1. 이미징 설정 많은 콤팩트하고 경량 필드 휴대용 아키텍처 8 조립 수 있으며, 또는 단면 이미징 능력 optofluidic 현미경으로 9.이 보고서에서, 그러나, 우리는 정적의 tomography 향해 벤치 상위 구현에 대한 기본 이미지 설정을 설명합니다 샘플. <ol><li> 조명 모듈 : 많은 경우에 이러한 발광 다이오드 - (LED가)와 같은 일부 코히어런트 광원을 활용할 수 있습니다. 실험적인 유연성을 위해 지논 램프 (초석 T260, 뉴 포트 사)와 단색을 사용. 단색은 예를 들어, 450-650 nm의의 중심 파장 주위 ~ 1-10 nm의 스펙트럼 폭 출력을 제공하도록 조정되었습니다. 이것은 부분적으로 일관된 출력 후 시스템에 부분적으로 일관된 빛을 제공하는 다중 모드 광섬유 (Thorlabs AFS105/125Y)에 결합됩니다. 광학 섬유 (Thorlabs 전동식 회전 무대에 마운트되어PRM-1Z8은 조명의 각도를 변경) Thorlabs TDC001 컨트롤러에 의해 구동. 동력 무대는 광원 붙어있는, 주어진 각도로 광원의 인 - 비행기 교대를 달성하는 데 사용된 2 차원 선형 XY 스테이지 (뉴 포트 ILS50CC 뉴 포트 MFA-PPD 컨트롤러에 의해 구동)에 마운트됩니다. </li><li> 탐지 : Lensfree 광학 tomography는 검출기 배열이 상당히 이미지 수집 단계 또는 데이터 처리를 변경하지 않고 응용 프로그램의 요구 사항에 따라 선택할 수있는 그러한 확장 기술입니다. 이 보고서에서는 2.2 μm의의 픽셀 크기 (IDS 이미징, UI-1485LE-M)로, 5 메가 픽셀을 갖는 CMOS 센서 배열을 채용. 검출기는 검출기의 활성 영역의 상단에 직접 배치되었다 샘플 와이드 뷰 필드 (예 : 24mm 2) 홀로그램 영상을 기록하는 데 사용됩니다. 이중 축 tomography 설정이 구현되어 7면 감지기가되어야합니다LSO는 광학 축에 대한 일반 비행기에서 샘플을 (함께 탐지기로) 회전 수평 (광학 테이블과 관련한) 회전 무대 (예 : Thorlabs RP-01)에 장착됩니다. 7 </li></ol> 2. 샘플 준비 lensfree 광학 tomography 이미지 등 세포 및 미생물과 같은 개체의 다양한 수 있지만, 우리는 C의 입체 현미경을 수행하여 기본 원칙을 보여주는 것입니다 elegans 샘플. <ol><li> 메스 또는 주걱 사용하여 C를 포함하는 배양 접시로부터 한천의 작은 조각을 elegans 문화. 각 차원을 따라 몇 mm의 큐빅 조각 nematodes 수백 포함됩니다. </li><li> 한 ML 드 이온화 (DI) 물을 포함하는 폴리 프로필렌의 유리병에 한천의 작은 청크를 놓습니다. </li><li> 0.5-1 분, 그리고 벌레는 DI 물에 한천 조각 기어 나와서 때까지 10-15 분 기다려야 위해 부드럽게 소용돌이. </li><li> ... 할 목적으로일시적으로, 벌레를 고정 5-10 밀리미터 levamisole 솔루션 (시그마 - 올드 리치) 1 ML을 추가하고 10 분 기다립니다. </li><li> 두건의 살인 사건이 은폐이 풀리지 사이 5-10 유리병의 바닥에서 샘플의 μl, 그리고 샌드위치를​​ 피펫. 일시적으로 고정화 웜 (예 : 50-100 웜)의 다수가 포함된이 샘플은, 데이터 수집을 시작 검출기에 둘 수 있습니다. </li></ol> 3. 데이터 수집 여기서는 LabView 인터페이스를 개발한 사용자 정의를 사용하여 자동화되었습니다 전형 많이 실험에 대한 이미지 수집 단계를 요약합니다. <ol><li> 0 °는 광원의 수직 위치에 해당 -50 °로 회전 무대의 초기 각도를 조정합니다. </li><li> (0,0)로 XY 스테이지의 초기 위치를 조정,이 홈 위치입니다. </li><li> 최고의 이미지가 어떠한들없이 최대한 밝은 것이 자사의 다이나믹 레인지와 같은 활용하는 감지기의 노출 시간을 조절aturated 픽셀. </li><li> 회전 무대의 각도를 변경하지 않고, 9 이미지를 캡처합니다. 각 이미지의 경우, 각 이미지가 이전에 비해 픽셀의 약 분기까지 바꿔 주던 그러한 3x3 사각형 격자의 새로운 위치로 XY 스테이지 이동합니다. 6μ6 그리드에 예를 들어, 각각의 각도에서 더 많은 이미지를 취득, 개체 및 신호 대 노이즈 비율 (SNR)의 종류에 따라 해상도를 향상시킬 수 있습니다. 10 </li><li> (0,0)로 XY 스테이지의 초기 위치를 조정,이 홈 위치입니다. </li><li> 50 °에 도달하기 전까지는, 2 ° 단위로 회전 무대의 각도를 높이십시오. 각 증가 후, 각각의 새 각도, 3-5 단계를 반복에서 즉. 각도 단위는 획득 시간 대 이미징 해상도의 최적화에 따라 미세한 또는 coarser 될 수 있습니다. </li><li> (이중 축 tomography 기법이 활용되는 경우 선택적 단계) 검출기가 90 °로 장착되는 수평 무대를 회전합니다. 그런 다음,1-6 단계를 반복합니다. </li></ol> 4. 데이터 분석 제 3 항, 조명의 51 각도의 각 9 서브 픽셀 이동 이미지가 포함되어 취득 459 이미지 세트에서 단계 1-6를 수행. 첫째, 9 이미지의 각 집합에 디지털 한 고해상도 (HR) 각도마다 프로젝션 홀로그램을 얻기 위해 픽셀 슈퍼 해상도 알고리즘 10, 사용, 처리됩니다. 이것은 화소 슈퍼 해상도 lensfree 이미지의 공간적 해상도에서 오히려 회절 한계보다 픽셀 크기의 한계를 극복이라는 점에 주목하여야한다. 픽셀 슈퍼 해결 홀로그램 그런 다음 디지털 51 프로젝션 이미지를 얻을 7-10 복원됩니다. 51 투영 화상의이 세트는 다음 TomoJ, 플러그인 ImageJ (오픈 소스 이미지 분석 소프트웨어)에 대한. 11이 백 프로젝션 작업이 샘플의 3 차원 이미지 (tomograms)를 출력합니다.를 사용하여 백 예상된다 여기에 구현되지 않지만,참조에서 설명한대로 이중 축 tomography 기법도 활용 수 있습니다. 7. 이 제도에서는 tomograms의 두 번째 세트는 샘플에 대한 자세한 공간적 정보를 제공하며 축 해상도를 향상 최초의 회전 축에 관하여 직교 축을 따라 광원을 회전하여 얻어진다. 5. 대표 결과 lensfree 광학 tomography의 넓은 뷰 필드 (FOV)은 그림 1에서 증명된다. 시료가 검출기 - 배열의 맨에 직접 배치되면서 개체의 홀로그램 이미지가 더 큰 활성 영역과 신흥 검출기 배열을 사용하여 증가 수 24 FOV mm 2 이상 기록될 수 있습니다. 검출기 - 배열의 픽셀 크기가 기록된 홀로그램 이미지의 해상도를 제한하지만, 화소 수퍼 해상도 기술이 문제를 완화. 그림 2는 함께 화소 슈퍼 해결 홀로그램을 보여줍니다와 조명의 세 가지 다른 각도, 하위 마이크로 미터 공간적 해상도를 제공하는 복원 이미지 (즉, 프로젝션 이미지). 프로젝션 이미지는 시편의 tomograms (즉, 입체 영상)을 계산하는 (예 : 여과 백 프로젝션) 단층 영상 재구성 기법을 사용하여 조합하여 사용할 수 있습니다. 그림 3은 웜의 앞부분을 통해 XY 평면에 세 슬라이스 이미지를 보여줍니다, 어디에 pharyngeal 튜브는 등 ~ 5 μm의 외경이 약 원통형 구조에서 예상 Z = 8 μm의를 통해 슬라이스에서 볼 수 있습니다. 또한, xz 평면에서 교차 단면 이미지 (그림 3 상단 패널에 삽입된 페이지) 명확 웜 경계와 인두의 성공적인 3D 영상을 보여주 pharyngeal 튜브 내부를 (화살표가 가리키는) 보여줍니다. <img src="/files/ftp_upload/4161/4161fig1.jpg" alt="그림 1" /> <str긴 사연&gt; 그림 1은 lensfree 광학 tomography (LOT) 수직 조명에 대한 설정을 사용하여 기록 풀 뷰 필드 (24mm 2) 홀로그램 이미지를 보여줍니다. 많은 대형 이미징 영역으로 인하여 많은 벌레들이 동시에 단일 데이터 수집 단계로 몇 군데하실 수 있습니다. <img src="/files/ftp_upload/4161/4161fig2.jpg" alt="그림 2" /> 그림 2는 슈퍼 해결 홀로그램 (왼쪽 패널)와 디지털 복원 프로젝션 이미지 (오른쪽 패널) C.위한 픽셀을 보여줍니다 세 가지 조명 각도에서 elegans 벌레 (대형 뷰 필드에서 잘립니다.) 각각의 투영 이미지는 필터링된 백 프로젝션 작업을 통해 3 차원 구조의 계산을 가능하게 다른 시야각을에 관한 정보가 포함되어 있습니다. <img src="/files/ftp_upload/4161/4161fig3.jpg" alt="그림 3" /> 그림 3은 C.에 대한 계산된 tomograms을 보여줍니다 elegans 벌레 OF 그림 2. (끝줄)는 Z = 3 μm의에서 전체 웜 슬라이스 이미지를 보여줍니다. 삽입된 페이지는 벌레의 앞부분에서 교차 단면 이미지를 보여줍니다. 삽입된 페이지의 스케일 바는 50 μm의입니다. (하단 행)은 lensfree 광학 tomography의 광학 sectioning 능력을 보여주 웜의 앞부분을 통해 세 슬라이스 이미지를 표시합니다. 그림 전체에 화살표가 웜의 pharyngeal 튜브에 동일한 지점을 나타냅니다. 현미경 이미지 (x40, 0.65-NA)도 시각적으로 비교 제공됩니다.

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관심의 어떠한 충돌 선언 없습니다.

그것은 lensfree 온칩 홀로그램 현미경의 독특한 기하학 픽셀 슈퍼 해상도와 단층 이미지를 달성하기위한 중요한 원동력임을 강조하는 것이 중요합니다. 녹화된 영상은 12에 도달 조리 연락처 이미징에서와 같이 프로젝션 이미지로 간주 아니지만 프로젝션 홀로그램, 전송 빛의 회절은 검출기에 impinges까지 디지털 홀로그램 재건축으로 해결할 수 있기 때문에. 따라서 샘플 - 투 - 센서 ...

<table border="1"><tbody><tr><td> 이름 </td><td> Compay </td><td> # 카탈로그 </td><td> 논평 </td></tr><tr><td> 선형 XY 스테이지 </td><td> 뉴 포트 주식 회사 </td><td> MFA-PP </td><td> 미니어처 리니어 스테이지 </td></tr><tr><td> 동력 회전 무대 </td><td> Thorlabs </td><td> PRM1Z8 </td><td> 동력 정밀 회전 마운트 </td></tr><tr><td> 다중 모드 광섬유 </td><td> Thorlabs </td><td> AFS105/125Y </td><td> 멀티모드 광섬유 </td></tr><tr><td> 광원 </td><td> 뉴 포트 주식 회사 </td><td> 6255 </td><td> 오존없는 크세논 램프 </td></tr><tr><td> 단색 화 </td><td> 뉴 포트 주식 회사 </td><td> 74,100 </td><td> 초석 260 4분의 1 m 단색 화 </td></tr><tr><td> CMOS 센서 어레이 </td><td> Aptina 주식 회사 </td><td> MT9P031STC </td><td> 5 메가픽셀의 CMOS 센서 </td></tr><tr><td> C. elegans 샘플 </td><td> 캐롤라이나 Biosupply </td><td> 173,500 </td><td> 야생 형 C. elegans </td></tr><tr><td> Levamisole </td><td> 시그마 알드리치 </td><td> L9756-5G </td><td> Tetramisole 하이드로 클로라이드 </td></tr></tbody></table>

lensfree on-chip tomographic microscopy employing multi-angle illumination and pixel super-resolution

그것이 서로 다른 크기의 비늘의 객체에 관한 입체 (3D) 구조적인 정보를 제공할 수있는 단층 영상 의학에서 널리 사용되는 도구가되었습니다. 마이크로 미터와 mm 스케일에서 광학 현미경 modalities는 가시 광선의 비 이온화 특성으로 증가 사용 인하여 찾고, 풍부한 조명 소스의 집합 (예 : 레이저와 발광 - 다이오드 등) 및 감지 요소 (예 :의 가용성 대형 CCD 및 CMOS 검출기 - 배열). 최근에 개발된 광학 단층 현미경 modalities 가운데 하나는 광학 일관성의 tomography, 광학 회절 tomography, 광학 프로젝션 tomography와 가벼운 시트 현미경을 포함할 수 있습니다. 1-6 이러한 플랫폼은 세포, 미생물 등 C. 등의 모델 동물의 단면 영상을 제공 elegans, zebrafish 및 마우스 배아. 기존 3D 광학 imagers는 일반적으로 일을 제한, 상대적으로 부피가 복잡한 아키텍처를전자는 첨단 실험실 이러한 장비의 가용성 및 실험실 - 온 - 칩 플랫폼과 microfluidic 칩을와의 통합을 방해한. 대안 단층 현미경을 제공하기 위해, 우리는 최근 일곱 많이 버리고 렌즈와 부피가 큰 광학 부품의 사용. 높은 처리량, 컴팩트하고 비용 효율적인 광학 tomography의 양상으로 lensfree 광학 tomography (LOT)를 개발하고, 대신 멀티 각도에 의존 조명과 대형 이미징 볼륨을 통해 마이크로 객체의 깊이 - 해결 이미징을 달성하기위한 디지털 계산. 많은에 대한 X &lt;1 μm의 X &lt;3 15~100mm 3 대형 이미징 볼륨여 각각 X, Y 및 Z 크기의 μm의,,, 그리고 될 수 있습니다 특히 유용 &lt;1 μm의의 공간적 해상도로 이미지를 생물 학적 시료 수 실험실 - 온 - 칩 플랫폼.

Watch this Scientific Journal Video about Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution at JoVE.com

Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution

Lensfree 광학 tomography은 &lt;1 μm의의 공간적 해상도를 제공하는 입체 현미경 기법이다 × &lt;1 μm의 × 15~1백밀리미터의 큰 이미지 볼륨여 각각 X, Y 및 Z 크기에서 &lt;3 μm의,<sup&gt; 3</sup&gt; 어느 실험실 - 온 - 칩 플랫폼 통합에 특히 유용할 수 있습니다.

멀티 앵글 조명 및 픽셀 슈퍼 해상도를 채용 Lensfree 온칩 단층 현미경

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different ...

lensfree-on-chip-tomographic-microscopy-employing-multi-angle

Research

JoVE Journal

Bioengineering

11.4K 조회수.  University of California, Los Angeles. Lensfree 광학 tomography은 

비디오: Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

Interferometric 반사율 이미징 센서 (IRIS)를 채용 Biomolecular 감지

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

연구

생체공학

On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic processes in simple, compact systems with no moving parts. Isotachophoresis (ITP) is a simple and very robust electrokinetic technique that can achieve million-fold preconcentration1,2 and efficient separation and extraction based on ionic mobility.3 For example, we have demonstrated the application of ITP to separation and sensitive detection of unlabeled ionic molecules (e.g. toxins, DNA, rRNA, miRNA) with little or no sample preparation4-8 and to extraction and purification of nucleic acids from complex matrices including cell culture, urine, and blood.9-12
ITP achieves focusing and separation using an applied electric field and two buffers within a fluidic channel system. For anionic analytes, the leading electrolyte (LE) buffer is chosen such that its anions have higher effective electrophoretic mobility than the anions of the trailing electrolyte (TE) buffer (Effective mobility describes the observable drift velocity of an ion and takes into account the ionization state of the ion, as described in detail by Persat et al.13). After establishing an interface between the TE and LE, an electric field is applied such that LE ions move away from the region occupied by TE ions. Sample ions of intermediate effective mobility race ahead of TE ions but cannot overtake LE ions, and so they focus at the LE-TE interface (hereafter called the "ITP interface"). Further, the TE and LE form regions of respectively low and high conductivity, which establish a steep electric field gradient at the ITP interface. This field gradient preconcentrates sample species as they focus. Proper choice of TE and LE results in focusing and purification of target species from other non-focused species and, eventually, separation and segregation of sample species.
We here review the physical principles underlying ITP and discuss two standard modes of operation: "peak" and "plateau" modes. In peak mode, relatively dilute sample ions focus together within overlapping narrow peaks at the ITP interface. In plateau mode, more abundant sample ions reach a steady-state concentration and segregate into adjoining plateau-like zones ordered by their effective mobility. Peak and plateau modes arise out of the same underlying physics, but represent distinct regimes differentiated by the initial analyte concentration and/or the amount of time allotted for sample accumulation.
We first describe in detail a model peak mode experiment and then demonstrate a peak mode assay for the extraction of nucleic acids from E. coli cell culture. We conclude by presenting a plateau mode assay, where we use a non-focusing tracer (NFT) species to visualize the separation and perform quantitation of amino acids.

Isotachophoresis (ITP) is a robust electrokinetic separation and preconcentration technique with applications ranging from toxin detection to sample preparation. We review the physical principles of ITP and the methodology of applying this technique to two specific example applications: separation and detection of small molecules and purification of nucleic acids from cell culture lysate.

이온의 분리 및 핵산의 정화를위한 온칩 Isotachophoresis

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic ...

On-Chip Endothelial Inflammatory Phenotyping

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial dysfunction. However, early functional changes in endothelium that signify an individual's level of risk are not directly assessed clinically to help guide therapeutic strategy. Moreover, the regulation of inflammation by local hemodynamics contributes to the non-random spatial distribution of atherosclerosis, but the mechanisms are difficult to delineate in vivo. We describe a lab-on-a-chip based approach to quantitatively assay metabolic perturbation of inflammatory events in human endothelial cells (EC) and monocytes under precise flow conditions. Standard methods of soft lithography are used to microfabricate vascular mimetic microfluidic chambers (VMMC), which are bound directly to cultured EC monolayers.1 These devices have the advantage of using small volumes of reagents while providing a platform for directly imaging the inflammatory events at the membrane of EC exposed to a well-defined shear field. We have successfully applied these devices to investigate cytokine-,2 lipid-3, 4 and RAGE-induced5 inflammation in human aortic EC (HAEC). Here we document the use of the VMMC to assay monocytic cell (THP-1) rolling and arrest on HAEC monolayers that are conditioned under differential shear characteristics and activated by the inflammatory cytokine TNF-&alpha;. Studies such as these are providing mechanistic insight into atherosusceptibility under metabolic risk factors.

Microfluidic flow chambers etched by photolithography and fabricated from PDMS are applied to probe functional outcomes associated with EC dysfunction and inflammation. In a representative experiment, the ability of differential shear stress to modulate monocytic cell adhesion to cytokine activated EC monolayers is demonstrated.

온칩 내피 염증성 Phenotyping

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial ...

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular level in the chemical and biomedical sciences. Although commercial SIM systems are available, systems that are custom designed in the laboratory can outperform commercial systems, the latter typically designed for ease of use and general purpose applications, both in terms of imaging fidelity and speed. This article presents an in-depth guide to building a SIM system that uses total internal reflection (TIR) illumination and is capable of imaging at up to 10 Hz in three colors at a resolution reaching 100 nm. Due to the combination of SIM and TIRF, the system provides better image contrast than rival technologies. To achieve these specifications, several optical elements are used to enable automated control over the polarization state and spatial structure of the illumination light for all available excitation wavelengths. Full details on hardware implementation and control are given to achieve synchronization between excitation light pattern generation, wavelength, polarization state, and camera control with an emphasis on achieving maximum acquisition frame rate. A step-by-step protocol for system alignment and calibration is presented and the achievable resolution improvement is validated on ideal test samples. The capability for video-rate super-resolution imaging is demonstrated with living cells.

This article provides an in depth guide for the assembly and operation of a structured illumination microscope operating with total internal reflection fluorescence illumination (TIRF-SIM) to image dynamic biological processes with optical super-resolution in multiple colors.

여러 색상과 고속에서 구조화 조명 TIRF 현미경에 가이드

Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

양자점 나노 입자를 사용하여 상호 빛 - 및 전자 현미경

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

드라이 필름 포토 레지스트 기반 전기 화학 미세 바이오 센서 플랫폼: 장치 제작, 온 칩 시험 준비 및 시스템 운영

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

제조 및 통합된 전극 Transendothelial 전기 저항을 직접 측정 하는 기관-온-칩 시스템의 유효성 검사

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies

Methanol is a well-established carbon source and inducer for efficient protein production employing Pichia pastoris (P. pastoris) as a host on micro-, lab and industrial scale. However, due to its toxicity and flammability, there is a desire to avoid methanol while maintaining the high productivity of P. pastoris. Small scale bioreactor cultivations (0.5-5 L working volume) are commonly used to evaluate a strain and its protein production characteristics since microscale cultivation in deep well plates can be hardly controlled or relies on expensive equipment. Furthermore, traditional protocols for the cultivation and induction of P. pastoris were established for constitutive expression or methanol induction and so far, no reliable protocols were described to screen P. pastoris expression strains with derepressible promoters in (controlled and monitored) parallel cultivations. To simplify such initial cultivations to characterize and compare new protein production strains, we established a simple shake flask cultivation system for methanol free expression that simulates bioreactor conditions including a constant slow glycerol feed and online monitoring, thereby coming closer to the real conditions in bioreactors compared to mostly applied small scale batch cultivations. To drive recombinant protein expression in P. pastoris, the carbon source repressed promoters PDC and PDF were applied. Polymer discs with embedded carbon source, releasing a constant amount of glycerol, assured a feed rate delivering the necessary energy for maintaining the promoters active while keeping the biomass generation low.

Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies

This method for the first time describes reliable experimental procedures for efficient inducible methanol-free recombinant protein production in Pichia pastoris (Komagataella phaffii), employing the carbon source regulated promoters PDCand PDF in small scale experiments while cultivation parameters can be monitored online, and a constant glycerol feed is applied.

Pichia Pastoris 탈 억제 기술을 채용 하 여 메탄올 독립 식

Methanol is a well-established carbon source and inducer for efficient protein production employing Pichia pastoris (P. pastoris) as a host on micro-, lab ...

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has facilitated advanced genetic modification of Saccharomyces cerevisiae, a model organism and workhorse in industrial biotechnology. CRISPR-associated protein 12a (Cas12a), an RNA-guided endonuclease with features distinguishable from Cas9 is applied in this work, further extending the molecular toolbox for genome editing purposes. A benefit of the CRISPR/Cas12a system is that it can be used in multiplex genome editing with multiple guide RNAs expressed from a single transcriptional unit (single CRISPR RNA (crRNA) array). We present a protocol for multiplex integration of multiple heterologous genes into independent loci of the S. cerevisiae genome using the CRISPR/Cas12a system with multiple crRNAs expressed from a single crRNA array construct. The proposed method exploits the ability of S. cerevisiae to perform in vivo recombination of DNA fragments to assemble the single crRNA array into a plasmid that can be used for transformant selection, as well as the assembly of donor DNA sequences that integrate into the genome at intended positions. Cas12a is pre-expressed constitutively, facilitating cleavage of the S.&#160;cerevisiae genome at the intended positions upon expression of the single crRNA array. The protocol includes the design and construction of a single crRNA array and donor DNA expression cassettes, and exploits an integration approach making use of unique 50-bp DNA connectors sequences and separate integration flank DNA sequences, which simplifies experimental design through standardization and modularization and extends the range of applications. Finally, we demonstrate a straightforward technique for creating yeast pixel art with an acoustic liquid handler using differently colored carotenoid producing yeast strains that were constructed.

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

The CRISPR/Cas12a system in combination with a single crRNA array enables efficient multiplex editing of the S.&#160;cerevisiae genome at multiple loci simultaneously. This is demonstrated by constructing carotenoid producing yeast strains which are subsequently used to create yeast pixel art.

CRISPR/Cas12a 멀티플렉스 게놈 편집 사카로미세세세레비시아와 이스트 픽셀 아트의 창조

High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) ...

Applications for Open Source Microplate-Compatible Illumination Panels

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well as large-scale high-throughput screening (HTS) campaigns. Though laboratory automation has greatly increased the utility of microplates there remain instances where automation-based instrumentation is not feasible, cost-effective or compatible with microplate formatting needs. In these cases, microplates must be manually prepared. Problematic to manual microplate manipulations is that a number of difficulties can arise pertaining to the accurate tracking of sample operations, data record keeping and quality control (QC) inspection for well artifacts or formatting errors. As microplate well densities increase (i.e., 96-well, 384-well, 1536-well) the potential for introducing errors also drastically increases.&#160; Moreover, for small bench-top laboratory operations there exists a need to improve the ease and accuracy of sample handling in a cost-effective fashion. Herein, we describe a system that acts as a semi-automated pipetting guide referred to as the Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.). &#160;M.A.P.L.E. has multiple uses for supporting compound hit-picking and microplate preparation for assay development in high-throughput screening or laboratory benchtop operations, as well as QC/quality assurance (QA) diagnostic evaluation of microplate quality or visualizing well formatting errors.

Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.) is a computer-driven device that systematically illuminates microtiter wells to provide guidance for the manual preparation of microplates.&#160; M.A.P.L.E. improves the accuracy of microplate preparation while automating data recordkeeping. &#160;In addition, it can assist with examining microplate quality or aid in the detection of errors.&#160; &#160;

오픈 소스 마이크로플레이트 호환 조명 패널용 애플리케이션

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well ...