Image scanning microscopy

1. Acknowledgements:
Deutsche Forschungsgesellschaft Sonderforschungsbereich 937 “Kollektives Verhalten von weicher und biologischer Materie”
Literature:
1Claus B. Müller, Jörg Enderlein, “Image Scanning Microscopy”, Phys. Rev. Lett., 104, 198101 (2010)
Image scanning microscopy
“super-resolution three-dimensional multi-color imaging”
Dirk Hähnel, Jörg Enderlein
III. Institute of Physics "Biophysics", Georg-August-University Göttingen, Friedrich-Hund-Platz 1, 37077 Göttingen, Germany
I (r ,s)=∫dr' U (r−r'+ s)⏟
PSF
E(r' s)⏟
EID
c(r')⏟
FDS
I eff (S)=∫dr∫dr ' U (
r
2
−r '+ s)
⏟
PSF
E(r '−s+
r
2
)
⏟
EID
c(r ' )⏟
FDS
3.Setup
ISM-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.
1 9
11
2
3
4
5
6
7
10
8
2.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 pixel’s 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
wave-length 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 µm. 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 2σ-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
fig.1
fig.2
fig.3
1.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 misalignment’s 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
.