DFG Advanced Microscopy Workshop Wuerzburg 2011

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Acknowledgements:Deutsche Forschungsgesellschaft Sonderforschungsbereich 937 Kollektives Verhalten von weicher und biologischer Materie Literature: 1Claus B. Mller, Jrg Enderlein, Image Scanning Microscopy, Phys. Rev. Lett., 104, 198101 (2010)

Image scanning microscopysuper-resolution three-dimensional multi-color imagingDirk Hhnel, Jrg EnderleinIII. Institute of Physics "Biophysics", Georg-August-University Gttingen, Friedrich-Hund-Platz 1, 37077 Gttingen, Germany

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3.Setup

\\JESRV\WG-Global\Seminar_Talks_Posters\Bernd Mller\2009-09-14_PicoQuant-Poster\Setup_PovRay\SIM_1.pngISM-Setup, (1) Excitation with super-continuum white light source and acousto-optic tunable filter, (2) 90/10 non-polarizing beam splitter cube, (3) major dichroic mirror, (4) piezo scan mirror, (5) 4f-telescope, (6) UPL APO 60x W microscope objective, (7) beam diagnostic camera, (8) confocal aperture, (9) EMCCD detection camera system, (10) intensity reference light path, and (11) intensity diode.19112345671082.Theory

The ISM method is a modified concept of SIM, using a CSLM, equipped with an EMCCD camera to record a whole image of the excited area. By composing these images in accordance with the scan position allows for achieving the same resolution as with SIM. The recorded information depends on two coordinates, pixel position r on the EMCCD Chip and scan position s on the sample.

By taking a image for each scanned pixel position r, moving it back by r/2 and superposing all these images, one obtains a composite image by the sample

This corresponds to taking an image with an effective point spread function given by the convolution of U with E and rescaling this convolution by the factor of two.

5.Results

Figure (1) shows the 17 x 15 scan images as recorded by different individual pixels of the EMCCD. By shifting back each of these images by an amount equal to half of the corresponding pixels position, and super-imposing all images, one instantly gains a factor of square root of two in image resolution

The image (2), shows a scan of a point-spread function bead, below aggregated fluorescent beads on a glass surface excitation wavelength was 630 nm, center emission wavelength was 670 nm. The yellow bar has a length of one micrometer.

Figure(3) shows an image of a single fluorescent bead of 100 nm diameter. Left panel: CLSM image; middle panel: ISM image; right panel: Fourier-weighted ISM image. The horizontal bar in the left panel has a length of 1 mm. The estimated resolution improvement of the cross sectioning through one of the bead images in the figure(3) is fitted as the two-dimensional intensity distributions with circular Gaussian distributions.

A typical linear cross-section along the horizontal axis with a Gaussian fit is shown in figure(4). We repeated the fit for ten different beads and found amazing homogeneity in peak width. The mean values and standard deviations for the 2s-widths of the CLSM, the ISM and the Fourier-filtered ISM images are 244 9 nm, 198 9 nm, and 150 10 nm, respectively. The total resolution enhancement was 1.63 0.08 when comparing the Fourier-filtered ISM image with the CLSM image

C:\Users\mueller\Desktop\2010-09-15_PicoQuant-Poster\ISM\BerndImage.pngC:\Users\mueller\Desktop\2010-09-15_PicoQuant-Poster\ISM\BerndPSF.pngfig.1fig.2fig.31.Introduction

Recently, we developed a new fluorescence microscopy method, termed image scanning microscopy (ISM), that enhances the spatial resolution of imaging approximately twofold. The basic principle is to combine focused laser excitation with wide-field detection camera. The physical basis of ISM is similar to structured illumination microscopy (SIM) which combines sinusoidally modulated excitation intensity distribution with wide-field imaging. By taking images at various positions and orientations of the excitation light pattern, SIM subsequently calculates an image with doubled resolution. The drawback is that the image quality becomes extremely sensitive to any optical imperfections or pattern misalignments In contrast to that, ISM can be adapted to any conventional confocal laser scanning microscope (CSLM) and is much more robust against potential aberrations and imperfections. The core challenge of the ISM system is the perfect automation of the imaging process: laser focus positioning, excitation light switching, and camera read-out have to be synchronized with nanosecond accuracy. The ISM setup acquires 3D images with four-colors. Additionally the ISM setup includes the option of excitation intensity modulation for further increasing spatial resolution by exploiting non-linear optical saturation.

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DFG Advanced Microscopy Workshop Wuerzburg 2011

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Transcript of DFG Advanced Microscopy Workshop Wuerzburg 2011