This work was supported by the Sheikh Zayed Institute for Pediatric Surgical Innovation (RAC Awards #30000174 and 30001489).

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The authors have nothing to disclose.

この記事では、biofunctionalizedプルシアンブルーのナノ粒子に基づくマルチモーダル、分子イメージング剤の新規クラスの合成方法を提示した。ナノ粒子に組み込まれた分子イメージングモダリティは、その補完的な特徴を、蛍光イメージングと分子MRIである。 biofunctionalizedプルシアンブルーのナノ粒子は、コア - シェル設計を有する。これらのナノ粒子の合成における重要なステップは次の?...

分子イメージングは、細胞の細胞内および分子レベルでの生物学的プロセスの非侵襲的な標的と可視化1である。分子イメージングは​​、その内因性経路およびメカニズムがリアルタイムで評価されている間、そのネイティブ微小環境のままに標本を許可します。典型的には、分子イメージングは2研究され、可視化対象、関連の生理学的プロセスを追跡する小分子、巨大分子、またはナノ粒子の形態の外因性造影剤の投与を含む。分子イメージングで検討されてきた様々な撮像モダリティは、MRI、CT、PET、SPECT、超音波、光音響、ラマン分光法、生物発光、蛍光、および生体顕微鏡3が含まれています。マルチモーダルイメージングの組み合わせは、様々な生物学的プロセス、イベント4を視覚化し、特徴付けする能力を増強する二つ以上の撮像モダリティを組み合わせたものである。 Multimoda個々の制限3を補償しながらリットルイメージングは、個々の画像化技術の強みを利用する。 マルチモーダル、分子イメージング剤の新規クラス - この記事では、biofunctionalizedプルシアンブルーナノ粒子の合成（PB NPS）のためのプロトコルを提示します。 PBのNPは、蛍光画像および分子MRIのために利用される。 PBは、面心立方ネットワーク鉄（II）、鉄（III）原 ​​子を交互からなる顔料（ 図1）である。 CN - -その三次元ネットワーク5内の電荷のバランスをとるためにカチオンを組み込ん鉄IIIリンケージ PB格子は、鉄IIの直線シアン化物配位子で構成されている。その格子に陽イオンを組み込むためにPBの能力は、別々にMRI造影のためにPBのNPへのガドリニウム及びマンガンイオンをロードすることによって利用される。 MRI造影のためのナノ粒子の設計を追求するための理論的根拠が原因である利点は、この設計は、現在のMRI造影剤と比較しています。 US FDAが承認したMRI造影剤の大部分は、本質的に常磁性であり、スピン格子緩和機構6,7,8正コントラストを提供ガドリニウムキレートである。独自に低い信号強度を提供する単一のガドリニウムキレートと比較して、ナノ粒子のPB格子内の複数のガドリニウムイオンの組み込みは、信号強度（ポジティブコントラスト）3,9が強化されています。さらに、PB格子内の複数のガドリニウムイオンの存在は、それによって、スピン - スピン緩和機構による負のコントラストを生成し、全体的なスピン密度及びその近傍に局所磁場を乱すナノ粒子の常磁性の大きさを増大させる。従ってガドリニウム含有ナノ粒子は、T 1（正）及びT 2（負の）造影剤10,11の両方として機能する。 腎機能障害を有する患者のサブセットでは、ガドリニウムベースの造影剤の投与は、腎性全身性線維症8,12、13の開発に関連している。この観察は、造影剤のような代替の常磁性イオンを使用するために調査を求めているMRI。したがって、ナノ粒子の多用途設計はPB格子内にマンガンイオンを組み込むように適合されている。ガドリニウムキレートと同様に、マンガンキレートはまた、常磁性であり、典型的にはMRI 7,14における正の信号強度を提供するために使用される。ガドリニウム含有PBのNPと同様に、マンガン含有PB NPはまた、（正の）T 1及びT 2（負の）造影剤として機能する。 蛍光イメージング機能を組み込むために、ナノ粒子「コア」は、蛍光標識された糖タンパク質アビジンからなる「生体機能性」シェルは（ 図1で被覆されている）。アビジンは、蛍光イメージングを可能にするだけでなく、特定の細胞や組織をターゲットに、ビオチン化リガンドのドッキングプラットフォームとして機能していないだけ。アビジン-ビオチン結合は、アビジンとビオチン15との間の非常に強い結合親和性によって特徴づけられる最強の既知の、非共有結合の一つです。アビジン被覆PBのNPへのビオチン化リガンドの取り付けは、PBのNPに分子標的の機能を付与する。 これらの画像診断法は、相補的な機能を有しているので、PB NPSを使用して蛍光とMRイメージングを追求するための動機はある。蛍光画像は、最も広く使用されている光学的な分子イメージング技術の一つであり、高感度1,16,17において、複数のオブジェクトを同時に可視化を可能にする。蛍光イメージングは、安全、非侵襲的様式であるが、浸透の深さおよび低い空間解像度1,3,16と関連している。一方、MRIは、高い時間ANを生成し、dの空間解像度、非侵襲的および放射線1,3,16をイオン化する必要がない。しかしMRIは低感度に苦しんでいる。したがって、蛍光画像化およびMRIは、浸透深さ、感度、および空間分解能のそれらの相補的な特徴に起因する分子イメージング技術として選択した。 この記事では、PB NPの合成と生体機能化するためのプロトコルを提示しガドリニウム含有PB NPを（GdPB）、およびマンガン含有PB NPを（MnPB）10,11。以下の方法が記載されている：1）サイズ、電荷の測定、およびナノ粒子の経時安定性、MRI緩和能の3）測定ナノ粒子の細胞毒性の2）評価、および蛍光分子のMRイメージングのためのナノ粒子の4）利用インビトロでの標的細胞の集団。これらの結果は、in vivoでのマルチモーダルの分子イメージング剤として使用するためのNPの可能性を示す。

<table border="0" cellpadding="0" cellspacing="0" width="854">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:455px;">Name of Material/ Equipment</td>
			<td style="width:115px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium hexacyanoferrate (II) trihydrate (K4Fe(CN)6 &#183; 3H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>P9387</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Manganese (II) chloride tetrahydrate (MnCl2 &#183; 4H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>221279</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gadolinium (III) nitrate hexahydrate (Gd(NO3)3 &#183; 6H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>211591</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iron (III) chloride hexahydrate (FeCl3 &#183; 6H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>236489</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-NG2 Chondroitin Sulfate Proteoglycan, Biotin Conjugate Antibody</td>
			<td>Millipore</td>
			<td>AB5320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biotinylated Anti-Human Eotaxin-3</td>
			<td>Peprotech</td>
			<td>500-P156GBT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neuro-2a Cell Line</td>
			<td>ATCC</td>
			<td>CCL-131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSG D10 Cell Line</td>
			<td>Lab stock</td>
			<td>---</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OE21 Cell Line</td>
			<td>Sigma-Aldrich</td>
			<td>96062201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SUDIPG1 Neurospheres</td>
			<td>Lab stock</td>
			<td>---</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eol-1 Cell Line</td>
			<td>Sigma-Aldrich</td>
			<td>94042252</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly(L-lysine) hydrobromide</td>
			<td>Sigma-Aldrich</td>
			<td>P1399</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aminoactinomycin D</td>
			<td>Sigma-Aldrich</td>
			<td>A9400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellTrace Calcein Red-Orange, AM</td>
			<td>Life Technologies</td>
			<td>C34851</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Avidin-Alexa Fluor 488</td>
			<td>Life Technologies</td>
			<td>A21370</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peristaltic Pump</td>
			<td>Instech</td>
			<td>P270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zetasizer Nano ZS</td>
			<td>Malvern</td>
			<td>ZEN3600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sonicator</td>
			<td>QSonica</td>
			<td>Q125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hot Plate/Magnetic Stirrer</td>
			<td>VWR</td>
			<td>97042-642</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra Clean Aluminum Foil</td>
			<td>VWR</td>
			<td>89107-732</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex Mixer</td>
			<td>VWR</td>
			<td>58816-121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.7 mL conical microcentrifuge tubes</td>
			<td>VWR</td>
			<td>87003-295</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 mL conical centrifuge tubes</td>
			<td>VWR</td>
			<td>21008-918</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tube holders</td>
			<td>VWR</td>
			<td>82024-342</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable plastic cuvettes</td>
			<td>VWR</td>
			<td>7000-590 (/586)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zetasizer capillary cell</td>
			<td>VWR</td>
			<td>DTS1070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifugal Filters, 0.2 micrometer spin column</td>
			<td>VWR</td>
			<td>82031-356</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-well cell culture tray</td>
			<td>VWR</td>
			<td>29442-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin EDTA 0.25% solution 1X</td>
			<td>JR Scientific</td>
			<td>82702</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Culture Grade PBS (1X)</td>
			<td>Life Technologies</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XTT Cell Proliferation Assay Kit</td>
			<td>Trevigen</td>
			<td>4891-025-K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T75 Flask</td>
			<td>89092-700</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td>Biowhitaker</td>
			<td>12-604Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>10437-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pen-Strep 1X</td>
			<td>Life Technologies</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluoview FV1200 Confocal Laser Scanning Microscope</td>
			<td>Olympus</td>
			<td>FV1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chambered Microscope Slides</td>
			<td>Thermo Scientific</td>
			<td>154534</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro Cover Glasses, Square, No. 1.5</td>
			<td>VWR</td>
			<td>48366-227</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope Slides</td>
			<td>VWR</td>
			<td>16004-368</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI</td>
			<td>Sigma-Aldrich</td>
			<td>R8758&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9539&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACSCalibur Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3 T Clinical MRI Magnet</td>
			<td>GE Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mL round-bottom flask</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

biofunctionalized prussian blue nanoparticles for multimodal molecular imaging applications

多峰性は、分子イメージングは​​、複数の相補的な画像化技術を使用して、細胞の細胞内および分子レベルの解像度での生物学的プロセスの可視化を可能にする。これらの造影剤は、診断と治療の両方の効果を増強インビボでの経路および機構のリアルタイム評価を容易にする。マルチモーダル、分子イメージングアプリケーションで使用するための薬剤の新規クラス - この記事では、biofunctionalizedプルシアンブルーナノ粒子の合成（PB NPS）のためのプロトコルを提示します。ナノ粒子は、蛍光イメージングおよび磁気共鳴イメージング（MRI）に組み込まれた撮像モダリティは、相補的な特徴を有している。 PB NPは、両方のT 1及びT 2強調シーケンスで、PB格子の格子間の空間内に組み込まれたガドリニウム、マンガンイオンがMRIコントラストを生成するコア-シェル設計を有する。 PBのNPは、静電用いた蛍光アビジンで被覆されている自己としてsembly、蛍光イメージングを可能にする。アビジン被覆ナノ粒子は、ナノ粒子に分子標的化能力を付与し、ビオチン化リガンドで修飾されている。ナノ粒子の安定性および毒性は、そのMRI緩和能、ならびに測定される。これらbiofunctionalized PB NPの多峰性の分子イメージング機能を蛍光イメージングおよびインビトロでの分子MRIのためにそれらを使用することによって実証される。

ワンポット合成スキームを使用して、PB NPのナノ粒子（直径78.8 nmの多分散指数（PDI）= 0.230を意味し、動的光散乱装置で計算して）、GdPB、またはMnPB（直径の164.2ナノメートル、PDI = 0.102を意味する）（直径122.4 nmの、PDI = 0.124）の平均（DLSにより測定）単分散であることを一貫して（ 図2A）を合成することができる。合成されたナノ粒子の測定ゼータ電位は、表面電荷に基づく粒子...

Watch this Scientific Journal Video about Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications at JoVE.com

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

マルチモーダル分子イメージングアプリケーションのためのBiofunctionalizedプルシアンブルーナノ粒子

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

biofunctionalized-prussian-blue-nanoparticles-for-multimodal

Research

JoVE Journal

Bioengineering

10.2K ビュー.  Children's National Medical Center. This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting. 

ビデオ: Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

生細胞における分子ローターの蛍光寿命イメージング

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

生物工学

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

のためのウイルス粒子<emインビボ&gt;</em&gt;腫瘍イメージング

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 &#181;m. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

医療画像処理アプリケーションのための超音波の広帯域光検出器

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

回生研究ハーモニックナノ粒子

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

商業顕微鏡でのライブ細胞膜上の分子拡散法への高速蛍光イメージングから

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe3O4) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

細胞標識および超常磁性酸化鉄ナノ粒子とターゲティング

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

細胞内ターゲティングアプリケーションのための固体脂質ナノ粒子（SLNs）

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Microwave-driven Synthesis of Iron Oxide Nanoparticles for Fast Detection of Atherosclerosis

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the synthesis of hydrophobic nanoparticles, our method is based on an adaptation from thermal decomposition method to microwave driven synthesis. The new methodology produces a decrease in the reaction times in comparison with traditional procedures. Moreover, the use of the microwave technology increases the reproducibility of the reactions, something important from the point of view of clinical applications. The novelty of this iron oxide nanoparticle is the attachment of Neridronate. The use of this molecule leads a bisphosphonate moiety towards the outside of the nanoparticle that provides Ca2+ binding properties in vitro and selective accumulation in vivo in the atheroma plaque. The protocol allows the synthesis and plaque detection in about 3 hr since the initial synthesis from organic precursors. Their accumulation in the atherosclerotic area in less than 1 hr provides a contrast agent particularly suitable for clinical applications.

Microwave technology enables extremely fast synthesis of iron oxide nanoparticles for atherosclerosis plaque characterization. The use of an aminobisphosphonate in the external side of the nanoparticle provides a fast accumulation in the atherosclerotic area.

アテローム性動脈硬化症の高速検出のための酸化鉄ナノ粒子のマイクロ波駆動型の合成

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the ...

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of spectroscopy techniques. Combining imaging and spectroscopy techniques into a single optical probe may provide a more complete analysis of tissue health. In this article, two dissimilar modalities are combined, high-resolution fluorescence microendoscopy imaging and diffuse reflectance spectroscopy, into a single optical probe. High-resolution fluorescence microendoscopy imaging is a technique used to visualize apical tissue micro-architecture and, although mostly a qualitative technique, has demonstrated effective real-time differentiation between neoplastic and non-neoplastic tissue. Diffuse reflectance spectroscopy is a technique which can extract tissue physiological parameters including local hemoglobin concentration, melanin concentration, and oxygen saturation. This article describes the specifications required to construct the fiber-optic probe, how to build the instrumentation, and then demonstrates the technique on in vivo human skin. This work revealed that tissue micro-architecture, specifically apical skin keratinocytes, can be co-registered with its associated physiological parameters. The instrumentation and fiber-bundle probe presented here can be optimized as either a handheld or endoscopically-compatible device for use in a variety of organ systems. Additional clinical research is needed to test the viability of this technique for different epithelial disease states.

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

The assembly and use of a multimodal microendoscope is described which can co-register superficial tissue image data with tissue physiological parameters including hemoglobin concentration, melanin concentration, and oxygen saturation. This technique can be useful for evaluating tissue structure and perfusion, and can be optimized for individual needs of the investigator.

マルチモーダルイメージングおよび非侵襲のための分光繊維束Microendoscopyプラットフォーム、<em&gt;インビボ</em&gt;組織分析

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of ...

Manufacture and Drug Delivery Applications of Silk Nanoparticles

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and biodegradability, as well its ability to protect and subsequently release its payload in response to a trigger. While silk can be formulated into various material formats, silk nanoparticles are emerging as promising drug delivery systems. Therefore, this article covers the procedures for reverse engineering silk cocoons to yield a regenerated silk solution that can be used to generate stable silk nanoparticles. These nanoparticles are subsequently characterized, drug loaded and explored as a potential anticancer drug delivery system. Briefly, silk cocoons are reverse engineered first by degumming the cocoons, followed by silk dissolution and clean up, to yield an aqueous silk solution. Next, the regenerated silk solution is subjected to nanoprecipitation to yield silk nanoparticles &#8211; a simple but powerful method that generates uniform nanoparticles. The silk nanoparticles are characterized according to their size, zeta potential, morphology and stability in aqueous media, as well as their ability to entrap a chemotherapeutic payload and kill human breast cancer cells. Overall, the described methodology yields uniform silk nanoparticles that can be readily explored for a myriad of applications, including their use as a potential nanomedicine.

Nanoparticles are emerging as promising drug delivery systems for a broad range of indications. Here, we describe a simple yet powerful method to manufacture silk nanoparticles using reverse engineered Bombyx mori silk. These silk nanoparticles can be readily loaded with a therapeutic payload and subsequently explored for drug delivery applications.