우리는 생물 공학 및 미주리 대학에서 크리스토퍼 S. 본드 생명 과학 센터의학과 지원을 인정합니다. 우리는 미주리 생명 과학 연구위원회 09-1034 및 NIH R21CA139186 - 0에서 교부금 지원을 인정합니다. 또한 재정 지원 미주리 미주리 분자 세포학 코어의 대학, 공학 대학 미주리 대학의 대학에서 생명 과학 학부 연구 기회 프로그램을 감사드립니다.

1. 혈액 샘플 준비 <ol><li> 이 5 ML vacutainers로 무대 IV의 암 환자에서 전체 혈액의 약 10 ML을 그립니다. </li><li> 분당 3,000 회전 (RPM) 10 분 동안 원심 튜브. </li><li> 혈액 샘플은 세 가지 다른 레이어로 분리됩니다 아래 계층은 또한 버피 코트라는 적혈구, 중간 계층, 구성되어 leukocytes와 흑색증 세포를 포함하고, 상위 레이어는 플라즈마와 혈소판이 포함되어 있습니다. 버피 코트를 방해하지 않고 가능한 한 많은 플라즈마로 제거합니다. </li><li> 이 한 ML의 Wintrobe 튜브에 Histopaque 1077의 장소 250 μL. </li><li> 9 &quot;파스퇴르 피펫을 사용하여 &lt;500 μL를 수집해야하고, 나머지 플라즈마, 전체 버피 코트, 심지어 적혈구의 상단 레이어를 추출합니다. </li><li> Histopaque 레이어 위에 솔루션을 놓고 3,000 rpm으로 10 분 15 ML 원뿔 튜브에 Wintrobe 튜브를 원심 분리기. </li><li> 피가 다시 것입니다별도의지만이 시간은 적혈구가 버피 코트 쉽게 추출 수있게 Histopaque로 구분됩니다. 전체 버피 코트를 구하여 2 ML eppendorf 튜브에 배치 9 &quot;파스퇴르 피펫을 사용합니다. </li><li> 2퍼센트 V / V 십대 초반에 20 PBS의 1.5 ML에서 버피 코트를 일시 중지합니다. </li></ol> 2. 흐름 상공 회의소 건설 <ol><li> 아크릴 실린더, 20mm 외경, 17mm 내경 및 높이 8mm를 구합니다. 서로 90도 각도에서 실린더의 측면을 통해 세 5mm 직경의 구멍을 드릴. 폴리머 튜브의 세 가지 (1.6 mm 내경, 외경 5mm)를 잘라 및 뚫고 구멍에 그들을 놓습니다. </li><li> 물이 꽉 도장을 만들기 위해 아크릴 반지의 맨 아래에 Parafilm을 부풀. parafilm은 아크릴 반지 얕은 그릇을 형성하고 흐름 챔버를 형성됩니다 아크릴 아미드 솔루션을 개최합니다. </li><li> 맞은편에 아르 튜브의 조각을 통해 1.6 mm 직경의 와이어를 일시 중지혼자 와이어 반지의 중간으로 볼 수 있는지 서로하고. 1.6 mm 직경의 와이어와 위치 떨어져 정지 와이어에서 튜브 1mm로 튜브의 세 번째 부분을 입력합니다. 이 전선은 튜브 입력에서 아크릴 아미드되지 않습니다. 아크릴 아미드가 굳은 후 와이어가 광섬유를 위치로 원통형 흐름 경로와 장소를 떠나, 추출됩니다 유동 경로에 초점을 맞추고 있습니다. </li><li> 준비 아크릴 아미드의 5 ML 추가 [20g의 아크릴 아미드 (시그마 알드리치), 0.7 g의 bisacrylamide (시그마 알드리치), 증류수 100 ML] 작은 비커 수 있습니다. 암모늄 persulfate (시그마)의 0.02 g을 측정하여 비커에 추가합니다. 3 분 저어 막대와 함께 섞는다. 비커 20 μL tetramethylethylenediamene (피셔 과학)를 추가합니다. 빨리 가기 작성, 아크릴 반지에 혼합물을 따라줘. 몇 초 후, 솔루션 젤, 흐름 챔버 금형을 만드는 것입니다. 조심스럽게 아크릴 아미드 젤을 방해하지 않도록하고, 전선을 빼낸다. 흐름 chamBER은 이제 사용할 준비가되었습니다. </li></ol> 3. Photoacoustic flowmetry 설정 <ol><li> 아쿠 스틱으로 몇 직접 유동 경로 위에 초음파 젤을 사용하여 흐름 챔버에 polyvinylidene 불소 압전 음향 변환기 (Panametrics). </li><li> 총검 닐 - Concelman (BNC) 케이블을 사용하여 RITEC 광대역 수신기 - 640A의 입력 (아웃, highpass 필터 : 1 MHz 이상, imput 임피던스 : 50 Ohms, 증폭 : 35dB 로우 패스 필터)로 변환기를 연결합니다. 오실로 스코프 (텍트로닉스 TDS 2024B)로 필터의 출력을 연결합니다. 오실로 스코프를 트리거하려면, photodiode (Thorlabs)은 변환기 근처에 설치되고 BNC 케이블을 사용하여 오실로 스코프에 연결됩니다. </li><li> T - 커넥터 (소형 부품)은 흐름 챔버의 팔 하나에 첨부되어 있습니다. 두 개의 주사기 펌프 (Braintree 과학)은 커넥터의 다른 지점으로 연결됩니다. 다른 부분은 1에서 준비한 세포 현탁액을 포함하면서 하나의 주사기, 공기가 포함되어 있습니다. 에 흐름 속도를 설정100 μL / 분. </li><li> 공기 형태의 작은 포켓까지 흐름 챔버의 작은 튜브를 추출, 그 슬롯에 0.37 수치 조리개와 1mm 직경 코어 광섬유를 삽입합니다. 인터페이스 신호를 줄이고 유동 경로에 적용되는 레이저 빛의 양을 증가 물로 주머니를 채우십시오. 광섬유와 흐름 경로로 짧은 거리의 수치 조리개로 인해, 레이저는 직접 레이저의 앞에 샘플 photoacoustic 신호를 만드는 동일한 예제입니다 있도록, 주어진 시간에 샘플 중 하나 탄환을 비추다 것입니다. 레이저 에너지는 7mm 2 스폿 크기가 약 28 MJ / cm 2되어야합니다. </li><li> 양쪽 주사기 펌프를 켜십시오. 두 혼합할 유체의 T - 접합에 도달했을 때 그들은 균질 슬러그와 변환기 과거 흐름으로 분할됩니다. </li><li> nanosecond가 흑색증 세포에서 photoacoustic 파도를 생성하는 레이저를 펄스로 흐르는 샘플을 비추다. 멜라닌은 broadb이므로그리고 흡수 광, 가시적인 레이저 파장을 사용하실 수 있습니다. </li><li> 탄환이 변환기 위에 때 신호가 오실로 스코프에 나타나는 경우, 총알은 흑색증가 포함되어 있으며, 수집 약병에 낙하 때까지 시스템을 통해 추적한다. </li></ol> 4. Immunocytochemistry <ol><li> 유리 슬라이드에 문제를 해결하기 위해 Cytospin 4 원심 분리기 (써모 과학)로 캡처한 총이 가져가라. 장소 유리 Cytofunnels로 슬라이드 (브라운 필터 카드 써모 과학로 Shandon EZ 싱글 Cytofunnel)하고 Cytospin 4 원심 분리기로 그들을로드합니다. 각 깔때기에 PBS에 (시그마) 1퍼센트 소 혈청 알부민의 100 μL를 넣고 다음 캡처한 세포가 유리 슬라이드에 충실 도와 삼분 600 rpm으로 원심 분리기. </li><li> 원심 분리 후, Cytofunnels에 세포 샘플을 추가하고 다음 3 분 600 rpm으로 원심 분리기. 그렇다면 에탄올 기반 정착액으로 슬라이드를 스프레이. </li><li> 두 번 10 분 동안 PBS에서 슬라이드를 씻으십시오. </li><li&gt;을, 특이 현상이 바인딩을 차단 염소 혈청 10 μL (시그마)과 실온에서 1 시간 동안 PBS의 90 μL의 혼합물로 각 슬라이드를 부화. </li><li> 부화 후, 실온에서 humidified 챔버 1 시간 동안 기본 항체 솔루션 슬라이드와 부화를 가만히 따르다. 각 슬라이드는 1 %의 토끼에서 제기된 마트 - 1 항체, 1 % 마우스에서 제기된 CD - 45 항체, 10 % 염소 혈청과 89% PBS를 포함해야합니다. </li><li> 부화 후 10 분 세 번이나 PBS에서 슬라이드를 씻으십시오. </li><li> 슬라이드는 다음 형광단 항체에 바인딩된있는 차 항체 용액에 incubated 수 있습니다. 혼합물은 안티 - 토끼 항체 0.05 % 염소, 0.05 %의 염소 안티 - 마우스 항체, 0.1 %의 염소 혈청과 99% PBS가 포함되어 있습니다. 슬라이드는 실온에서 1 시간 동안 어둠 속에서 incubated 수 있습니다. </li><li> 부화 후, 슬라이드 5 분 세 번위한 PBS로 세척 가만히 따르다. </li><li> 핵에 대한 얼룩, DAPI의 0.1 μg / ML (Invitrogen)에있는 세포를 품어일분. 다음 슬라이드를 가만히 따르다하고 PBS로 세척. </li><li> 슬라이드 당 수지 기반 설치 미디어를 20 μL를 사용하여 세포 샘플을 통해 coverslip를 탑재합니다. 슬라이드를 보존하기 위해 투명 매니큐어로 coverslip을 봉쇄해. 이제 슬라이드는 형광 현미경으로 몇 군데 준비하고 있습니다. </li></ol> 5. 대표 결과 : <img src="/files/ftp_upload/3559/3559fig1.jpg" alt="그림 1"/> 그림 1. 백혈구 및 흑색증 세포에서 photoacoustic 파형이 여기에 표시됩니다. 백혈구는 아무 고유의 착색 필요 없다, 전자 노이즈의 평평한 라인 (왼쪽)와 같은 더 photoacoustic 파도와 매니 페스트를 생성하지 않습니다. 색소 흑색증 세포는 강력한 photoacoustic 웨이브 (오른쪽) 생산하고 있습니다. <img src="/files/ftp_upload/3559/3559fig2.jpg" alt="그림 2"/> 그림 2. immunocytochemical 얼룩 후, 교양 흑색증 전지는 BL에 DAPI 신호를 표시UE 동안 MART1는 녹색으로 강조 표시됩니다. <img src="/files/ftp_upload/3559/3559fig3.jpg" alt="그림 3"/> 그림 3. leukocytes의 지표로서 CD45와 그림 2에서, DAPI와 MART1에 대한 이미지를 오버레이,이 수치는 여러 가지 백혈구 사이 흑색증 세포를 보여줍니다.

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A., Gupta, S. K., Goldschmidt, B. S., Bhattacharyya, K., Raghuraman, R., Shukla, R., Dale, P. S., Boote, E., Katti, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+gold+nanoparticle+enhanced+prostate+cancer+cells+using+photoacoustic+flowmetry+with+optical+reflectance.">Detection of gold nanoparticle enhanced prostate cancer cells using photoacoustic flowmetry with optical reflectance.</a> J. Biomed. Nanotech. 6, 187-191 (2010).</li></ol>

존 A. Viator는 설립자이자 Viator 기술 Inc의 인간의 건강의 개선을위한 photoacoustic 기술을 상용화하기 위해 형성된 회사의 사장입니다.

CTCs을 감지하면 드문 종양 세포를 분리의 어려운 문제로 인해 거의 임상 응용 프로그램과 함께 여전히 연구 집약적인 분야입니다. 많은 다른 기술은 RT - PCR, microfluidic 세포 캡처, immunomagnetic 캡처 및 기타 방법 8-12 포함하여 CTC 탐지에 대한 평가되고있다. 그것은 무료 레이블이고 작은 빛을 흡수 입자를 포착하는 신속하고 정확한 방법을 제공하는 그러나, CMCs 쇼 photoacoustic 감지 약속 해요. 프로토콜 백혈구를 분리하기 위해 설명하는 것은 매우 효율적이며, 아직 적혈구의 낮은 숫자가 샘플에 존재한다. 이 루즈 오염 세포는 레이저 빛을 흡수하지만 크게 감소 수준. 적혈구 우리 흑색증 탐지 시스템을 방해하지 않도록하기 위해 다섯 건강한 환자 샘플은 시스템을 통해 소개되었으며 아무도 적혈구가 낮은 정도의 농도에서 존재하는 나타내는 신호를 준 없습니다무시됩니다. 집중 연구의 일련 교양 흑색증 세포를 사용하고 photoacoustic 흐름 시스템이 민감한 아래 1 셀 / μL (게시되지 않은 데이터)에 입증되었습니다 실시되었습니다. 이러한 연구는 가고있다 곧 암 환자 샘플을 사용하여 시험이 포함됩니다. 이 photoacoustic 기술은 또한 외인성 chromophores을 첨부하여 유방암과 전립선 암 등 비 색소 CTCs에 사용하기 위해 평가되고 있습니다. 우리는 암 cells13에 금 nanoparticles를 사용하여 예비 작업을 수행했습니다. 이 테스트는 nanoparticles가 photoacoustic 파도를 생성하는 데 필요한 광학 흡수를 제공할 수 것으로 나타났습니다. 나머지 기술 과제는 선택적으로 암세포에 이러한 입자를 첨부하는 것입니다.

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> ABCD1234 </td><td></td></tr></tbody></table>

detection and isolation of circulating melanoma cells using photoacoustic flowmetry

순환 종양 세포 (CTCs)는 매크로 종양에서 분리하고 종자 보조 종양 1,2,3로 혈액과 림프 시스템을 통해 확산이있는 세포입니다. CTCs는 전이성 질환과 혈액 샘플에서 검출의 지표가 암을 진단하고 치료에 대한 환자의 반응을 모니터하는 데 사용할 수 있습니다되었습니다. CTCs가 희귀하기 때문에, 하나 종양의 첨단 암 환자에서 정상 혈액 세포 수십억 사이 세포들이 감지 및 열거에 대해 구성된 것은 어려운 작업입니다. 우리는 photoacoustic 생성하는 대부분의 흑색증 세포에서 색소의 존재를 이용하거나, 레이저 순환 흑색증 세포 (CMCs) 4,5의 검출에 대한 사용자 지정 흐름 cytometer에 초음파를 유도. 이 과정은 원심 분리를 사용하여 백혈구 층을 얻기 전체 혈액 샘플을 분리 알아볼 수 있습니다. 전체 혈액에 존재하는 경우 CMCs 비슷한 밀도로 인해 백혈구로 구분됩니다. 이러한 세포에 resuspended 아르인산염은 식염수 (PBS)를 버퍼와 유량계에 도입했다. 오히려 혈액 세포 현탁액의 지속적인 흐름보다, 우리는 더 나은 연구를 위해서 이러한 세포를 캡처하기 위해서는 두 단계 흐름을 유도. 2 상류에서 microfluidic 시스템에서 두 혼합할 액체는 액체 6,7의 총이 번갈아 가며 교차점과 양식에서 만나십시오. PBS는 순차적으로 레이저 빛이 조사 아르 백혈구 및 공기 양식 microliter의 총이 정지. 액상에 대한 계면 활성제의 또한 균일한 슬러그 형성이 가능하고 사용자는 두 단계의 흐름 속도를 변경하여 다른 크기의 굼벵이를 만들 수 있습니다. 백혈구와 공기와 PBS의 굼벵이의 총이 더 밝은 absorbers을 포함하지 않으며 따라서, photoacoustic 파도를 생성하지 않습니다. 그러나, 심지어 하나의 CMCs를 포함하는 백혈구의 슬러그 레이저 빛을 흡수하고 고주파 음향 파를 생성합니다. photoacoustic 파도를 생성하는이 굼벵이는 verific에 대한 압수 및 cytochemical 얼룩을 위해 수집됩니다CMCs의 ation.

Watch this Scientific Journal Video about Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry at JoVE.com

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

우리는 전이성 질환의 조기 지표로 흑색증 세포 순환 감지하는 레이저 유도 초음파를 사용하여 플로우 cytometer을 개발했습니다.

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 ...

detection-isolation-circulating-melanoma-cells-using-photoacoustic

Research

JoVE Journal

Bioengineering

15.7K 조회수.  University of Missouri. 우리는 전이성 질환의 조기 지표로 흑색증 세포 순환 감지하는 레이저 유도 초음파를 사용하여 플로우 cytometer을 개발했습니다.

비디오: Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

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

Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary tumors at distant sites. CTC count can be used to follow disease progression based on the correlation between CTC concentration in blood and disease severity1. As a treatment tool, CTC could be studied in the laboratory to develop personalized therapies. To this end, CTC isolation must cause no cellular damage, and contamination by other cell types, particularly leukocytes, must be avoided as much as possible2. Many of the current techniques, including the sole FDA-approved device for CTC enumeration, destroy CTC as part of the isolation process (for more information see Ref. 2). A microfluidic device to capture viable CTC is described, consisting of a surface functionalized with E-selectin glycoprotein in addition to antibodies against epithelial markers3. To enhance device performance a nanoparticle coating was applied consisting of halloysite nanotubes, an aluminosilicate nanoparticle harvested from clay4. The E-selectin molecules provide a means to capture fast moving CTC that are pumped through the device, lending an advantage over alternative microfluidic devices wherein longer processing times are necessary to provide target cells with sufficient time to interact with a surface. The antibodies to epithelial targets provide CTC-specificity to the device, as well as provide a readily adjustable parameter to tune isolation. Finally, the halloysite nanotube coating allows significantly enhanced isolation compared to other techniques by helping to capture fast moving cells, providing increased surface area for protein adsorption, and repelling contaminating leukocytes3,4. This device is produced by a straightforward technique using off-the-shelf materials, and has been successfully used to capture cancer cells from the blood of metastatic cancer patients. Captured cells are maintained for up to 15 days in culture following isolation, and these samples typically consist of &gt;50% viable primary cancer cells from each patient. This device has been used to capture viable CTC from both diluted whole blood and buffy coat samples. Ultimately, we present a technique with functionality in a clinical setting to develop personalized cancer therapies.

Circulating tumor cells are isolated from the blood of cancer patients without inflicting cellular damage. Isolation of tumor cells is accomplished using a bimolecular surface of E-selectin in addition to antibodies against epithelial markers. A nanotube coating specifically promotes cancer cell adhesion resulting in high capture purities.

환자의 혈액 샘플에서 가능한 순환 종양 세포의 신속한 분리

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary ...

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

전기 화학 센서를 사용하여 세균의 검출 및 식별

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

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 ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

근육 재생의 마우스 모델에서 유도 만능 줄기 세포 유래 메소안지오블라스트 와 같은 근생 성 전구의 이식

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

Microtoroid 광 공진기를 사용하여 라벨이없는 단일 분자 검출

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

금 나노 막대를 포함하는 세포 차량의 제조 및 광 음향 분석

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

마이크로 RNA 정량는 DNA 결합 염료 화학, 물방울 디지털 PCR을 사용하여 순환

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

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 ...