이 연구는 폴란드 국립과학센터가 자금을 지원한 쏘나타 비스9 프로젝트(2019/34/E/ST5/00276)의 지원을 받았다.

Polarization-Sensitive Two-Photon Microscopy를 이용한 구형석 특성화

<ol>
	<li>Petazzi, R. A., Aji, A. K., Chiantia, S., Giraldo, J., Ciruela, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Progress in Molecular Biology and Translational Science. 169, 1-41 (2020).</li><li>Kress, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+orientational+behavior+of+MHC+class+I+protein+and+lipid+probes+in+cell+membranes+by+fluorescence+polarization-resolved+imaging.">Probing orientational behavior of MHC class I protein and lipid probes in cell membranes by fluorescence polarization-resolved imaging.</a> Biophysical Journal. 101 (2), 468-476 (2011).</li><li>Duboisset, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thioflavine-T+and+Congo+Red+reveal+the+polymorphism+of+insulin+amyloid+fibrils+when+probed+by+polarization-resolved+fluorescence+microscopy.">Thioflavine-T and Congo Red reveal the polymorphism of insulin amyloid fibrils when probed by polarization-resolved fluorescence microscopy.</a> The Journal of Physical Chemistry B. 117 (3), 784-788 (2013).</li><li>De Luca, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+ensemble+polymorphism+and+single+aggregate+structural+heterogeneity+in+insulin+amyloid+self-assembly.">Probing ensemble polymorphism and single aggregate structural heterogeneity in insulin amyloid self-assembly.</a> Journal of Colloid and Interface Science. 574, 229-240 (2020).</li><li>Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+tissue+two-photon+microscopy.">Deep tissue two-photon microscopy.</a> Nature Methods. 2 (12), 932-940 (2005).</li><li>Obstarczyk, P., Lipok, M., Grelich-Mucha, M., Samo&#263;, M., Olesiak-Ba&#324;ska, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+excited+polarization-dependent+autofluorescence+of+amyloids+as+a+label-free+method+of+fibril+organization+imaging.">Two-photon excited polarization-dependent autofluorescence of amyloids as a label-free method of fibril organization imaging.</a> The Journal of Physical Chemistry Letters. 12 (5), 1432-1437 (2021).</li><li>Obstarczyk, P., Lipok, M., &#379;ak, A., Cwynar, P., Olesiak-Ba&#324;ska, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+fibrils+in+superstructures+-+local+ordering+revealed+by+polarization+analysis+of+two-photon+excited+autofluorescence.">Amyloid fibrils in superstructures - local ordering revealed by polarization analysis of two-photon excited autofluorescence.</a> Biomaterials Science. 10 (6), 1554-1561 (2022).</li><li>Le Floc'h, V., Brasselet, S., Roch, J. -. F., Zyss, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+orientation+in+molecular+ensembles+by+polarization+sensitive+nonlinear+microscopy.">Monitoring of orientation in molecular ensembles by polarization sensitive nonlinear microscopy.</a> The Journal of Physical Chemistry B. 107 (45), 12403-12410 (2003).</li><li>Chen, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+near-infrared+two-photon+imaging+of+amyloid+plaques+in+deep+brain+of+Alzheimer's+disease+mouse+model.">In vivo near-infrared two-photon imaging of amyloid plaques in deep brain of Alzheimer's disease mouse model.</a> ACS Chemical Neuroscience. 9 (12), 3128-3136 (2018).</li><li>Gasecka, P., Balla, N. K., Sison, M., Brasselet, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipids-fluorophores+interactions+probed+by+combined+nonlinear+polarized+microscopy.">Lipids-fluorophores interactions probed by combined nonlinear polarized microscopy.</a> The Journal of Physical Chemistry B. 125 (50), 13718-13729 (2021).</li><li>Mojzisova, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarization-sensitive+two-photon+microscopy+study+of+the+organization+of+liquid-crystalline+DNA.">Polarization-sensitive two-photon microscopy study of the organization of liquid-crystalline DNA.</a> Biophysical Journal. 97 (8), 2348-2357 (2009).</li><li>Olesiak-Banska, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+crystal+phases+of+DNA:+Evaluation+of+DNA+organization+by+two-photon+fluorescence+microscopy+and+polarization+analysis.">Liquid crystal phases of DNA: Evaluation of DNA organization by two-photon fluorescence microscopy and polarization analysis.</a> Biopolymers. 95 (6), 365-375 (2011).</li><li>Olesiak-Banska, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+nanorods+as+multifunctional+probes+in+a+liquid+crystalline+DNA+matrix.">Gold nanorods as multifunctional probes in a liquid crystalline DNA matrix.</a> Nanoscale. 5 (22), 10975-10981 (2013).</li><li>Grelich-Mucha, M., Lipok, M., R&#243;&#380;ycka, M., Samo&#263;, M., Olesiak-Ba&#324;ska, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-+and+two-photon+excited+autofluorescence+of+lysozyme+amyloids.">One- and two-photon excited autofluorescence of lysozyme amyloids.</a> The Journal of Physical Chemistry Letters. 13 (21), 4673-4681 (2022).</li><li>Brasselet, S., M&#233;ly, Y., Duportail, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fluorescent Methods to Study Biological Membranes. , 311-337 (2013).</li><li>Krebs, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+formation+of+spherulites+by+amyloid+fibrils+of+bovine+insulin.">The formation of spherulites by amyloid fibrils of bovine insulin.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (40), 14420-14424 (2004).</li><li>A&#239;t-Belkacem, D., Gasecka, A., Munhoz, F., Brustlein, S., Brasselet, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+birefringence+on+polarization+resolved+nonlinear+microscopy+and+collagen+SHG+structural+imaging.">Influence of birefringence on polarization resolved nonlinear microscopy and collagen SHG structural imaging.</a> Optics Express. 18 (14), 14859-14870 (2010).</li><li>Sch&#246;n, P., Munhoz, F., Gasecka, A., Brustlein, S., Brasselet, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarization+distortion+effects+in+polarimetric+two-photon+microscopy.">Polarization distortion effects in polarimetric two-photon microscopy.</a> Optics Express. 16 (25), 20891-20901 (2008).</li><li>Svoboda, K., Yasuda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+two-photon+excitation+microscopy+and+its+applications+to+neuroscience.">Principles of two-photon excitation microscopy and its applications to neuroscience.</a> Neuron. 50 (6), 823-839 (2006).</li><li>Zipfel, W. R., Williams, R. M., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+magic:+multiphoton+microscopy+in+the+biosciences.">Nonlinear magic: multiphoton microscopy in the biosciences.</a> Nature Biotechnology. 21 (11), 1369-1377 (2003).</li><li>Ferrand, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultimate+use+of+two-photon+fluorescence+microscopy+to+map+orientational+behavior+of+fluorophores.">Ultimate use of two-photon fluorescence microscopy to map orientational behavior of fluorophores.</a> Biophysical Journal. 106 (11), 2330-2339 (2014).</li><li>Chipman, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depolarization+index+and+the+average+degree+of+polarization.">Depolarization index and the average degree of polarization.</a> Applied Optics. 44 (13), 2490-2495 (2005).</li><li>de Reguardati, S., Pahapill, J., Mikhailov, A., Stepanenko, Y., Rebane, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-accuracy+reference+standards+for+two-photon+absorption+in+the+680&amp;#x2013;1050+nm+wavelength+range.">High-accuracy reference standards for two-photon absorption in the 680&amp;#x2013;1050 nm wavelength range.</a> Optics Express. 24 (8), 9053-9066 (2016).</li><li>Mansfield, J. C., Mandalia, V., Toms, A., Winlove, C. P., Brasselet, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+reorganization+in+cartilage+under+strain+probed+by+polarization+sensitive+second+harmonic+generation+microscopy.">Collagen reorganization in cartilage under strain probed by polarization sensitive second harmonic generation microscopy.</a> Journal of The Royal Society Interface. 16 (150), 20180611 (2019).</li><li>Duboisset, J., A&#239;t-Belkacem, D., Roche, M., Rigneault, H., Brasselet, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generic+model+of+the+molecular+orientational+distribution+probed+by+polarization-resolved+second-harmonic+generation.">Generic model of the molecular orientational distribution probed by polarization-resolved second-harmonic generation.</a> Physical Review A. 85 (4), 043829 (2012).</li><li>Loksztejn, A., Dzwolak, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chiral+Bifurcation+in+aggregating+insulin:+an+induced+circular+dichroism+study.">Chiral Bifurcation in aggregating insulin: an induced circular dichroism study.</a> Journal of Molecular Biology. 379 (1), 9-16 (2008).</li></ol>

저자는 공개할 이해 상충이 없습니다.

편광에 민감한 이광자 현미경은 아밀로이드 상부구조 내부의 원섬유의 국부적 순서를 연구하는 데 유용한 도구로, 표준 다광자 설정을 약간만 수정하면 됩니다. 비선형 광학 현상에서 작동하기 때문에 단광자 여기 형광 현미경 방법에 비해 각도 광선택을 줄이고 축 분해능을 향상시킬 수 있습니다. 또한, 단일 광자 여기 형광 현미경 기술19,20과 비교할 ...

지난 수십 년 동안 단백질과 그 응집체1의 바이오이미징을 위한 수많은 형광 현미경 기술이 크게 발전했지만, 샘플 2,3 내에서 국소 순서를 확인하는 데 사용된 것은 소수에 불과합니다. 형광 수명 이미징 현미경4는 아밀로이드 상부 구조-구형석의 고유한 구조적 이질성을 연구하는 데 사용되었습니다. 또한, 구형석(spherulites)과 같은 복잡하고 조밀하게 포장된 생물구조 내부의 국소 질서에 대한 정량적 결정은 편광에 민감한 방법을 사용하여 해결할 수 있습니다3. 그러나 UV-VIS 파장을 사용하여 생체 내 형광단을 여기시키면 조직 광 산란이 높기 때문에 표면 조직 침투를 사용하는 표준 형광 기술은 제한적입니다5. 또한 이러한 이미징은 종종 특정 형광 프로브를 설계하고 표적 생체 분자에 바인딩해야 하므로 이미징을 수행하는 데 필요한 비용과 작업량이 증가합니다.
최근 이러한 문제를 해결하기 위해 우리 팀은 생물학적 구조의 무표지 이미징을 위해 편광 민감성 이광자 여기 형광 현미경(ps-2PFM)을 채택했습니다 6,7. Ps-2PFM은 여기 빔의 선형 편광 방향에 대한 이광자 형광 강도의 의존성을 측정하고 방출된 형광8의 편광을 분석할 수 있습니다. 이 기술을 구현하려면 표준 다광자 현미경 설정 여기 경로(그림 1)를 반파장 플레이트로 보완하여 광 편광면을 제어해야 합니다. 그런 다음 여기 레이저 빔의 편광에 대한 이광자 여기 형광 강도의 의존성을 나타내는 극좌표 그래프가 두 개의 애벌랜치 포토다이오드에 의해 수집된 신호에서 생성되어 형광 편광의 두 개의 상호 수직 구성 요소를 수집합니다.
마지막 단계는 데이터 분석 프로세스로, dichroic mirrors 또는 high numerical aperture objective와 같은 광학 요소가 편광에 미치는 영향을 고려합니다. 이광자 공정의 특성으로 인해 이 방법은 초점면 외부의 형광단의 이광자 여기가 확률적으로 제한되기 때문에 각도 광선택이 감소하고 축 분해능이 향상됩니다. 또한 심부 조직 이미징을 위한 근적외선 프로브(NIR)의 생체 내 이미징에 대해 유사한 방법을 성공적으로 구현할 수 있음이 입증되었습니다9. Ps-2PFM은 이전에 세포막10 및 DNA11,12의 이미지 형광단뿐만 아니라 금 나노 입자13과 같은 생물학적 시스템의 비표준 형광 마커에 적용되었습니다. 그러나, 이 모든 예에서, 생체 분자의 조직에 관한 정보는 간접적으로 얻어졌고 형광단과 생체 분자 사이의 사전 정의된 상호 배향을 필요로 했습니다.
최근 논문 중 하나에서 우리는 ps-2PFM이 구형석의 아밀로이드 피브릴에 결합된 아밀로이드 특이적 염료인 Thioflavin T의 아밀로이드 상부 구조 및 형광의 자가형광 국소 분극을 결정하는 데 성공적으로 적용될 수 있음을 보여주었습니다6. 또한, 또 다른 연구에서는 ps-2PFM을 서브미크론 크기 영역에서 아밀로이드 구형 구형 내부의 아밀로이드 피브릴 배향을 검출하는 데 사용할 수 있음을 입증했으며, 이는 투과 전자 현미경(TEM) 이미징7과 연관시켜 확인되었습니다. 이 결과의 달성은 i) 하나 또는 두 개의 광자로 여기될 때 구형석-아밀로이드의 고유 자가형광이 450에서 500nm 범위에 위치한 방출 최대값 및 표준 형광 염료14와 유사한 이광자 흡수 단면을 나타내는 고유 자가형광을 나타내고, ii) 생물학적 막 및 DNA 구조를 표지하는 염료의 ps-2PEF가 어떻게 적용될 수 있는지 설명하기 위해 이전에 소개된 수학적 모델 덕분에 가능했습니다. 구형석과 염료에 의해 표시되는 형광 8,11,15. 따라서 분석을 진행하기 전에 이 주제6에 관한 첫 번째 논문의 본문과 지원 정보 모두에 설명된 필수 이론을 살펴보는 것이 좋습니다. 여기에서는 소 인슐린 구형석의 무표지 아밀로이드 구조적 특성 분석을 위해 ps-2PFM 기법을 적용하는 방법에 대한 프로토콜을 제시합니다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sample preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips, 24 x 24 mm</td>
			<td>Chemland</td>
			<td>04-298.202.04</td>
			<td></td>
		</tr>
		<tr>
			<td>DPX mountant for histology</td>
			<td>Sigma-Aldrich</td>
			<td>6522</td>
			<td>Slide mountant</td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock tubes, 1.5 mL, polypropylene</td>
			<td>Chemland</td>
			<td>02-63102</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf ThermoMixer&#160;C</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Used for spherulite incubation</td>
		</tr>
		<tr>
			<td>HLP 5UV Water purification system</td>
			<td>Hydrolab</td>
			<td></td>
			<td>Source of dionized water used in sample preparation</td>
		</tr>
		<tr>
			<td>Hydrochloric acid (&#8805;37%, APHA &#8804;10),</td>
			<td>Sigma-Aldrich</td>
			<td>30721-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin powder from the bovine pancreas (&#8805;25 units/mg (HPLC))</td>
			<td>Sigma-Aldrich</td>
			<td>I5500</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol (HPLC grade)</td>
			<td>Sigma-Aldrich</td>
			<td>270474</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides with a concave, 76 x 26 x 1 mm</td>
			<td>Chemland</td>
			<td>04-296.202.09</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus BX60</td>
			<td>Olympus</td>
			<td></td>
			<td>Polarized Optical Microscope used in Figure 2</td>
		</tr>
		<tr>
			<td>PTFE thread seal tape, 12 mm x 12 mm x 0.1 mm, 60 gm2</td>
			<td>Chemland</td>
			<td>VIT131097</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope ps-2PFM setup</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chameleon Ultra II</td>
			<td>Coherent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FELH0800 - &#216;25.0 mm Longpass Filter</td>
			<td>Thorlabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FESH0700 - &#216;25.0 mm Shortpass Filter</td>
			<td>Thorlabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IDQ100 photon-counting avalanche photodiodes&#160;</td>
			<td>ID Quantique</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multiphoton short-pass emission filter 720 nm&#160;</td>
			<td>Semrock</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mounted Achromatic Half-Wave Plate, 690-1200 nm</td>
			<td>Thorlabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Plan Apo Oil Immersion 100x/1.4 NA</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>piezo 3D stage</td>
			<td>Piezosystem Jena</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polarizing Beamsplitter</td>
			<td>Thorlabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>S130C - Slim Photodiode Power Sensor, Si, 400 - 1100 nm, 500 mW</td>
			<td>Thorlabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LabView 2018</td>
			<td>National Instruments</td>
			<td></td>
			<td>Version 18.0.1f2</td>
		</tr>
		<tr>
			<td>Matplotlib library</td>
			<td></td>
			<td></td>
			<td>Version 3.3.2</td>
		</tr>
		<tr>
			<td>NumPy library</td>
			<td></td>
			<td></td>
			<td>Version 1.19.2</td>
		</tr>
		<tr>
			<td>SciPy library</td>
			<td></td>
			<td></td>
			<td>Version 1.5.2</td>
		</tr>
		<tr>
			<td>Spyder Python 3 IDE</td>
			<td></td>
			<td></td>
			<td>Version 4.1.5</td>
		</tr>
	</tbody>
</table>

polarization-sensitive two-photon microscopy for a label-free amyloid structural characterization

1광자에 비해 2광자 여기(excitation)는 광독성이 낮고, 조직 침투가 깊고, 조밀하게 밀집된 시스템에서 효율적으로 작동하고, 형광단의 각도 광선택이 감소하기 때문에 바이오이미징 실험에 유용합니다. 따라서 이광자 형광 현미경(2PFM)에 편광 분석을 도입하면 선형 광학 프로세스를 기반으로 하는 표준 이미징 방법에 비해 샘플의 분자 조직을 보다 정밀하게 측정할 수 있습니다. 이 연구에서는 편광에 민감한 2PFM(ps-2PFM)과 복잡한 생체 구조-아밀로이드 구형 입자 내 분자 순서 결정에 대한 응용에 중점을 둡니다. 알츠하이머병이나 파킨슨병과 같은 신경퇴행성 질환은 종종 손상된 단백질 접힘 과정으로 인해 형성된 아밀로이드 단백질 응집체의 검출을 통해 진단됩니다. 이들의 구조를 탐구하면 생성 경로를 더 잘 이해할 수 있고, 결과적으로 더 민감한 진단 방법을 개발할 수 있습니다. 이 논문은 소 인슐린 구형석 및 구형 아밀로이드 단백질 응집체 내부의 국소 피브릴 순서 측정에 적합한 ps-2PFM을 제시합니다. 또한, 우리는 제안된 기술이 구형석 내부의 피브릴의 3차원 조직을 해결할 수 있음을 증명합니다.

Over the past decades, although there has been significant development of numerous fluorescence microscopy techniques for bioimaging of proteins and their aggregates1, only a few have been used to resolve their local ordering within the sample2,3. Fluorescence lifetime imaging microscopy4 was used to study the intrinsic structural heterogeneity of amyloid superstructures-spherulites. Moreover, quantitative determination of the local ordering inside complex and densely-packed biostructures such as spherulites could be resolved using polarization-sensitive methods3. However, standard fluorescence techniques with superficial tissue penetration are limited since using UV-VIS wavelengths to excite fluorophores in vivo leads to high tissue light scattering5. In addition, such imaging often requires designing and binding specific fluorescent probes to a targeted biomolecule, thereby increasing the cost and amount of work needed to perform imaging.
Recently, to address these issues, our team has adapted polarization-sensitive two-photon excited fluorescence microscopy (ps-2PFM) for label-free imaging of biological structures6,7. Ps-2PFM allows for the measurement of the dependence of two-photon fluorescence intensity on the direction of linear polarization of the excitation beam and analysis of the polarization of the emitted fluorescence8. The implementation of this technique requires supplementation of the standard multi-photon microscope setup excitation path (Figure 1) with a half-wave plate to control the light polarization plane. Then, polar graphs, depicting the dependence of two-photon excited fluorescence intensity on the polarization of the excitation laser beam, are created from signals collected by two avalanche photodiodes, thus collecting the two, mutually perpendicular components of fluorescence polarization.
The last step is the data analysis process, taking into account the impact of the optical elements, such as dichroic mirrors or a high numerical aperture objective, on polarization. Because of the nature of the two-photon process, this method provides both reduced angular photo-selection and enhanced axial resolution, as two-photon excitation of fluorophores outside the focal plane is probabilistically limited. It was also proven that similar methods could be successfully implemented for in vivo imaging of near-infrared probes (NIR) for deep-tissue imaging9. Ps-2PFM has been previously applied to image fluorophores in cell membranes10 and DNA11,12, as well as non-standard fluorescent markers of biological systems, such as gold nanoparticles13. However, in all these examples, the information about the organization of biomolecules was obtained indirectly and required a predefined mutual orientation between a fluorophore and a biomolecule.
In one of our recent papers, we have shown that ps-2PFM could be successfully applied for determining the local polarization of the autofluorescence of amyloid superstructures and fluorescence from Thioflavin T, an amyloid-specific dye, bound to amyloid fibrils in spherulites6. Furthermore, in another one, we have proven that ps-2PFM could be utilized to detect amyloid fibril orientation inside amyloid spherulites in a sub-micron size regime, which was confirmed by correlating it with transmission electron microscopy (TEM) imaging7. The achievement of this outcome was possible thanks to i) intrinsic autofluorescence of spherulites-amyloids, when excited with either one or two photons, exhibit intrinsic autofluorescence with emission maxima located in a range from 450 to 500 nm and two-photon absorption cross sections comparable with standard fluorescent dyes14, ii) mathematical models introduced previously to describe how ps-2PEF of dyes labeling biological membranes and DNA structures could be applied to fluorescence exhibited by spherulites and dyes bound to them8,11,15. Thus, before proceeding with the analysis, we highly recommend looking through the required theory described in both, the main text and supporting information of our first paper concerning this topic6. Here, we present the protocol for how to apply the ps-2PFM technique for a label-free amyloid structural characterization of bovine insulin spherulites.

저자 스포트라이트: 편광에 민감한 이광자 현미경을 사용한 복잡한 생체 구조의 비침습적 이미징 

Label-free Amyloid Structural Characterization을 위한 편광에 민감한 2광자 현미경

Our goal is to develop new optical markers and techniques for the detection of protein aggregates called amyloids. They are involved in a range of diseases such as Alzheimer's disease and type two diabetes. So in order to cure these diseases, we need to have techniques to visualize amyloids.We demonstrated that two-photon microscopy can be used to detect amyloid fibril orientation inside amyloid superstructures. It can be achieved not only using amyloid-specific dyes, but also by utilizing the autofluorescence of amyloids. Since our technique operates on nonlinear optical phenomena, it can achieve reduced angular photoselection, enhance axial resolution, lower light scattering, lower phototoxicity, and deeper sample penetration when compared to the one-photon fluorescence microscopic technique.Polarization-sensitive two-photon fluorescence microscopy is a promising tool to image internal structure of various complex biological structures, not only protein aggregates, but also DNA vesicles and lipid membranes.

제시된 프로토콜은 ps-2PFM을 사용한 테스트를 위한 아밀로이드 상부 구조 준비, 현미경 시스템 구성 및 적절한 샘플 측정에 대한 단계별 지침을 제공합니다. 그러나 최종 측정 세트 전에 APD를 등방성 기준과 적절하게 정렬하는 것이 중요하며, 이를 통해 두 검출기에서 유사한 모양과 강도의 대칭 신호를 수집할 수 있습니다(그림 4C). X축과 Y축의 검출기에서 측정된 강도 간의 ?...

Watch this Scientific Journal Video about Non-Invasive Imaging of Complex Bio-Structures Using Polarization-Sensitive Two-Photon Microscopy at JoVE.com

이 논문은 편광에 민감한 이광자 현미경을 적용하여 표지가 없는 아밀로이드 상부구조-구형석 내의 국소 조직을 특성화하는 방법을 설명합니다. 또한 샘플을 준비 및 측정하고, 필요한 설정을 조립하고, 데이터를 분석하여 아밀로이드 섬유의 국소 조직에 대한 정보를 얻는 방법을 설명합니다.

Compared to its one-photon counterpart, two-photon excitation is beneficial for bioimaging experiments because of its lower phototoxicity, deeper tissue ...

우리의 목표는 아밀로이드라고 하는 단백질 응집체의 검출을 위한 새로운 광학 마커와 기술을 개발하는 것입니다. 그들은 알츠하이머병 및 제2형 당뇨병과 같은 다양한 질병에 관여합니다. 따라서 이러한 질병을 치료하기 위해서는 아밀로이드를 시각화하는 기술이 필요합니다.우리는 이광자 현미경을 사용하여 아밀로이드 상부 구조 내부의 아밀로이드 피브릴 방향을 검출할 수 있음을 입증했습니다. 아밀로이드 특이적 염료를 사용할 뿐만 아니라 아밀로이드의 자가형광을 이용해서도 달성할 수 있습니다. 우리의 기술은 비선형 광학 현상에서 작동하기 때문에 단광자 형광 현미경 기술과 비교할 때 각도 광선택을 줄이고 축 분해능을 향상시키며 광 산란을 낮추고 광독성을 낮추고 더 깊은 샘플 침투를 달성할 수 있습니다.편광에 민감한 이광자 형광 현미경은 단백질 응집체뿐만 아니라 DNA 소포 및 지질막과 같은 다양한 복잡한 생물학적 구조의 내부 구조를 이미지화하는 데 유망한 도구입니다.

polarization-sensitive-two-photon-microscopy-for-label-free-amyloid

Research

JoVE Journal

Chemistry

Polarization-Sensitive Two-Photon Microscopy for a Label-Free Amyloid Structural Characterization

665 조회수.  Wrocław University of Science and Technology. 우리의 목표는 아밀로이드라고 하는 단백질 응집체의 검출을 위한 새로운 광학 마커와 기술을 개발하는 것입니다. 그들은 알츠하이머병 및 제2형 당뇨병과 같은 다양한 질병에 관여합니다. 따라서 이러한 질병을 치료하기 위해서는 아밀로이드를 시각화하는 기술이 필요합니다.우리는 이광자 현미경을 사용하여 아밀로이드 상부 구조 내부의 아밀로이드 피브릴 방향을 ...

비디오: 저자 스포트라이트: 편광에 민감한 이광자 현미경을 사용한 복잡한 생체 구조의 비침습적 이미징 

Compared to its one-photon counterpart, two-photon excitation is beneficial for bioimaging experiments because of its lower phototoxicity, deeper tissue penetration, efficient operation in densely packed systems, and reduced angular photoselection of fluorophores. Thus, the introduction of polarization analysis in two-photon fluorescence microscopy (2PFM) provides a more precise determination of molecular organization in a sample compared to standard imaging methods based on linear optical processes. In this work, we focus on polarization-sensitive 2PFM (ps-2PFM) and its application in the determination of molecular ordering within complex bio-structures-amyloid spherulites. Neurodegenerative diseases such as Alzheimer's or Parkinson's are often diagnosed through the detection of amyloids-protein aggregates formed due to an impaired protein misfolding process. Exploring their structure leads to a better understanding of their creation pathway and consequently, to developing more sensitive diagnostic methods. This paper presents the ps-2PFM adapted for the determination of local fibril ordering inside the bovine insulin spherulites and spherical amyloidogenic protein aggregates. Moreover, we prove that the proposed technique can resolve the three-dimensional organization of fibrils inside the spherulite.

This paper describes how polarization-sensitive two-photon microscopy could be applied to characterize the local organization within label-free amyloid superstructures-spherulites. It also describes how to prepare and measure the sample, assemble the required setup, and analyze the data to obtain information about the local organization of amyloid fibrils.

연구

화학

Preparing Microscope Slide with Fully-Grown Amyloid Spherulite

To begin, thoroughly wash the microscope glass slide with water. Then, after dipping the slides in methanol, allow them to dry under ambient conditions on a dust-free wipe. Next, using an automatic pipette, transfer 100 microliters of the insulin spherulite solution to the well in the center area of the microscope slide.Carefully cover the solution with a cover slip, avoiding air bubble formation. Then, using an automatic pipette, deposit the mountant along the edges of the cover slip on the slide to seal the native spherulite solution. Leave the sample under ambient conditions for the mountant to harden.Using a polarized optical microscope with crossed polarizers, confirm if insulin spherulites have formed correctly. Scan through the sample, looking for a characteristic pattern of bright areas, known as a Maltese cross. The polarized optical microscopy revealed the presence of the Maltese cross in the spherulites.

완전히 성장한 아밀로이드 구형암으로 현미경 슬라이드 준비

Polarization-Sensitive Two-Photon Fluorescence Microcopy of Bovine Insulin Spherulites

After building and aligning the polarization-sensitive two-photon microscope system, according to the displayed schematic representation, test the optics alignment quality with an isotropic reference sample like fluorescein embodied in an amorphous polymer. To do so, switch on the excitation laser and measure the 360 degree polar graphs of the reference sample for a zero half-wave plate. Adjust the half-wave plate angle to correspond to the X and Y axes of the sample plane, and check if both photodiodes collect similar intensities of two photon-excited emission components.To analyze the sample, mount the glass slide with this spherulite specimen on the Piezoelectric Stage, ensuring the face of the slide containing the cover slip faces the high numerical aperture immersion objective. Then secure the glass slide with tape. Next, by adjusting the X, Y, and Z positions using the microscope knobs, focus the objective on the spherulite in the solution.Focus specifically on the central area of the entire structure, as spherulite diameters are usually in the range of tens of microns. Then center this spherulite in the field of view in the microscope plane. Determine the size of the XY scan by adjusting the number of Piezo Stage steps in X and Y directions to cover the entire structure's area.Adjust the Piezo Stage parameters such as scanning speed, step size, and range to cover the area of the whole spherulite. Open the shutter, turn on the photodiodes, and collect the X and Y emission components of two-photon excited fluorescence intensity for every single step of the selected scanning area for the excitation beam polarized corresponding to the X and then the Y axes. Then scan this spherulite sample, resulting in two-photon excited fluorescence.To perform full polarization analysis from the specific spot on the sample, turn off the excitation beam. Take specific coordinates of Piezo Stage corresponding to the chosen location on this spherulite where the information about the orientation of molecules is required with the submicron resolution. Adjust the Piezoelectric Stage to center the field of view at indicated X and Y coordinates.After turning on the excitation beam, turn on the 180 degree rotation of the half-wave plate to perform a full 360 degree polarization analysis of the X and Y emission components of the two-photon excited fluorescence emission. The image of the insulin spherulite obtained after performing a 2PFM raster scan was consistent with those reported for a spherulite. The position of the half-wave plate corresponding to the excitation of the sample along the X and Y axes was also verified.The polar graphs changed while performing a full polarization analysis from different selected spots. Outside the spherulite, the PLO graph looked like a collection of artifacts and random signal noise spikes. Properly measured polygraphs from highly ordered locations along the X and Y axes had different shapes and geometries depending on the local orientation and organization of fluorophores.