Name: simultaneous evaluation of cerebral hemodynamics and light scattering properties of the in vivo rat brain using multispectral diffuse reflectance imaging
Uploaded: 2017-05-07T15:00:00.000+00:00
Duration: 7 min 6 s
Description: Watch this Scientific Journal Video about Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging at J

Part of this work was supported by a Grant-in-Aid for Scientific Research (C) from the Japanese Society for the Promotion of Science (25350520, 22500401, 15K06105) and the US-ARMY ITC-PAC Research and Development Project (FA5209-15-P-0175).

동물 관리, 준비, 실험 프로토콜은 도쿄 농공 대학의 동물 연구위원회에 의해 승인되었다. 이 방법의 경우, 래트는 음식과 물을 사용할 수의와, 제어 된 환경 (24 ° C, 12 시간 광 / 암주기)에 수용된다. 광파 멀티 스펙트럼 확산 반사율 이미징 시스템의 1. 건설 <ol><li> 산의 중심 파장 협 아홉 광학 간섭 필터 500, 520, 540, 560, 570, 580, 600, 730, 및 전동 필터 휠의 필터 구멍 760 내지. </li><li> 광대역 백색 광원, 협 대역 간섭 필터들의 상기 세트, 광 가이드, 포집 렌즈, 비디오 줌 렌즈, 및 모노크롬 CCD 카메라와 전동 필터 휠을 사용하여 멀티 스펙트럼 영상 획득 시스템을 구축. 도 2에 도시 된 광학 구성 요소의 배치는 차에 대해 참조 할 수있다건설 절차입니다. 참고 : 조명 각도는 시료 표면에 대해 약 45 °입니다. </li><li> 할로겐 램프 광원을 켜서 간섭 필터, 광 가이드 및 수집 렌즈를 통해 시료 표면을 비 춥니 다. </li><li> CCD 카메라의 운영 소프트웨어를 엽니 다. </li></ol> 2. 동물 준비 참고 :이 프로토콜에서 쥐는 미래의 실험에 사용되지 않았으며 그것은 multispectral 이미지의 측정 직후 희생되었다. <ol><li> 유도 챔버의 입구 포트를 마취 기계의 출구 포트에 튜브로 연결하십시오. 유도 챔버의 출구 포트를 두 번째 튜브가있는 마취 기계의 흡입 포트에 연결하십시오. </li><li> 쥐를 유도 챔버에 넣고 5.0 % 이소 플루 란으로 마취를 유도하십시오. 쥐가 발가락 핀치에 반응하지 않도록 깊이 마취를 유지하십시오. 엘마취 기계의 회전식 손잡이를 사용하여 2.0 % isoflurane까지 세척하십시오. </li><li> stereotaxic 프레임에 쥐 머리를 수정. stereotaxic 프레임에 마 취용 마우스 피스를 연결합니다. </li><li> 마우스 피스의 입구 포트를 마취 기계의 출구 포트에 튜브로 연결하십시오. 마우스 피스의 출구 포트를 마취 기계의 흡입구에 튜브로 연결하십시오. </li><li> 피부 표면이 나타날 때까지 헤어 클리퍼를 사용하여 절개 부위를 넘어 머리 부위를 면도하십시오. </li><li> 외과 용 메스 ( 그림 1 (a) )를 사용하여 머리의 중간 선을 따라 약 20mm 길이로 세로 절개를하고 피하 결합 조직 ( 그림 1 (b) )을 노출 시키십시오. </li><li> 날카로운 curette 또는 협공을 사용하여 피하 결합 조직을 제거하고 두개골 뼈 ( 그림 1 (c) )를 노출하기 위해 머리의 양쪽으로 당겨. </li><li> 두개골 sutur 안에 두개골 뼈에 타원 도랑을 파다.es (coronal suture, sagittal suture, lambdoid 봉합사)를 사용 하였다 ( 그림 1 (d) ). </li><li> 고속 드릴을 사용하여 도랑 속의 두개골 뼈를 천천히 그리고 균일하게 굴착하십시오. </li><li> 뇌 혈관이 나타난 후 뼈의 두께와 강도를 추정하기 위해 협공의 끝으로 얇아진 두개골의 표면을 가볍게 누릅니다. 만약 얇아진 두개골 부위가 쉽게 눌려지면, 고속 드릴로 두개골 뼈의 축소를 종료하십시오. </li><li> 족집게 또는 작은 외과 용 가위를 사용하여 얇은 두개골 조각의 타원형 경계선을 자릅니다. </li><li> 협지를 사용하여 뇌 표면에서 얇은 두개골을 천천히 부드럽게 제거하십시오. </li><li> 생리 식염수로 두개골 창을 부드럽게 목욕하고 약 0.1mm 두께의 투명 유리판으로 덮습니다. </li></ol> 3. 영감을받은 산소의 분율 조절 참고 : 호흡기 상태기 영감 산소의 분율 (FIO 2)를 조절하여 변경할 수있다. <ol><li> 튜브를 사용하여, 다른 Y 형 튜브 커넥터 (커넥터 (2))의 제 포트에 Y 형 튜브 커넥터 (커넥터 (1))의 첫 번째 포트를 연결한다. </li><li> 튜브 커넥터 (1)의 두 번째 포트에 마우스 피스의 입구를 연결한다. </li><li> 튜브를 사용하여, 산소 농도를 모니터 장치에 튜브 커넥터 (1)의 제 3 포트를 연결한다. </li><li> 튜브를 통해 마취 기계의 출구 포트 튜브 커넥터 (2)의 두 번째 포트를 연결한다. </li><li> 튜브를 사용하는 가스 혼합물을 장치의 출구 포트 튜브 커넥터 (2)의 제 3 포트를 연결한다. </li><li> 5 %의 튜브를 사용하여 CO2 가스 실린더 - 고압 95 % O 2 가스 혼합 장치의 하나 개의 입구 포트를 연결한다. </li><li> 튜브를 사용하여 5 % CO 2 가스 실린더 - 고압 95 % N 2 가스 혼합물을 장치의 다른 입구 포트를 연결한다. </li><li> 오 가스 유량을 변경가스 혼합물을 장치에 로터리 노브를 사용하여 F의 O 2 N 2. </li><li> 확인 및 산소 농도를 모니터 장치를 사용하여 2 FIO을 조절한다. </li></ol> 멀티 스펙트럼 확산 반사율 이미지 4. 취득 <ol><li> 참조 이미지의 취득 참고 : 같은 광원, 광학 섬유 등이 실험에 사용 된 광학 부품, 및 감지기는 자신의 스펙트럼 특성을 가지고있다. 따라서, 이들 구성 요소를 통과하는 빛의 강도가 참조 화상으로서 기록한다. 참조 화상은 광원으로부터의 광으로 조명하는 표준 화이트 확산기로 찍은 이미지이다. <ol><li> 수평으로 무대에 표준 흰색 확산을 넣습니다. </li><li> 배럴의 줌 링을 돌려 흰색 확산 판의 표면에 카메라 렌즈를 초점을 맞 춥니 다. </li><li> 로부터 적절한 값을 선택하여 카메라의 적분 시간을 조정드롭 다운 통합 시간의리스트를 카메라의 운영 소프트웨어에서 광의 최대량이 최대 카운트의 약 75 % 인 신호를 생성하도록. 화소 값의 히스토그램을 보면서 신호 강도 레벨이 최대 카운트의 약 75 %가 될 때까지 적분 시간을 조정한다. </li><li> 파일에 이미지를 저장하려면 파일 메뉴에서 &quot;저장&quot;명령을 선택합니다. </li><li> 필터 휠을 돌려 필터 위치를 변경합니다. </li><li> 상술 한 방법에 따라, 다른 파장의 이미지를 저장한다. 파일 이름은 시료와 사용 파장 (예를 들어, W500, W520, W540 ... W760)를 식별해야합니다. </li></ol></li><li> 샘플 이미지의 취득 참고 : 구 파장에 노출 된 쥐의 뇌의 확산 반사 된 빛의 세기의 이미지 캡처와 같은 인수 조건을 사용하여 개인 컴퓨터의 하드 드라이브에 저장됩니다. <ol><li> 조심스럽게 연구를 배치카메라가 쥐의 뇌의 표면에 초점을 맞출 수 있도록 무대에서 천천히 무대 레벨을 조절합니다. </li><li> 파일에 이미지를 저장하려면 파일 메뉴에서 &quot;저장&quot;명령을 선택합니다. </li><li> 필터 휠을 돌려 필터 위치를 변경합니다. </li><li> 상술 한 방법에 따라, 다른 파장의 이미지를 저장한다. 파일 이름은 샘플과 파장 (예를 들어, R500, R520, R540 ... R760)를 식별해야합니다. </li></ol></li><li> 어두운 이미지의 취득 주 : CCD 카메라는 전기 신호에 따라 광 세기를 생성 할 수있다. 그러나, 광이 검출기에 입력하지 않더라도 때문에 전기 회로와 검출기의 잡음에 약간의 출력이있다; 이 어두운 전류 잡음이라고합니다. 정확하게 광의 분광 강도를 측정하기 위해, 암전류 성분이 어두운 화상으로서 기록하고 측정 된 신호로부터 감산한다. 어두운 화상은 화상 포획이다N 개의 광로로 막았다. <ol><li> 할로겐 램프 광원을 끕니다. </li><li> 차폐 판을 이용하여 CCD 카메라 시스템의 광 경로를 차단. </li><li> 파일에 이미지를 저장하려면 파일 메뉴에서 &quot;저장&quot;명령을 선택합니다. 파일 이름은 샘플 (예를 들어, 다크)를 식별해야합니다. </li></ol></li></ol> 5. 헤모글로빈 콘텐츠 및 빛 산란 매개 변수를 시각화 참고 : 다중 스펙트럼 확산 반사율 이미지의 집합은 개인용 컴퓨터의 하드 드라이브에 저장하고 오프라인으로 분석된다. 아홉 개 파장들 (500, 520, 540, 560, 570, 580, 600, 730, 및 760 ㎚)에서 멀티 스펙트럼 확산 반사율 영상의 몬테카를로 시뮬레이션 (19)를 이용한 다중 회귀 분석은 다음 두 차원 시각화 수행 산소화 헤모글로빈 농도, 탈산 소화 헤모글로빈 농도, 총 헤모글로빈 농도 지역 뇌 산소 포화도, 및 배설물의지도전원을 tering. 자세한 알고리즘은 문헌 17, 18 년에 출판되었다. <ol><li> 참조 화상과 각 파장에서의 샘플 화상 모두로부터 어두운 화상을 뺀다. </li><li> 각 파장 λ에서 기준 화상에 의한 샘플 화상을 정규화한다. 확산 반사율 화상 R로서 표준화 이미지를 처리한다. </li><li> 각 파장 λ에서의 확산 반사율 화상 R의 역수의 대수를 취함으로써 흡광도 (또는 광학 밀도) 영상 A를 계산한다 : <img alt="수학 식 1" src="/files/ftp_upload/55399/55399eq1.jpg" /> (1) </li><li> y 평면 뇌 표면 얻어진 구조 정보를 표시하고, Z는시킴으로써 행한다 스펙트럼 정보를 표시 -은 X가 파장의 순서에서 흡광도 이미지를 적층하여 입체 매트릭스를 생성한다. </li><li> 체육각각의 XY 좌표를 흡광도 스펙트럼 A (λ)에 대한 다중 회귀 분석을 rform. </li><li> 단계 5.5 독립 변수와 종속 변수 HBO (λ) ε 산소화 헤모글로빈의 몰 흡광 계수 스펙트럼 및 탈산 소화 헤모글로빈 ε의 HBR (λ)로 흡광도 스펙트럼 A (λ)를 사용하여 (HBO ε의 값을 문서 (λ) 및 ε HBR (λ)의) 표 1에 제공된다. </li><li> 세 회귀의 2 차원지도 (화상)을 확인하는 것은 HBO하는 HBR 및 0 계수. </li><li> 순서에 HBO하는 HBR, 및 0, 회귀 계수의 이미지를 적층하여 입체 매트릭스를 생성 어디 <em&gt; X - Y 평면 뇌 표면의 Z시킴으로써 행한다 대해 얻어진 구조 정보를 나타낸다는 회귀 계수를 나타낸다. </li><li> 산소화 헤모글로빈 농도 C HBO, 탈산 소화 헤모글로빈 농도 C의 HBR, 각각 XY에서 HBO하는 HBR 및 0 계수 그가 β 값 (다음의 실험식을 이용하여 좌표 회귀 세트로부터 산란 전원 (B)을 계산 HBO, I, β HBR, I 및 β 0, I는 (i = 0,1,2,3)는 표 2에 제공된다) : <img alt="수학 식 2" src="/files/ftp_upload/55399/55399eq2.jpg" /> (2) <img alt="식 (3)" src="/files/ftp_upload/55399/55399eq3.jpg" /> (삼) <img alt="수학 식 4" src="/files/ftp_upload/55399/55399eq4.jpg" /> (4) </li><li> 산소화 헤모글로빈 농도 C HBO, 탈산 소화 헤모글로빈 농도 C HBR 및 산란 전원 (B)의 2 차원지도 (화상)을 확인한다. </li><li> 각 X에서 C 및 C HBO HBr을 합산하여 총 헤모글로빈 농도 C의 HBT의 2 차원지도를 계산 - y 좌표. </li><li> Y 좌표 - 각각 X에서 총 헤모글로빈 농도 C의 HBT하여 산소화 헤모글로빈 농도 C HBO 나누어 지역 뇌 산소 포화도 RSO 2의 2 차원지도를 계산한다. </li></ol>

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Express. 14 (3), 1125-1144 (2006).</li><li>Bouchard, M. B., Chen, B. R., Burgess, S. A., Hillman, E. M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-fast+multispectral+optical+imaging+of+cortical+oxygenation,+blood+flow,+and+intracellular+calcium+dynamics.">Ultra-fast multispectral optical imaging of cortical oxygenation, blood flow, and intracellular calcium dynamics.</a> Opt. Express. 17 (18), 15670-15678 (2009).</li><li>Jones, P. B., 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=Simultaneous+multispectral+reflectance+imaging+and+laser+speckle+flowmetry+of+cerebral+blood+flow+and+oxygen+metabolism+in+focal+cerebral+ischemia.">Simultaneous multispectral reflectance imaging and laser speckle flowmetry of cerebral blood flow and oxygen metabolism in focal cerebral ischemia.</a> J. Biomed. Opt. 13 (4), 044007 (2008).</li><li>Kawauchi, S., 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=Diffuse+light+reflectance+signals+as+potential+indicators+of+loss+of+viability+in+brain+tissue+due+to+hypoxia:+charge-coupled-device-based+imaging+and+fiber-based+measurement.">Diffuse light reflectance signals as potential indicators of loss of viability in brain tissue due to hypoxia: charge-coupled-device-based imaging and fiber-based measurement.</a> J. Biomed. Opt. 18 (1), 015003 (2013).</li><li>Yoshida, 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=Multispectral+imaging+of+absorption+and+scattering+properties+of+in+vivo+exposed+rat+brain+using+a+digital+red-green-blue+camera.">Multispectral imaging of absorption and scattering properties of in vivo exposed rat brain using a digital red-green-blue camera.</a> J. Biomed. Opt. 20 (5), 051026 (2015).</li><li>Nishidate, I., 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=Evaluation+of+Cerebral+Hemodynamics+and+Tissue+Morphology+of+In+Vivo+Rat+Brain+Using+Spectral+Diffuse+Reflectance+Imaging.">Evaluation of Cerebral Hemodynamics and Tissue Morphology of In Vivo Rat Brain Using Spectral Diffuse Reflectance Imaging.</a> Appl. Spectrosc. , (2016).</li><li>Wang, L. -. H., Jacques, S. L., Zheng, L. -. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MCML-Monte+Carlo+modeling+of+photon+transport+in+multi-layered+tissues.">MCML-Monte Carlo modeling of photon transport in multi-layered tissues.</a> Comput. Methods Programs Biomed. 47 (2), 131-146 (1995).</li><li>Tsytsarev, V., Premachandra, K., Takeshita, D., Bahar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cortical+electrical+stimulation+in+vivo:+Fast+intrinsic+optical+signal+versus+voltage-sensitive+dyes.">Imaging cortical electrical stimulation in vivo: Fast intrinsic optical signal versus voltage-sensitive dyes.</a> Opt. Lett. 33 (9), 1032-1034 (2008).</li><li>Arnold, T., Biasio, M. D., Leitner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyper-spectral+video+endoscope+for+intra-surgery+tissue+classification+using+auto-fluorescence+and+reflectance+spectroscopy.">Hyper-spectral video endoscope for intra-surgery tissue classification using auto-fluorescence and reflectance spectroscopy.</a> Proc. SPIE. 8087, 808711 (2011).</li><li>Basiri, A., 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=Use+of+a+multi-spectral+camera+in+the+characterization+of+skin+wounds.">Use of a multi-spectral camera in the characterization of skin wounds.</a> Opt. Express. 18 (4), 3244-3257 (2010).</li><li>Nishidate, I., Maeda, T., Niizeki, K., Aizu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+melanin+and+hemoglobin+using+spectral+reflectance+images+reconstructed+from+a+digital+RGB+image+by+the+Wiener+estimation+method.">Estimation of melanin and hemoglobin using spectral reflectance images reconstructed from a digital RGB image by the Wiener estimation method.</a> Sensors. 13 (6), 7902-7915 (2013).</li></ol>

The authors have nothing to disclose.

이 프로토콜에서 가장 중요한 단계는 두개골 창을 만들 수있는 얇아 두개골 지역의 제거이다; 이 예기치 않은 출혈을 방지하기 위해주의 깊게 수행해야합니다. 이 단계는 높은 정밀도로 반사 이미지를 확산 다중 분광 높은 품질을 얻는 것이 중요하다. 실체 현미경의 사용은 수술 가능한 경우에 권장됩니다. 젤라틴 스폰지의 작은 조각 지혈하는 데 유용합니다. 이 문서에서 ?...

멀티 스펙트럼 확산 반사율 영상은 피질 조직에서 고유의 광 신호 (손실이 최소화)의 공간지도를 획득하기위한 가장 일반적인 방법이다. 형태 학적 변화에 의해 유발 된 광 산란 특성의 광 흡수 변화 의한 피질 혈류 역학에 대한 산란 성질, 감소 또는 미토콘드리아 내의 사이토 크롬의 산화에 따라 흡수 편차 및 변형 : 손실이 최소화 주로 세 현상에 기인하는 생체의 뇌에서 관찰 1. 근적외선 (NIR) 분광 범위 (VIS)에 보이는 빛을 효과적으로 흡수 및 생체 조직에 의해 산란된다. 생체의 뇌의 확산 반사율 스펙트럼은 흡수 및 산란 스펙트럼을 특징으로한다. 감소 된 스 캐터링 계수는 단조로운 산란 스펙트럼 전시에 VIS 투 NIR 파장 범위의 결과 뇌 조직들 &#39;μ긴 파장에서 더 작은 크기를 보내고. 감소 된 산란 스펙트럼 계수 μ는 S &#39;(λ)는 s의 μ로서 멱 함수 (2), (3)의 형태로 근사 할 수있는&#39;(λ)는 λ × -b =. 산란 파워는 조직 B (2, 3) 살아있는 생물에서 산란의 크기에 관련된다. 조직 및 대뇌 피질의 생체 조직의 생존을 감소 형태 학적 변화는 생물학적 산란 4, 5, 6, 7, 8, 9의 크기에 영향을 미칠 수있다. 멀티 스펙트럼 확산 반사율 촬상 용 광학 시스템을 용이 백열 리로 구성 될 수있다GHT 원본, 간단한 광학 구성 요소 및 단색 충전 결합 소자 (CCD). 따라서, 다양한 알고리즘과 멀티 스펙트럼 확산 반사율 촬상 광학 시스템은 피질 혈류 역학 및 / 또는 티슈 형태 10, 11, 12, 13, 14, 15, 16, 17, 18을 평가하기 위해 사용되었다. 본 문서에 기재된 방법은 혈류 역학과 종래의 멀티 스펙트럼 확산 반사율 이미징 시스템을 사용하여 생체 내에서 래트 뇌 조직의 산란 특성 모두를 시각화하는데 사용된다. 대안 기술을 통해이 방법의 장점은 대뇌 혈류 역학과 대뇌 피질의 조직 모두의 시공간적 변화를 평가 할 수있는 기능입니다형태뿐만 아니라 다양한 뇌 기능 장애의 동물 모델에의 적용. 따라서, 방법은 외상성 뇌 손상, 간질 발작, 뇌졸중, 허혈의 조사에 적합합니다.

<table><tbody><tr><td>150-W halogen-lamp light source</td><td>Hayashi Watch Works Co., Ltd, Tokyo, Japan</td><td>LA-150SAE</td><td></td></tr><tr><td>Light guide</td><td>Hayashi Watch Works Co., Ltd, Tokyo, Japan</td><td>LGC1-5L1000</td><td></td></tr><tr><td>Collecting lens</td><td>Hayashi Watch Works Co., Ltd, Tokyo, Japan</td><td>SH-F16</td><td></td></tr><tr><td>Interference filters l@ 500 nm</td><td>Edmund Optics Japan Ltd, Tokyo, Japan</td><td>#65088</td><td></td></tr><tr><td>Interference filters l@ 520 nm</td><td>Edmund Optics Japan Ltd, Tokyo, Japan</td><td>#65093</td><td></td></tr><tr><td>Interference filters l@ 540 nm</td><td>Edmund Optics Japan Ltd, Tokyo, Japan</td><td>#65096</td><td></td></tr><tr><td>Interference filters l@ 560 nm</td><td>Edmund Optics Japan Ltd, Tokyo, Japan</td><td>#67766</td><td></td></tr><tr><td>Interference filters l@ 570 nm</td><td>Edmund Optics Japan Ltd, Tokyo, Japan</td><td>#67767</td><td></td></tr><tr><td>Interference filters l@ 580 nm</td><td>Edmund Optics Japan Ltd, Tokyo, Japan</td><td>#65646</td><td></td></tr><tr><td>Interference filters l@ 600 nm</td><td>Edmund Optics Japan Ltd, Tokyo, Japan</td><td>#65102</td><td></td></tr><tr><td>Interference filters l@ 730 nm</td><td>Edmund Optics Japan Ltd, Tokyo, Japan</td><td>#65115</td><td></td></tr><tr><td>Interference filters l@ 760 nm</td><td>Edmund Optics Japan Ltd, Tokyo, Japan</td><td>#67777</td><td></td></tr><tr><td>Motorized filter wheel&amp;nbsp;</td><td>Andover Corporation, NH, USA</td><td>FW-MOT-12.5</td><td></td></tr><tr><td>8-bit monochromatic CCD camera</td><td>THE IMAGINGSOURCE, Germany</td><td>DMK21BU618.H</td><td></td></tr><tr><td>Video zoom lens</td><td>Edmund Optics Japan Ltd, Tokyo, Japan</td><td>VZM&lt;sup&gt;TM&lt;/sup&gt;300i</td><td></td></tr><tr><td>Spectralon white standard with 99% diffuse reflectance</td><td>Labsphere Incorporated, North Sutton, NH, USA</td><td>SRS-99-020</td><td></td></tr></tbody></table>

simultaneous evaluation of cerebral hemodynamics and light scattering properties of the in vivo rat brain using multispectral diffuse reflectance imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system. This system is constructed from a broadband white light source, a motorized filter wheel with a set of narrowband interference filters, a light guide, a collecting lens, a video zoom lens, and a monochromatic charged-coupled device (CCD) camera. An ellipsoidal cranial window is made in the skull bone of a rat under isoflurane anesthesia to capture in vivo multispectral diffuse reflectance images of the cortical surface. Regulation of the fraction of inspired oxygen using a gas mixture device enables the induction of different respiratory states such as normoxia, hyperoxia, and anoxia. A Monte Carlo simulation-based multiple regression analysis for the measured multispectral diffuse reflectance images at nine wavelengths (500, 520, 540, 560, 570, 580, 600, 730, and 760 nm) is then performed to visualize the two-dimensional maps of hemodynamics and the light scattering properties of the in vivo rat brain.

생체 래트 뇌로부터 취득한 확산 반사율의 대표적인 분광 화상 500, 520, 540, 560, 570에서도 3 이미지 표시되었으며 580 명확 대뇌 피질에서 혈관의 밀도 네트워크 시각화 nm의. 혈관 (600), (730)에서 이미지에 주변 조직 관찰, 및 760 nm의 사이의 콘트라스트의 저하는 이상과 NIR 파장의 광에 의한 헤모글로빈의 낮은 흡수를 반영한다. <p class="jove_content" fo:keep-to...

Watch this Scientific Journal Video about Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging at J

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

뇌 혈류 역학 및 생체 내 쥐의 뇌 조직의 산란 특성의 동시 평가는 종래의 멀티 스펙트럼 확산 반사율 촬상 시스템을 이용하여 설명된다.

대뇌 혈류와의 광 산란 특성을 동시에 평가<em&gt; 생체</em&gt; 다중 스펙트럼 확산 반사 이미징을 이용한 쥐 뇌

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional ...

simultaneous-evaluation-cerebral-hemodynamics-light-scattering

Research

JoVE Journal

Neuroscience

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

7.6K 조회수.  Tokyo University of Agriculture & Technology.  뇌 혈류 역학 및 생체 내 쥐의 뇌 조직의 산란 특성의 동시 평가는 종래의 멀티 스펙트럼 확산 반사율 촬상 시스템을 이용하여 설명된다. 

비디오: Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

How to Build a Laser Speckle Contrast Imaging (LSCI) System to Monitor Blood Flow

Laser Speckle Contrast Imaging (LSCI) is a simple yet powerful technique that is used for full-field imaging of blood flow. The technique analyzes fluctuations in a dynamic speckle pattern to detect the movement of particles similar to how laser Doppler analyzes frequency shifts to determine particle speed. Because it can be used to monitor the movement of red blood cells, LSCI has become a popular tool for measuring blood flow in tissues such as the retina, skin, and brain. It has become especially useful in neuroscience where blood flow changes during physiological events like functional activation, stroke, and spreading depolarization can be quantified. LSCI is also attractive because it provides excellent spatial and temporal resolution while using inexpensive instrumentation that can easily be combined with other imaging modalities. Here we show how to build a LSCI setup and demonstrate its ability to monitor blood flow changes in the brain during an animal experiment.

How to Build a Laser Speckle Contrast Imaging LSCI System to Monitor Blood Flow

This video demonstrates how to build a Laser Speckle Contrast Imaging (LSCI) system that can easily be used to monitor blood flow.

어떻게 레이저를 작성하는 것은 혈액 흐름을 모니터링하는 콘트라스트 이미징 (LSCI) 시스템 스페클

Laser Speckle Contrast Imaging (LSCI) is a simple yet powerful technique that is used for full-field imaging of blood flow.  The technique analyzes ...

연구

신경과학

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

유아의 뇌 신진 대사와 혈류 역학의 비 침습적 광학 측정

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

의학

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics disturbance in affected cells. Diffusion weighted magnetic resonance imaging (MRI) identifies regions that are damaged, potentially irreversibly, by hypoxia-ischemia. Alterations in glucose utilization in the affected tissue may be detectable by positron emission tomography (PET) imaging of 2-deoxy-2-(18F)fluoro-&#7429;-glucose ([18F]FDG) uptake. Due to the rapid and variable nature of injury in this animal model, acquisition of both modes of data must be performed simultaneously in order to meaningfully correlate PET and MRI data. In addition, inter-animal variability in the hypoxic-ischemic injury due to vascular differences limits the ability to analyze multi-modal data and observe changes to a group-wise approach if data is not acquired simultaneously in individual subjects. The method presented here allows one to acquire both diffusion-weighted MRI and [18F]FDG uptake data in the same animal before, during, and after the hypoxic challenge in order to interrogate immediate physiological changes.

The method presented here uses simultaneous positron emission tomography and magnetic resonance imaging. In the cerebral hypoxia-ischemia model, dynamic changes in diffusion and glucose metabolism occur during and after injury. The evolving and irreproducible damage in this model necessitates simultaneous acquisition if meaningful multi-modal imaging data are to be acquired.

마우스 대뇌 저산소증 - 허혈 동안 동시 PET / MRI 영상

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics ...

Cerebral Blood Flow-Based Resting State Functional Connectivity of the Human Brain using Optical Diffuse Correlation Spectroscopy

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a key hemodynamic parameter related to cerebral oxygen supply. Resting state low frequency fluctuations based on oxygenation contrast have been shown to provide correlations between functionally connected regions. The presented protocol uses optical diffuse correlation spectroscopy (DCS) to assess blood flow-based resting state functional connectivity (RSFC) in the human brain. Results of CBF-based RSFC in human frontal cortex indicate that intra-regional RSFC is significantly higher in the left and right cortices compared to inter-regional RSFC in both cortices. This protocol should be of interest to researchers who employ multi-modal imaging techniques to study human brain function, especially in the pediatric population.

This protocol demonstrates how to measure resting state functional connectivity in the human prefrontal cortex using a custom-made diffuse correlation spectroscopy instrument. The report also discuss practical aspects of the experiment as well as detailed steps for analyzing the data.

광학 확산 상관 분광법을 사용하여 인간의 뇌의 대뇌 혈류 기반 휴식 상태 기능 적 연결

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a ...

생체공학

Real-Time, Two-Color Stimulated Raman Scattering Imaging of Mouse Brain for Tissue Diagnosis

Stimulated Raman scattering (SRS) microscopy has emerged as a powerful optical imaging technique for tissue diagnosis. In recent years, two-color SRS has been shown to be able to provide hematoxylin and eosin (H&amp;E)-equivalent images that allow fast and reliable diagnosis of brain cancer. Such capability has enabled exciting intraoperative cancer diagnosis applications. Two-color SRS imaging of tissue can be done with either a picosecond or femtosecond laser source. Femtosecond lasers have the advantage of enabling flexible imaging modes, including fast hyperspectral imaging and real-time, two-color SRS imaging. A spectral-focusing approach with chirped laser pulses is typically used with femtosecond lasers to achieve high spectral resolution. 
Two-color SRS acquisition can be realized with orthogonal modulation and lock-in detection. The complexity of pulse chirping, modulation, and characterization is a bottleneck for the widespread adoption of this method. This article provides a detailed protocol to demonstrate the implementation and optimization of spectral-focusing SRS and real-time, two-color imaging of mouse brain tissue in the epi-mode. This protocol can be used for a broad range of SRS imaging applications that leverage the high speed and spectroscopic imaging capability of SRS.

Stimulated Raman scattering (SRS) microscopy is a powerful, nondestructive, and label-free imaging technique. One emerging application is stimulated Raman histology, where two-color SRS imaging at the protein and lipid Raman transitions are used to generate pseudo-hematoxylin and eosin images. Here, we demonstrate a protocol for real-time, two-color SRS imaging for tissue diagnosis.

조직 진단을 위한 마우스 뇌의 실시간 두 가지 색상 자극 라만 산란 영상

Stimulated Raman scattering (SRS) microscopy has emerged as a powerful optical imaging technique for tissue diagnosis. In recent years, two-color SRS has ...

Advanced Diffusion Imaging in The Hippocampus of Rats with Mild Traumatic Brain Injury

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous variability and heterogeneity (age, gender, type of trauma, other possible pathologies, etc.), animal models play a key role in unraveling factors that are limitations in clinical research. They provide a standardized and controlled setting to investigate the biological mechanisms of injury and repair following TBI. However, not all animal models mimic the diffuse and subtle nature of mTBI effectively. For example, the commonly used controlled cortical impact (CCI) and lateral fluid percussion injury (LFPI) models make use of a craniotomy to expose the brain and induce widespread focal trauma, which are not commonly seen in mTBI. Therefore, these experimental models are not valid to mimic mTBI. Thus, an appropriate model should be used to investigate mTBI. The Marmarou weight drop model for rats induces similar microstructural alterations and cognitive impairments as seen in patients who sustain mild trauma; therefore, this model was selected for this protocol. Conventional computed tomography and magnetic resonance imaging (MRI) scans commonly show no damage following a mild injury, because mTBI induces often only subtle and diffuse injuries. With diffusion weighted MRI, it is possible to investigate microstructural properties of brain tissue, which can provide more insight into the microscopic alterations following mild trauma. Therefore, the goal of this study is to obtain quantitative information of a selected region-of-interest (i.e., hippocampus) to follow up disease progression after obtaining a mild and diffuse brain injury.

The overall goal of this procedure is to obtain quantitative microstructural information of the hippocampus in a rat with mild traumatic brain injury. This is done using an advanced diffusion-weighted magnetic resonance imaging protocol and region-of-interest based analysis of parametric diffusion maps.

가벼운 외상성 뇌 손상을 가진 쥐의 해마에 있는 향상된 확산 화상 진찰

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous ...

Systems Analysis of the Neuroinflammatory and Hemodynamic Response to Traumatic Brain Injury

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional deficits. Numerous studies by our group and others have shown that mTBI stimulates cytokine expression and activates microglia, decreases cerebral blood flow and metabolism, and impairs cerebrovascular reactivity. Moreover, several works have reported an association between derangements in these neuroinflammatory and hemodynamic markers and cognitive impairments. Herein we detail methods to characterize the neuroinflammatory and hemodynamic tissue response to mTBI in mice. Specifically, we describe how to perform a weight-drop model of mTBI, how to longitudinally measure cerebral blood flow using a non-invasive optical technique called diffuse correlation spectroscopy, and how to perform a Luminex multiplexed immunoassay on brain tissue samples to quantify cytokines and immunomodulatory phospho-proteins (e.g., within the MAPK and NF&#954;B pathways) that respond to and regulate activity of microglia and other neural immune cells. Finally, we detail how to integrate these data using a multivariate systems analysis approach to understand the relationships between all of these variables. Understanding the relationships between these physiologic and molecular variables will ultimately enable us to identify mechanisms responsible for mTBI.

This protocol presents methods to characterize the neuroinflammatory and hemodynamic response to mild traumatic brain injury and to integrate these data as part of a multivariate systems analysis using partial least squares regression.

외상성 뇌 손상에 대한 신경 염증 및 혈역학 반응의 시스템 분석

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional ...

Quantification of Cerebral Perfusion using Laser Speckle Imaging and Infarct Volume using MRI in a Pre-clinical Model of Posterior Circulation Stroke

Background: Basilar artery occlusion (BAO) is a subset of posterior circulation stroke that carries a mortality as high as 90%.&#160; The current clinical standard to diagnose ischemic stroke include computerized tomography (CT), CT angiography and perfusion and magnetic resonance imaging (MRI). Large animal pre-clinical models to accurately reflect the clinical disease as well as methods to assess stroke burden and evaluate treatments are lacking.
Methods: We describe a canine model of large vessel occlusion (LVO) stroke in the posterior circulation, and developed a laser speckle imaging (LSI) protocol to monitor perfusion changes in real time.&#160; We then utilized high b-value DWI (b=1800s/mm2) MRI to increase detection sensitivity. We also evaluated the ability of magnetic resonance angiography (MRA) to assess arterial occlusion and correlate with DSA. Finally, we verified infarct size from apparent diffusion coefficient (ADC) mapping with histology.&#160;
Results:&#160; Administration of thromboembolism occluded the basilar artery as tracked by DSA (n=7).&#160; &#160;LSI correlated with DSA, demonstrating a reduction in perfusion after stroke onset that persisted throughout the experiment, allowing us to monitor perfusion in real time.&#160; DWI with an optimized b-value for dogs illustrated the stroke volume and allowed us to derive ADC and magnetic resonance angiography (MRA) images. The MRA performed at the end of the experiment correlated with DSA performed after occlusion. Finally, stroke burden on MRI correlated with histology.
Conclusions: Our studies demonstrate real time perfusion imaging using LSI of a canine thromboembolic LVO model of posterior circulation stroke, which utilizes multimodal imaging important in the diagnosis and treatment of ischemic stroke.

A canine model of LVO stroke was utilized to develop laser speckle imaging to monitor cerebral perfusion in real-time.&#160; Diffusion-weighted MRI was optimized to image infarct volume utilizing a high b-value, enabling ADC and MRA, correlated with DSA at the time of stroke.&#160; Finally, ADC reconstructions correlated with histological findings.

Background: Basilar artery occlusion (BAO) is a subset of posterior circulation stroke that carries a mortality as high as 90%.&#160; The current clinical ...

Two-Dimensional Super-Resolution Visualization of Rat Brain Microvasculature Using Ultrasound Localization Microscopy

The cerebral microvasculature forms a complex network of vessels essential for maintaining brain function. Diseases such as stroke, Alzheimer's disease, gliomas, and vascular dementia can profoundly disrupt the microvascular system. Unfortunately, current medical imaging modalities offer only indirect observations at this scale. Inspired by optical microscopy, ultrasound localization microscopy (ULM) overcomes the classical trade-off between penetration depth and spatial resolution. By localizing and tracking individual injected microbubbles (MBs) with sub-wavelength precision, vascular and velocity maps can be generated at the micrometer scale. Here, we present a robust protocol for super-resolution imaging of the brain microvasculature in vivo in rats using a commercial ultrasound platform. This method achieves 12.5 &#181;m spatial resolution, reconstructing the microvascular architecture and providing detailed information on blood flow direction and velocity, greatly enhancing our understanding of cerebral microcirculation. The protocol can be extended to rat disease models, offering a powerful tool for the early diagnosis and treatment of neurovascular diseases.

Here, we describe a protocol for ultrasound localization microscopy (ULM), which achieves 12.5 &#181;m spatial resolution to image the brain microvasculature in rats. It enables detailed visualization of blood flow direction and velocity, offering a powerful tool for advancing studies of cerebral circulation and vascular disorders.

초음파 국소화 현미경을 사용한 쥐 뇌 미세혈관 구조의 2차원 초해상도 시각화

The cerebral microvasculature forms a complex network of vessels essential for maintaining brain function. Diseases such as stroke, Alzheimer's disease, ...

Real-Time Monitoring of Neurocritical Patients with Diffuse Optical Spectroscopies

Neurophysiological monitoring is an important goal in the treatment of neurocritical patients, as it may prevent secondary damage and directly impact morbidity and mortality rates. However, there is currently a lack of suitable non-invasive, real-time technologies for continuous monitoring of cerebral physiology at the bedside. Diffuse optical techniques have been proposed as a potential tool for bedside measurements of cerebral blood flow and cerebral oxygenation in case of neurocritical patients. Diffuse optical spectroscopies have been previously explored to monitor patients in several clinical scenarios ranging from neonatal monitoring to cerebrovascular interventions in adults. However, the feasibility of the technique to aid clinicians by providing real-time information at the bedside remains largely unaddressed. Here, we report the translation of a diffuse optical system for continuous real-time monitoring of cerebral blood flow, cerebral oxygenation, and cerebral oxygen metabolism during intensive care. The real-time feature of the instrument could enable treatment strategies based on patient-specific cerebral physiology rather than relying on surrogate metrics, such as arterial blood pressure. By providing real-time information on the cerebral circulation at different time scales with relatively cheap and portable instrumentation, this approach may be especially useful in low-budget hospitals, in remote areas and for&#160;monitoring in open fields (e.g., defense and sports).

Presented here is a protocol for non-invasively monitoring cerebral hemodynamics of neurocritical patients in real-time and at the bedside using diffuse optics. Specifically, the proposed protocol uses a hybrid diffuse optical systems to detect and display real-time information on cerebral oxygenation, cerebral blood flow and cerebral metabolism.

확산 광학 분광법을 이용한 신경중증 환자의 실시간 모니터링

Neurophysiological monitoring is an important goal in the treatment of neurocritical patients, as it may prevent secondary damage and directly impact ...

대뇌 혈류와의 광 산란 특성을 동시에 평가

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