Микроскопия Лекция 2 А П Савицкий Институт биохимии

Микроскопия Лекция 2 А. П. Савицкий Институт биохимии им. А. Н. Баха РАН Московский Государственный университет им. М. В. Ломоносова

Субдифракционная микроскопия Наноскопия

Fluorescence imaging with one-nanometer accuracy (FIONA) Stepping traces of three different myosin V molecules displaying 74 -nm steps and histogram (inset) of a total of 32 myosin V's taking 231 steps.

Photoconversion green to red Spectra of green and red states of Kaede. (A) Absorption spectra of the unconverted green (green line) and converted red (red line) states. Spectra obtained during the greenred conversion are displayed as thin black lines. (B) G 380 and G 480: emission spectra of the green state (green line) on excitation at 380 and 480 nm, respectively Ando et al. PNAS, 2002 vol. 99, 12651 -12656

Photoactivated localization microscopy (PALM) sx, y=s/(N 1/2) S – standard deviation of Gaussian aproximating the true PSF, N – total number of detected photons

The principle behind PALM. A sparse subset of PA-FP molecules that are attached to proteins of interest and then fixed within a cell are activated (A and B) with a brief laser pulse at lact =405 nm and then imaged at lexc = 561 nm until most are bleached (C). This process is repeated many times (C and D) until the population of inactivated, unbleached molecules is depleted. Summing the molecular images across all frames results in a diffraction-limited image (E and F). However, if the location of each molecule is first determined by fitting the expected molecular image given by the PSF of the microscope [(G), center] to the actual molecular image [(G), left], the molecule can be plotted [(G), right] as a Gaussian that has a standard deviation equal to the uncertainty sx, y in the fitted position. Repeating with all molecules across all frames (A’ through D’) and summing the results yields a superresolution image (E’ and F’) in which resolution is dictated by the uncertainties sx, y as well as by the density of localized molecules. Scale: 1 x 1 mm in (F) and (F’), 4 x 4 mm elsewhere.

COS-7 cell expressing the lysosomal transmembrane protein CD 63

COS-7 cell expressing d. Eos. FP-tagged cytochrome-C oxidase import ssequence Comparative summed-molecule TIRF (A), PALM (B), TEM (C), and PALM/TEM overlay (D)

$Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)$

Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)

Multi-color STORM imaging microtubules (green) and clathrin-coated pits (red) in fixed BSC 1 cells

Three-dimensional STORM imaging Three-dimensional STORM image of microtubules in a cell

High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Single particle traking spt. PALM imaging of Gag and VSVG expressed in live COS 7 cells

$Super-resolution Optical Imaging Methods • Optical Diffraction Limit ~ 250 nanometers • cannot resolve$

Super-resolution Optical Imaging Methods • Optical Diffraction Limit ~ 250 nanometers • cannot resolve fluorescent-labeled molecules located within proximity to interact • Förster distance (for fluorescence resonance energy transfer) usually less than 10 to 12 nm STimulated Emission. Depletion (STED): reduces size of fluorescent “spot” to scan Photo. Activated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM): sequential excitation of fluorescent molecules, precise location determined by fitting point spread functions

Stimulated emission depletion (STED)

Stimulated emission depletion image. Revealing the nanopattern of the SNARE protein SNAP-25 on the plasma membrane of a mammalian cell. Confocal (a) vs. STED image (b) of the antibody-tagged proteins. The encircled areas show linearly deconvolved data. STED microscopy provides a substantial leap forward in the imaging of protein selfassembly; here it reveals for the first time that SNAP-25 is ordered in clusters of <60 nm average size.

as. CP 595 TRITC filter set, 30 sec, 530 -570 nm TRITC filter set, 30 sec; FITC filter set, 1 sec, 450 -480 nm Lukyanov, K. A. et. al. . (2000) J. Biol. Chem. 275, 25879– 25882

as. FP 595 Photoswitching in the Context of RESOLFT Microscopy M. Hofmann et. al Proc. Natl. Acad. Sci. U. S. A. 102, 17565 (2005).

Structured-Illumination Microscopy (SIM) • • • widefield fluorescence imaging lateral resolution between 80 -120 nm z-resolution between 250350 nm http: //www. api. com/deltavision-omx. asp

Super-resolution optical imaging using structured illumination microscopy

Comparison of four major superresolution approaches in terms of resolution in X-Y, Z and time.

- Источник света работает по специальному алгоритму - Особые требования к флуоресцентной метки, состояния on-off - Компьютерный сбор (накопление) данных и их последующая визуализация

Molecular imaging in living subjects

Nature Reviews 7: 591 -606; J. K. Willmann et al. , Molecular imaging in drug development

Pysz M. A. , Gambhir S. S. , Willmann J. K. . Clinical Radiology 65 (2010) 500 -516

Fluorescent Small animal whole body imaging AFM, atomic-force microscopy; AOH, all-optical histology; BL, bioluminescence; FL, fluorescence microscopy at visible wavelengths; MEG, magnetoencephalography; MRI, functional magnetic esonance imaging; OCT, optical coherence tomography; PET, positron emission tomography; TIR-FM, total internal reflection fluorescence microscopy; US, ultrasound. Tsien R. Nature Reviews Molecular Cell Biology, Vol. 4 (2003)pages SS 16–SS 21.

Fluorescen t Small animal whole body imaging AFM, atomic-force microscopy; AOH, all-optical histology; BL, bioluminescence; FL, fluorescence microscopy at visible wavelengths; MEG, magnetoencephalography; MRI, functional magnetic esonance imaging; OCT, optical coherence tomography; PET, positron emission tomography; TIR-FM, total internal reflection fluorescence microscopy; US, ultrasound. Tsien R. Nature Reviews Molecular Cell Biology, Vol. 4 (2003)pages SS 16–SS 21.

Клеточный и молекулярный in vivo имиджинг (Институт биохимии им. А. Н. Баха РАН, Москва) Вектор Изолированная ДНК ФБ Ген флуоресцирующего белка (ФБ) встраивают в опухолевые клетки человека Рекомбинантная молекула Опухолевая клетка опухолевая клетка трансформированная во флуоресцирующую Флуоресцирующие опухолевые клетки человека прививаются мышам линии Nu На животных – биомоделях проводится изучение опухолевого роста, метастазирования, ангиогенеза, оценка эффективности новых противоопухолевых Флуоресцентный томограф средств Трехмерная реконструкция флуоресцирующей опухоли Развитие опухоли детектируется и количественно оценивается по флуоресценции

FLIM-FRET на опухолевых моделях target DNA m. RNA fusion proteins reporter stop

FLIM-FRET опухолевых ксенографтов Hep 2 TR 23 K на мышах линии nude 1 Нер. TR 23 Kдо введения цисплатина/э топозида, 16 ый день роста 2 Нер. TR 23 K на 7 -ой день после введения цисплатина/этопозида, 23 -ий день роста 3 Нер. TR 23 K контрольная, 23 -ий день роста 4 Нер. Tag. RFP

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