can be a powerful tool in noninvasive and longitudinal preclinical studies of ischemic stroke.

저자 스포트라이트: 허혈성 뇌졸중의 비침습적 전뇌 영상을 위한 통합 광음향, 초음파 및 혈관 조영 단층 촬영(PAUSAT)  

This work demonstrates the use of a multimodal ultrasound-based imaging platform for noninvasive imaging of ischemic stroke. This system allows for the quantification of blood oxygenation through photoacoustic imaging and impaired perfusion in the brain through acoustic angiography.

허혈성 뇌졸중의 비침습적 전뇌 영상을 위한 통합 광음향, 초음파 및 혈관조영 단층촬영(PAUSAT)

Presented here is an experimental ischemic stroke study using our newly developed noninvasive imaging system that integrates three acoustic-based imaging ...

다음을위한 고성능 컴팩트 광 음향 단층 촬영 시스템

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