Name: three-dimensional optical-resolution photoacoustic microscopy
Uploaded: 2011-05-03T17:00:00
Description: Watch this Scientific Journal Video about Three-dimensional Optical-resolution Photoacoustic Microscopy at JoVE.com

저자는 원고의 박사 Lynnea 브롬바 클로즈 읽어 주셔서 감사합니다. 이 작품은 보건 보조금 R01 EB000712, R01 EB008085, R01 CA134539, U54 CA136398 및 5P60 DK02057933 국립 연구소에 의해 후원되었다. 교수 Lihong V. 왕 그러나이 작품을 지원하지 않았 Microphotoacoustics, 주식 및 Endra 주식 회사에 금융 관심을했습니다.

1. 시스템 구성 <ol><li> 광학 조사 <ol><li> 광학 방사선 출처 : 고체 펄스 레이저 (INNOSLAB, Edgewave)과 염료 레이저 (CBR - D, 시라)을 펌핑 - 다이오드. </li><li> 출력 레이저 빔을는 (펄스 폭 : 7 NS) (P50C, Thorlabs) 50 μm의 핀홀 통과하는 콘덴서 렌즈 (LA1131, Thorlabs)로 초점을 맞추고 있습니다. </li><li> 핀홀은 효율적인 공간 필터링을위한 기본 모드 빔 직경 핀홀 직경과 일치하도록 콘덴서 렌즈의 초점에서 약간 ?...

<ol>
	<li>Maslov, K., Zhang, H. F., Hu, S., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical-resolution+photoacoustic+microscopy+for+in+vivo+imaging+of+single+capillaries.">Optical-resolution photoacoustic microscopy for in vivo imaging of single capillaries.</a> Opt. Lett. 33, 929-931 (2008).</li><li>Zhang, C., Maslov, K., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subwavelength-resolution+label-free+photoacoustic+microscopy+of+optical+absorption+in+vivo.">Subwavelength-resolution label-free photoacoustic microscopy of optical absorption in vivo.</a> Opt. Lett. 35, 3195-3197 (2010).</li><li>Wang, Y., Maslov, K., Kim, C., Hu, S., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+photoacoustic+and+fluorescence+confocal+microscopy.">Integrated photoacoustic and fluorescence confocal microscopy.</a> IEEE. Trans. Biomed. Eng. 57, 2576-2578 (2010).</li><li>Jiao, S., Xie, Z., Zhang, H. F., Puliafito, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+multimodal+imaging+with+integrated+photoacoustic+microscopy+and+optical+coherence+tomography.">Simultaneous multimodal imaging with integrated photoacoustic microscopy and optical coherence tomography.</a> Opt. Lett. 34, 2961-2963 (2009).</li><li>Hu, S., Maslov, K., Tsytsarev, V., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+transcranial+brain+imaging+by+optical-resolution+photoacoustic+microscopy.">Functional transcranial brain imaging by optical-resolution photoacoustic microscopy.</a> J. Biomed. Opt. 14, 040503-040503 (2009).</li><li>Hu, S., Yan, P., Maslov, K., Lee, J. M., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+amyloid+plaques+in+a+transgenic+mouse+model+using+optical-resolution+photoacoustic+microscopy.">Intravital imaging of amyloid plaques in a transgenic mouse model using optical-resolution photoacoustic microscopy.</a> Opt. Lett. 34, 3899-3901 (2009).</li><li>Hu, S., Rao, B., Maslov, K., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+Photoacoustic+Ophthalmic+Angiography.">Label-free Photoacoustic Ophthalmic Angiography.</a> Opt. Lett. 35, 1-3 (2010).</li><li>Jiao, S. L., Jiang, M. S., Hu, J. M., Fawzi, A., Zhou, Q. F., Shung, K. K., Puliafito, C. A., Zhang, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoacoustic+ophthalmoscopy+for+in+vivo+retinal+imaging.">Photoacoustic ophthalmoscopy for in vivo retinal imaging.</a> Opt. Express. 18, 3967-3972 (2010).</li><li>Oladipupo, S., Hu, S., Santeford, A., Yao, J., Kovalski, J. R., Shohet, R., Maslov, K., Wang, L. V., Arbeit, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+HIF-1+induction+produces+multistage+neovascularization+with+stage-specific+sensitivity+to+VEGFR+inhibitors+and+myeloid+cell+independence.">Conditional HIF-1 induction produces multistage neovascularization with stage-specific sensitivity to VEGFR inhibitors and myeloid cell independence.</a> Blood. , .</li><li>Hu, S., Maslov, K., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+functional+chronic+imaging+of+a+small+animal+model+using+optical-resolution+photoacoustic+microscopy.">In vivo functional chronic imaging of a small animal model using optical-resolution photoacoustic microscopy.</a> Med. Phys. 36, 2320-2323 (2009).</li><li>Hu, S., Maslov, K., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Second-generation+optical-resolution+photoacoustic+microscopy+with+improved+sensitivity+and+speed.">Second-generation optical-resolution photoacoustic microscopy with improved sensitivity and speed.</a> Opt. Lett. 36, 1134-1136 (2011).</li><li>Wang, X., Pang, Y., Ku, G., Xie, X., Stoica, G., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+laser-induced+photoacoustic+tomography+for+structural+and+functional+in+vivo+imaging+of+the+brain.">Noninvasive laser-induced photoacoustic tomography for structural and functional in vivo imaging of the brain.</a> Nat. Biotechnol. 21, 803-806 (2003).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laser Institute of America, American National Standard for Safe Use of Lasers ANSI Z136. , (2007).</li><li>Jacques, S. L., Prahl, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Absorption+of+Hemoglobin+.">Optical Absorption of Hemoglobin .</a> Oregon Medical Laser Center [Internet]. , (1999).</li></ol>

이 비디오에서는, 우리는 시스템 구성, 시스템 정렬하고, 전형적인 실험 절차를 포함 또는 - PAM의 실험 프로토콜에 대한 자세한 지침을 제공합니다. 라벨 - 무료 비침 투 또는 - PAM은 단일 모세 기준 microvascular 기능 및 대사 연구를 활성화시키고 엑기스 관련 생리와 병리에 대한 우리의 이해를 확장 가능성을 가지고있다. Microphotoacoustics 현재이 또는 - PAM 시스템을 생산합니다.

집에서 만든 음향 - 광학 빔 결합기 : <ul><li> 마우스 오른쪽 단추로 각도 프리즘 (NT32 - 545, 에드먼드 광학) </li><li> 편능형 프리즘 (NT49 - 419, 에드먼드 광학) </li><li> 실리콘 오일 (1000cSt, Clearco 제품) </li><li> 또는 - PAM 시스템 (Microphotoacoustics) </li></ul>

three-dimensional optical-resolution photoacoustic microscopy

세포와 organelle 수준에서 귀중한 통찰력을 제공하는 광학 현미경은, 널리 사용 생물 의학 기술로 인정되었습니다. 마찬가지로 생체내 (3 - D) 입체은 광학 현미경, 형광 ​​현미경 single-/multi-photon 및 광학 일관성의 tomography (OCT)는 각각 형광 및 광학 산란 대비하기 위해 특별한 감성을 입증의 mainstays. 그러나, 필수 생리 / 병리 학적 정보를 인코딩 생물 조직,의 광 흡수 대비는 아직 평가할 수있는가되지 않았습니다. 의생명 photoacoustics의 출현은 광학 방사선이 세포 1도 subcellular 2 수준 측면 해상도를 달성하기 위해 회절 한계에 초점을 맞추고 있습니다 광학 현미경 광학 해상도 photoacoustic 현미경 (OR - PAM) 1의 새로운 지점되었다. 기존의 광학 현미경 기술에 대한 가치있는 보완으로, 또는 - PAM은 적어도 두 개의 보지 못하던 상품으로 제공합니다. 가장 중요한 첫 번째 AND, OR - PAM은 뛰어난 감도 (즉, 100 %)과 광학 흡수 대조를​​ 감지합니다. 형광 현미경 또는 광학 3 - 산란 기반 10월 4일 (또는 모두) 또는 - PAM을 결합하는 것은 생물 학적 조직의 포괄적인 광학 특성을 제공합니다. 둘째, 또는 - PAM은 형광 현미경과 OCT에서 순수한 광학 프로세스와는 대조적으로, 음향 파도에 광학 흡수를 인코딩하고, 배경이없는 감지 기능을 제공합니다. OR - PAM의 음향 탐지 신호 저하에 대한 광 산란의 영향을 mitigates하고 자연스럽게 여기와 여기와 형광 스펙트럼 사이의 중복으로 인해 형광 현미경에서 일반적인 문제 탐지, 사이 가능한 interferences (즉, crosstalks)를 제거합니다. 광학 흡수 이미징에 대해 고유한, OR - PAM을 포함한 자사의 발명 이후 광범위한 생물 의학 응용 프로그램을 시연지만, 신경 5, 6, 안과 7, 8, 혈관 생물학 9, 피부과 10에 국한되지 않았습니다. 이 비디오에서는, 우리는 시스템 구성과 OR - PAM의 정렬뿐 아니라 생체내 기능 microvascular 이미징에 대한 실험 절차를 가르칩니다.

Watch this Scientific Journal Video about Three-dimensional Optical-resolution Photoacoustic Microscopy at JoVE.com

Three-dimensional Optical-resolution Photoacoustic Microscopy

광학 해상도 photoacoustic 현미경 (또는 - PAM)는 영상 광학 흡수 능력이 새로운 기술을 대조합니다<em&gt; 생체내에</em&gt; 셀룰러 해상도와 감도와 함께. 여기, 우리는 시스템 구성, 시스템 정렬을 포함 또는 - PAM의 실험 프로토콜에 시각 명령을 제공 전형<em&gt; 생체내에</em&gt; 실험 절차 및 기능 이미징 방식.

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 ...

three-dimensional-optical-resolution-photoacoustic-microscopy

Research

JoVE Journal

Bioengineering

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

난소와 Oviducts의 3 차원 조직 문화 알긴산의 Hydrogels

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

연구

생체공학

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

전도성 잉크의 평면 및 3 차원 인쇄

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

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 ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

입체 잉크젯 인쇄 기술을 사용하여 인간의 연골 조직 제작

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

세 가지 차원 콜라겐 매트릭스 내에서 세포의 이동 분석

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

준비 및 분석<em&gt; 체외</em&gt; 3 차원 유방 암 서로 게이트

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

세포 외 매트릭스 내에서 임베디드 공학 3 차원 상피 조직

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

면역 측정법에 대한 3 차원 종이 기반 미세 유체 장치의 제작

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

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 ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.