Advanced methods of fluorescence microscopybiph.kiev.ua/images/f/f4/2_Sheremet.pdf · 3D...

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Advanced methods of fluorescence microscopy Jenia Sheremet

Transcript of Advanced methods of fluorescence microscopybiph.kiev.ua/images/f/f4/2_Sheremet.pdf · 3D...

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Advanced methods of fluorescence microscopy

Jenia Sheremet

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Флуоресценция

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http://www.olympusconfocal.com/theory/

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Спектр(ы) флуоресценции

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Базовые методы: Widefield

20μm

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airydiskformation.swf

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3D Deconvolution

bigwww.epfl.ch

Point spread function

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Widefield RAW vs Deconvolved

http://www.svi.nl

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Confocal Laser Scanning Microscope (CLSM)

http://www.olympusconfocal.com/theory/

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Universal confocal/STED

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Widefield vs Confocal

http://www.andor.com

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Multiphoton

Hellen C. Ishikawa-Ankerhold, Molecules 2012

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Multiphoton z-depth

www.nikoninstruments.com

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http://www.leica-microsystems.com/superres-infographic/

Superresolution

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http://www.leica-microsystems.com/superres-infographic/

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GSDIM/PALM/STORM - стохастическиеВажно:Флуорофоры должны «мигать», т.е. одновременно не должны излучать два флуорофора, расстояние между которыми меньше 300нм.Картинка получается путем вычисления координат центра пятна.Для хорошего изображения нужно снять от 10 до 100 тысяч кадров.

Разрешение(XYZ): 20х20х50 нм

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Confocal vs STORM

www.nikon.com

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SIM - Structured illumination microscopy

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SIM – Structured Illumination Microscopy

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STED – Stimulated Emission Depletion

http://www.leica-microsystems.com/superres-infographic/

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У всех методов есть преимущества и недостатки, зная их, нужно оптимально выбирать метод исследования для конкретной цели.

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Seeing is not always believing!

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Спасибо за внимание