Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf ·...
Transcript of Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf ·...
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Microscopy lecture 14. June 2019
Structured Illumination Microscopy (plus a little bit of 4Pi…)
Dr. Kai Wicker Team Head – Imaging and Smart Sensor Systems Carl Zeiss AG, Corporate Research and Technology
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Revision: - McCutchen Aperture - Incoherent wide-field OTF Increasing the NA: - 4Pi Microscopy Beyond the Abbe-limit: Structured Illumination Microscopy (SIM) - Basic setup - PSF and OTF - SIM image formation and reconstruction
Today:
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Quick revision: the incoherent wide-field OTF (and the missing cone)
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Inverse
Fourier
Transform
Fourier
Transform
Inverse
Fourier
Transform
Fourier
Transform
Absolute square Convolution (Auto-correlation)
PSF
APSF
OTF
ATF
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Optical Transfer Function (OTF): For incoherent microscopy techniques, e.g. fluorescence microscopy
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Missing cone
Optical Transfer Function (OTF): For incoherent microscopy techniques, e.g. fluorescence microscopy
Lateral support
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Missing cone
Optical Transfer Function (OTF): For incoherent microscopy techniques, e.g. fluorescence microscopy
Lateral support
Axial support
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Missing cone
Optical Transfer Function (OTF): For incoherent microscopy techniques, e.g. fluorescence microscopy
Lateral support
Axial support
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The missing cone and optical sectioning:
Missing cone
Lateral support
Axial support
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The missing cone and optical sectioning:
Missing cone
Lateral support
Axial support
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The missing cone and optical sectioning:
Missing cone
Lateral support
Axial support
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Enlarging the NA:
4Pi Microscopy
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Aperture increase: 4 Pi Microscope (Type C)
Fluorescence Intensity
z
z
Stefan W. Hell Max Planck Institute of Biophysical Chemistry
Göttingen, Germany
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Aperture increase: 4 Pi Microscope (Type C)
Fluorescence Intensity
z
z
Stefan W. Hell Max Planck Institute of Biophysical Chemistry
Göttingen, Germany
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Aperture increase: 4 Pi Microscope (Type C)
Fluorescence Intensity
z
z
Stefan W. Hell Max Planck Institute of Biophysical Chemistry
Göttingen, Germany
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Aperture increase: 4 Pi Microscope (Type C)
Fluorescence Intensity
z
z
Stefan W. Hell Max Planck Institute of Biophysical Chemistry
Göttingen, Germany
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Aperture increase: 4 Pi Microscope (Type C)
Sample between Coverslips
Fluorescence Intensity
z
z
Stefan W. Hell Max Planck Institute of Biophysical Chemistry
Göttingen, Germany
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Aperture increase: 4 Pi Microscope (Type C)
Sample between Coverslips
Fluorescence Intensity
z
z
Stefan W. Hell Max Planck Institute of Biophysical Chemistry
Göttingen, Germany
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Aperture increase: 4 Pi Microscope (Type C)
Sample between Coverslips
Illumination Emission
Fluorescence Intensity
z
z
Stefan W. Hell Max Planck Institute of Biophysical Chemistry
Göttingen, Germany
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Aperture increase: 4 Pi Microscope (Type C)
Sample between Coverslips
Illumination Emission
Fluorescence Intensity
z
z
Stefan W. Hell Max Planck Institute of Biophysical Chemistry
Göttingen, Germany
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Aperture increase: 4 Pi Microscope (Type C)
Sample between Coverslips
Illumination Emission
Fluorescence Intensity
z
z
Stefan W. Hell Max Planck Institute of Biophysical Chemistry
Göttingen, Germany
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Aperture increase: 4 Pi Microscope (Type C)
Sample between Coverslips
Illumination Emission
Fluorescence Intensity
z
z
Stefan W. Hell Max Planck Institute of Biophysical Chemistry
Göttingen, Germany
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Aperture increase: 4 Pi Microscope (Type C)
Sample between Coverslips
Illumination Emission
Fluorescence Intensity
z
z
Stefan W. Hell Max Planck Institute of Biophysical Chemistry
Göttingen, Germany
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Aperture increase: 4 Pi Microscope (Type C)
Sample between Coverslips
Illumination Emission
Fluorescence Intensity
z
z
Dichromatic Beamsplitter
Stefan W. Hell Max Planck Institute of Biophysical Chemistry
Göttingen, Germany
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Aperture increase: 4 Pi Microscope (Type C)
Sample between Coverslips
Illumination Emission
Detector Pinhole
Fluorescence Intensity
z
z
Dichromatic Beamsplitter
Stefan W. Hell Max Planck Institute of Biophysical Chemistry
Göttingen, Germany
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Aperture increase: 4 Pi Microscope (Type C)
Sample between Coverslips
Illumination Emission
Detector Pinhole
High Sidelobes
Fluorescence Intensity
z
z
Dichromatic Beamsplitter
Stefan W. Hell Max Planck Institute of Biophysical Chemistry
Göttingen, Germany
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Aperture increase: 4 Pi Microscope (Type C)
Sample between Coverslips
Illumination Emission
Detector Pinhole
High Sidelobes
Fluorescence Intensity
z
z
Dichromatic Beamsplitter
Stefan W. Hell Max Planck Institute of Biophysical Chemistry
Göttingen, Germany
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Aperture increase: 4 Pi Microscope (Type C)
Sample between Coverslips
Illumination Emission
Detector Pinhole
High Sidelobes
Fluorescence Intensity
z
z
Dichromatic Beamsplitter
Stefan W. Hell Max Planck Institute of Biophysical Chemistry
Göttingen, Germany
2 Photon Effect
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Aperture increase: 4 Pi Microscope (Type C)
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ATF OTF
widefield
4Pi
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widefield, l=500nm 4Pi, l=500nm
widefield, l=1000nm 4Pi, l=1000nm
4Pi PSFs
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widefield, l=500nm 4Pi, l=500nm
widefield, l=1000nm 4Pi, l=1000nm
4Pi PSFs
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widefield, l=500nm 4Pi, l=500nm
widefield, l=1000nm 4Pi, l=1000nm 2-photon, l=1000nm 4Pi, l=1000nm, 2-photon
4Pi PSFs
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widefield, l=500nm 4Pi, l=500nm
widefield, l=1000nm 4Pi, l=1000nm
4Pi PSFs
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widefield, l=500nm 4Pi, l=500nm
widefield, l=1000nm 4Pi, l=1000nm
4Pi PSFs
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widefield, l=500nm 4Pi, l=500nm
widefield, l=1000nm 4Pi, l=1000nm 2-photon, l=1000nm 4Pi, l=1000nm, 2-photon
4Pi PSFs
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Leica 4Pi
http://www.leica-microsystems.com
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4Pi images Deviding Escherichia Coli
From: Bahlmann, K., S. Jakob, and S. W. Hell (2001). Ultramicr. 87: 155-164.
Image: Joerg Bewersdorf, Max-Planck-Institute for Biophysical Chemistry, Goettingen, Germany
Confocal
4Pi
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Beyond the Abbe limit:
Structured Illumination Microscopy (SIM)
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Real space Fourier space
Sample Sample frequencies
Structured Illumination Microscopy How it works
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Real space Fourier space
Sample Sample frequencies
Structured Illumination Microscopy How it works
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The moiré effect
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The moiré effect
moiré patterns
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high frequency
detail
Structured Illumination Microscopy How it works
The Moiré effect
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high frequency
detail
high frequency
grid
Structured Illumination Microscopy How it works
The Moiré effect
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high frequency
detail
high frequency
grid
low frequency
Moiré fringes
Structured Illumination Microscopy How it works
The Moiré effect
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The Moiré effect
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Sample
Illumination
The Moiré effect
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Real space Real space
Sample Illumination
Structured Illumination Microscopy How it works
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Real space Real space
Sample Illumination
Structured Illumination Microscopy How it works
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Real space Real space
Sample Illumination ∙
Structured Illumination Microscopy How it works
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Real space Real space
Sample Illumination ∙
Emission pattern
Structured Illumination Microscopy How it works
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Sample ∙ Illumination
Real space Fourier space
„Convolution theorem“
Structured Illumination Microscopy How it works
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Sample ∙ Illumination
Real space Fourier space
„Convolution theorem“
Structured Illumination Microscopy How it works
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Sample ∙ Illumination
Real space Fourier space
FT
„Convolution theorem“
Multiplication
Structured Illumination Microscopy How it works
![Page 56: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/56.jpg)
Sample ∙ Illumination
Real space Fourier space
FT
„Convolution theorem“
Multiplication
Structured Illumination Microscopy How it works
![Page 57: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/57.jpg)
Sample ∙ Illumination
Real space Fourier space
FT
„Convolution theorem“
Multiplication
Sample Illumination
Convolution
Structured Illumination Microscopy How it works
![Page 58: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/58.jpg)
Structured Illumination Microscopy How it works
Fourier space Fourier space
Sample Illumination
Convolution: Take the sample as a brush to „paint“ the illumination.
![Page 59: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/59.jpg)
Structured Illumination Microscopy How it works
Fourier space Fourier space
Sample Illumination
Convolution: Take the sample as a brush to „paint“ the illumination.
![Page 60: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/60.jpg)
Structured Illumination Microscopy How it works
Fourier space Fourier space
Sample Illumination
Convolution: Take the sample as a brush to „paint“ the illumination.
![Page 61: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/61.jpg)
Structured Illumination Microscopy How it works
Fourier space Fourier space
Sample Illumination
Convolution: Take the sample as a brush to „paint“ the illumination.
![Page 62: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/62.jpg)
Structured Illumination Microscopy How it works
Fourier space Fourier space
Sample Illumination
Convolution: Take the sample as a brush to „paint“ the illumination.
![Page 63: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/63.jpg)
Real space Fourier space
Emission pattern Emission pattern frequencies
Structured Illumination Microscopy How it works
![Page 64: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/64.jpg)
Real space Fourier space
Emission pattern Emission pattern frequencies
Structured Illumination Microscopy How it works
![Page 65: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/65.jpg)
Real space Fourier space
Emission pattern Emission pattern frequencies SIM raw image
Structured Illumination Microscopy How it works
![Page 66: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/66.jpg)
Real space Fourier space
Emission pattern Emission pattern frequencies SIM raw image SIM raw image frequencies
Structured Illumination Microscopy How it works
![Page 67: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/67.jpg)
Real space Fourier space
SIM raw image SIM raw image frequencies
enhanced contrast
Moiré fringes
Structured Illumination Microscopy How it works
![Page 68: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/68.jpg)
Structured Illumination Microscopy How it works
Real space Real space
Phase stepping of illumination pattern provides information for linear unmixing of components.
ZOOM
![Page 69: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/69.jpg)
Structured Illumination Microscopy How it works
Real space Real space
Phase stepping of illumination pattern provides information for linear unmixing of components.
ZOOM
![Page 70: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/70.jpg)
Structured Illumination Microscopy How it works
Real space Real space
Phase stepping of illumination pattern provides information for linear unmixing of components.
ZOOM
![Page 71: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/71.jpg)
Structured Illumination Microscopy How it works
Real space Real space
Phase stepping of illumination pattern provides information for linear unmixing of components.
ZOOM
![Page 72: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/72.jpg)
Structured Illumination Microscopy How it works
Real space Real space
Phase stepping of illumination pattern provides information for linear unmixing of components.
ZOOM
![Page 73: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/73.jpg)
Structured Illumination Microscopy How it works
Real space Real space
Phase stepping of illumination pattern provides information for linear unmixing of components.
ZOOM
![Page 74: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/74.jpg)
Structured Illumination Microscopy How it works
Real space Real space
Phase stepping of illumination pattern provides information for linear unmixing of components.
ZOOM
![Page 75: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/75.jpg)
Fourier space
1. Separate the 3 components
Structured Illumination Microscopy How it works
![Page 76: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/76.jpg)
Fourier space
1. Separate the 3 components
Structured Illumination Microscopy How it works
![Page 77: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/77.jpg)
Fourier space
1. Separate the 3 components
Structured Illumination Microscopy How it works
![Page 78: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/78.jpg)
Fourier space
1. Separate the 3 components
Structured Illumination Microscopy How it works
![Page 79: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/79.jpg)
Fourier space
1. Separate the 3 components 2. Shift them to the correct frequencies
Structured Illumination Microscopy How it works
![Page 80: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/80.jpg)
Fourier space
1. Separate the 3 components 2. Shift them to the correct frequencies 3. Recombine them (weighted averaging)
Structured Illumination Microscopy How it works
![Page 81: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/81.jpg)
Fourier space
1. Separate the 3 components 2. Shift them to the correct frequencies 3. Recombine them (weighted averaging)
Structured Illumination Microscopy How it works
![Page 82: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/82.jpg)
Fourier space
1. Separate the 3 components 2. Shift them to the correct frequencies 3. Recombine them (weighted averaging)
Structured Illumination Microscopy How it works
![Page 83: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/83.jpg)
Fourier space
1. Separate the 3 components 2. Shift them to the correct frequencies 3. Recombine them (weighted averaging)
Real space
SIM image (x only)
Structured Illumination Microscopy How it works
![Page 84: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/84.jpg)
Fourier space
1. Separate the 3 components 2. Shift them to the correct frequencies 3. Recombine them (weighted averaging)
Real space
SIM image (x only)
Structured Illumination Microscopy How it works
![Page 85: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/85.jpg)
Real space Fourier space
SIM image (x only) SIM image frequencies
Structured Illumination Microscopy How it works
![Page 86: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/86.jpg)
Real space Fourier space
SIM image (x only) SIM image frequencies
Structured Illumination Microscopy How it works
![Page 87: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/87.jpg)
Real space Fourier space
SIM image (x only) SIM image frequencies Wide field image Wide field image frequencies
Structured Illumination Microscopy How it works
![Page 88: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/88.jpg)
Full-field illumination
1 focus in back focal plane
![Page 89: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/89.jpg)
Missing cone – no optical sectioning
Full-field illumination
1 focus in back focal plane
![Page 90: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/90.jpg)
2-beam structured illumination
2 foci in back focal plane
![Page 91: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/91.jpg)
Missing cone – no optical sectioning
2-beam structured illumination
2 foci in back focal plane
![Page 92: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/92.jpg)
3-beam structured illumination
3 foci in back focal plane
![Page 93: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/93.jpg)
Missing cone filled – optical sectioning
3-beam structured illumination
3 foci in back focal plane
![Page 94: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/94.jpg)
Missing cone filled – optical sectioning
3-beam structured illumination
3 foci in back focal plane better
z-resolution
![Page 95: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/95.jpg)
SIM-Setup
![Page 96: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/96.jpg)
SIM-Setup
![Page 97: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/97.jpg)
SIM-Setup
![Page 98: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/98.jpg)
Image formation in SIM
![Page 99: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/99.jpg)
Raw image: 𝐸 𝒓 = {𝐼 𝑆} ⊗ ℎ 𝒓
In real space
![Page 100: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/100.jpg)
Raw image: 𝐸 𝒓 = {𝐼 𝑆} ⊗ ℎ 𝒓
Illumination pattern: 𝐼 𝒓 = 1 + cos 𝒌𝑔𝒓 + 𝜃
grating vector grating position
In real space
![Page 101: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/101.jpg)
Raw image: 𝐸 𝒓 = {𝐼 𝑆} ⊗ ℎ 𝒓
Illumination pattern: 𝐼 𝒓 = 1 + cos 𝒌𝑔𝒓 + 𝜃
grating vector grating position
nth illumination pattern: 𝐼𝑛 𝒓 = 1 + cos 𝒌𝑔𝒓 + 𝜃𝒏
In real space
![Page 102: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/102.jpg)
Raw image: 𝐸 𝒓 = {𝐼 𝑆} ⊗ ℎ 𝒓
Illumination pattern: 𝐼 𝒓 = 1 + cos 𝒌𝑔𝒓 + 𝜃
grating vector grating position
nth illumination pattern: 𝐼𝑛 𝒓 = 1 + cos 𝒌𝑔𝒓 + 𝜃𝒏
= 1 +1
2exp 𝑖{𝒌𝑔𝒓 + 𝜃𝒏} +
1
2exp −𝑖{𝒌𝑔𝒓 + 𝜃𝒏}
In real space
![Page 103: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/103.jpg)
nth illumination pattern: 𝐼𝑛 𝒓 = 1 + cos 𝒌𝑔𝒓 + 𝜃𝒏
Raw image: 𝐸 𝒓 = {𝐼 𝑆} ⊗ ℎ 𝒓
= 1 +1
2exp 𝑖{𝒌𝑔𝒓 + 𝜃𝒏} +
1
2exp −𝑖{𝒌𝑔𝒓 + 𝜃𝒏} = 1 +
1
2exp 𝑖{𝒌𝑔𝒓 + 𝜃𝒏} +
1
2exp −𝑖{𝒌𝑔𝒓 + 𝜃𝒏}
Raw image: 𝐸 𝒓 = {𝐼 𝑆} ⊗ ℎ 𝒓
nth illumination pattern: 𝐼𝑛 𝒓 = 1 + cos 𝒌𝑔𝒓 + 𝜃𝒏
Illumination pattern: 𝐼 𝒓 = 1 + cos 𝒌𝑔𝒓 + 𝜃
grating vector grating position
In real space
![Page 104: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/104.jpg)
nth illumination pattern: 𝐼𝑛 𝒓 = 1 + cos 𝒌𝑔𝒓 + 𝜃𝒏
Raw image: 𝐸 𝒓 = {𝐼 𝑆} ⊗ ℎ 𝒓
= 1 +1
2exp 𝑖{𝒌𝑔𝒓 + 𝜃𝒏} +
1
2exp −𝑖{𝒌𝑔𝒓 + 𝜃𝒏} = 1 +
1
2exp 𝑖{𝒌𝑔𝒓 + 𝜃𝒏} +
1
2exp −𝑖{𝒌𝑔𝒓 + 𝜃𝒏}
Raw image: 𝐸 𝒓 = {𝐼 𝑆} ⊗ ℎ 𝒓
nth illumination pattern: 𝐼𝑛 𝒓 = 1 + cos 𝒌𝑔𝒓 + 𝜃𝒏
Illumination pattern: 𝐼 𝒓 = 1 + cos 𝒌𝑔𝒓 + 𝜃
grating vector grating position
In real space In Fourier space
![Page 105: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/105.jpg)
nth illumination pattern: 𝐼𝑛 𝒓 = 1 + cos 𝒌𝑔𝒓 + 𝜃𝒏
Raw image: 𝐸 𝒓 = {𝐼 𝑆} ⊗ ℎ 𝒓
= 1 +1
2exp 𝑖{𝒌𝑔𝒓 + 𝜃𝒏} +
1
2exp −𝑖{𝒌𝑔𝒓 + 𝜃𝒏} = 1 +
1
2exp 𝑖{𝒌𝑔𝒓 + 𝜃𝒏} +
1
2exp −𝑖{𝒌𝑔𝒓 + 𝜃𝒏}
Raw image: 𝐸 𝒓 = {𝐼 𝑆} ⊗ ℎ 𝒓
nth illumination pattern: 𝐼𝑛 𝒓 = 1 + cos 𝒌𝑔𝒓 + 𝜃𝒏
Illumination pattern: 𝐼 𝒓 = 1 + cos 𝒌𝑔𝒓 + 𝜃
grating vector grating position
In real space In Fourier space
𝐸 𝑛 𝒌 = 𝐼 𝑛 ⊗ 𝑆 ℎ 𝒌
𝐼 𝑛 𝒌 = 𝛿 𝒌 +1
2𝒆𝑖𝜃𝒏 𝛿 𝒌 − 𝒌𝑔 +
1
2𝒆−𝑖𝜃𝒏 𝛿 𝒌 + 𝒌𝑔
![Page 106: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/106.jpg)
In Fourier space
𝐸 𝑛 𝒌 = 𝐼 𝑛 ⊗ 𝑆 ℎ 𝒌
𝐼 𝑛 𝒌 = 𝛿 𝒌 +1
2𝒆𝑖𝜃𝒏 𝛿 𝒌 − 𝒌𝑔 +
1
2𝒆−𝑖𝜃𝒏 𝛿 𝒌 + 𝒌𝑔
![Page 107: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/107.jpg)
In Fourier space
𝐸 𝑛 𝒌 = 𝐼 𝑛 ⊗ 𝑆 ℎ 𝒌
𝐼 𝑛 𝒌 = 𝛿 𝒌 +1
2𝒆𝑖𝜃𝒏 𝛿 𝒌 − 𝒌𝑔 +
1
2𝒆−𝑖𝜃𝒏 𝛿 𝒌 + 𝒌𝑔
![Page 108: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/108.jpg)
In Fourier space
𝐸 𝑛 𝒌 = 𝐼 𝑛 ⊗ 𝑆 ℎ 𝒌
𝐼 𝑛 𝒌 = 𝛿 𝒌 +1
2𝒆𝑖𝜃𝒏 𝛿 𝒌 − 𝒌𝑔 +
1
2𝒆−𝑖𝜃𝒏 𝛿 𝒌 + 𝒌𝑔
𝐸 𝑛 𝒌 = 𝑆 𝒌 ℎ 𝒌 + 𝒆𝑖𝜃𝒏1
2𝑆 𝒌 − 𝒌𝑔 ℎ 𝒌 + 𝒆−𝑖𝜃𝒏
1
2𝑆 𝒌 + 𝒌𝑔 ℎ 𝒌
![Page 109: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/109.jpg)
In Fourier space
𝐸 𝑛 𝒌 = 𝐼 𝑛 ⊗ 𝑆 ℎ 𝒌
𝐼 𝑛 𝒌 = 𝛿 𝒌 +1
2𝒆𝑖𝜃𝒏 𝛿 𝒌 − 𝒌𝑔 +
1
2𝒆−𝑖𝜃𝒏 𝛿 𝒌 + 𝒌𝑔
𝐸 𝑛 𝒌 = 𝑆 𝒌 ℎ 𝒌 + 𝒆𝑖𝜃𝒏1
2𝑆 𝒌 − 𝒌𝑔 ℎ 𝒌 + 𝒆−𝑖𝜃𝒏
1
2𝑆 𝒌 + 𝒌𝑔 ℎ 𝒌
𝐶 0 𝒌 𝐶 1 𝒌 𝐶 −1 𝒌 Components:
Images:
![Page 110: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/110.jpg)
In Fourier space
𝐸 𝑛 𝒌 = 𝐼 𝑛 ⊗ 𝑆 ℎ 𝒌
𝐼 𝑛 𝒌 = 𝛿 𝒌 +1
2𝒆𝑖𝜃𝒏 𝛿 𝒌 − 𝒌𝑔 +
1
2𝒆−𝑖𝜃𝒏 𝛿 𝒌 + 𝒌𝑔
𝐸 𝑛 𝒌 = 𝑆 𝒌 ℎ 𝒌 + 𝒆𝑖𝜃𝒏1
2𝑆 𝒌 − 𝒌𝑔 ℎ 𝒌 + 𝒆−𝑖𝜃𝒏
1
2𝑆 𝒌 + 𝒌𝑔 ℎ 𝒌
𝐶 0 𝒌 𝐶 1 𝒌 𝐶 −1 𝒌 Components:
Images:
![Page 111: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/111.jpg)
In Fourier space
𝐸 𝑛 𝒌 = 𝐼 𝑛 ⊗ 𝑆 ℎ 𝒌
𝐼 𝑛 𝒌 = 𝛿 𝒌 +1
2𝒆𝑖𝜃𝒏 𝛿 𝒌 − 𝒌𝑔 +
1
2𝒆−𝑖𝜃𝒏 𝛿 𝒌 + 𝒌𝑔
𝐸 𝑛 𝒌 = 𝑆 𝒌 ℎ 𝒌 + 𝒆𝑖𝜃𝒏1
2𝑆 𝒌 − 𝒌𝑔 ℎ 𝒌 + 𝒆−𝑖𝜃𝒏
1
2𝑆 𝒌 + 𝒌𝑔 ℎ 𝒌
𝐶 0 𝒌 𝐶 1 𝒌 𝐶 −1 𝒌 Components:
Images:
![Page 112: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/112.jpg)
In Fourier space
𝐸 𝑛 𝒌 = 𝐼 𝑛 ⊗ 𝑆 ℎ 𝒌
𝐼 𝑛 𝒌 = 𝛿 𝒌 +1
2𝒆𝑖𝜃𝒏 𝛿 𝒌 − 𝒌𝑔 +
1
2𝒆−𝑖𝜃𝒏 𝛿 𝒌 + 𝒌𝑔
𝐸 𝑛 𝒌 = 𝑆 𝒌 ℎ 𝒌 + 𝒆𝑖𝜃𝒏1
2𝑆 𝒌 − 𝒌𝑔 ℎ 𝒌 + 𝒆−𝑖𝜃𝒏
1
2𝑆 𝒌 + 𝒌𝑔 ℎ 𝒌
𝐶 0 𝒌 𝐶 1 𝒌 𝐶 −1 𝒌 Components:
Images:
![Page 113: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/113.jpg)
In Fourier space
𝐸 𝑛 𝒌 = 𝐼 𝑛 ⊗ 𝑆 ℎ 𝒌
𝐼 𝑛 𝒌 = 𝛿 𝒌 +1
2𝒆𝑖𝜃𝒏 𝛿 𝒌 − 𝒌𝑔 +
1
2𝒆−𝑖𝜃𝒏 𝛿 𝒌 + 𝒌𝑔
𝐸 𝑛 𝒌 = 𝑆 𝒌 ℎ 𝒌 + 𝒆𝑖𝜃𝒏1
2𝑆 𝒌 − 𝒌𝑔 ℎ 𝒌 + 𝒆−𝑖𝜃𝒏
1
2𝑆 𝒌 + 𝒌𝑔 ℎ 𝒌
𝐶 0 𝒌 𝐶 1 𝒌 𝐶 −1 𝒌 Components:
Images:
![Page 114: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/114.jpg)
In Fourier space
𝐸 𝑛 𝒌 = 𝐼 𝑛 ⊗ 𝑆 ℎ 𝒌
𝐼 𝑛 𝒌 = 𝛿 𝒌 +1
2𝒆𝑖𝜃𝒏 𝛿 𝒌 − 𝒌𝑔 +
1
2𝒆−𝑖𝜃𝒏 𝛿 𝒌 + 𝒌𝑔
𝐸 𝑛 𝒌 = 𝑆 𝒌 ℎ 𝒌 + 𝒆𝑖𝜃𝒏1
2𝑆 𝒌 − 𝒌𝑔 ℎ 𝒌 + 𝒆−𝑖𝜃𝒏
1
2𝑆 𝒌 + 𝒌𝑔 ℎ 𝒌
𝐶 0 𝒌 𝐶 1 𝒌 𝐶 −1 𝒌 Components:
Images:
𝐶 0 𝒌
𝐶 1 𝒌
𝐶 −1 𝒌
![Page 115: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/115.jpg)
In Fourier space
𝐸 𝑛 𝒌 = 𝐼 𝑛 ⊗ 𝑆 ℎ 𝒌
𝐼 𝑛 𝒌 = 𝛿 𝒌 +1
2𝒆𝑖𝜃𝒏 𝛿 𝒌 − 𝒌𝑔 +
1
2𝒆−𝑖𝜃𝒏 𝛿 𝒌 + 𝒌𝑔
𝐸 𝑛 𝒌 = 𝑆 𝒌 ℎ 𝒌 + 𝒆𝑖𝜃𝒏1
2𝑆 𝒌 − 𝒌𝑔 ℎ 𝒌 + 𝒆−𝑖𝜃𝒏
1
2𝑆 𝒌 + 𝒌𝑔 ℎ 𝒌
𝐶 0 𝒌 𝐶 1 𝒌 𝐶 −1 𝒌 Components:
Images:
![Page 116: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/116.jpg)
In Fourier space
𝐸 𝑛 𝒌 = 𝐼 𝑛 ⊗ 𝑆 ℎ 𝒌
𝐼 𝑛 𝒌 = 𝛿 𝒌 +1
2𝒆𝑖𝜃𝒏 𝛿 𝒌 − 𝒌𝑔 +
1
2𝒆−𝑖𝜃𝒏 𝛿 𝒌 + 𝒌𝑔
𝐸 𝑛 𝒌 = 𝑆 𝒌 ℎ 𝒌 + 𝒆𝑖𝜃𝒏1
2𝑆 𝒌 − 𝒌𝑔 ℎ 𝒌 + 𝒆−𝑖𝜃𝒏
1
2𝑆 𝒌 + 𝒌𝑔 ℎ 𝒌
𝐶 0 𝒌 𝐶 1 𝒌 𝐶 −1 𝒌 Components:
Images:
Matrix notation:
![Page 117: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/117.jpg)
In Fourier space
𝐸 𝑛 𝒌 = 𝐼 𝑛 ⊗ 𝑆 ℎ 𝒌
𝐼 𝑛 𝒌 = 𝛿 𝒌 +1
2𝒆𝑖𝜃𝒏 𝛿 𝒌 − 𝒌𝑔 +
1
2𝒆−𝑖𝜃𝒏 𝛿 𝒌 + 𝒌𝑔
𝐸 𝑛 𝒌 = 𝑆 𝒌 ℎ 𝒌 + 𝒆𝑖𝜃𝒏1
2𝑆 𝒌 − 𝒌𝑔 ℎ 𝒌 + 𝒆−𝑖𝜃𝒏
1
2𝑆 𝒌 + 𝒌𝑔 ℎ 𝒌
𝐶 0 𝒌 𝐶 1 𝒌 𝐶 −1 𝒌 Components:
Images:
Matrix notation:
𝑬 𝒌 =
𝐸 1 𝒌
𝐸 2 𝒌
𝐸 3 𝒌
Vector of images:
![Page 118: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/118.jpg)
In Fourier space
𝐸 𝑛 𝒌 = 𝐼 𝑛 ⊗ 𝑆 ℎ 𝒌
𝐼 𝑛 𝒌 = 𝛿 𝒌 +1
2𝒆𝑖𝜃𝒏 𝛿 𝒌 − 𝒌𝑔 +
1
2𝒆−𝑖𝜃𝒏 𝛿 𝒌 + 𝒌𝑔
𝐸 𝑛 𝒌 = 𝑆 𝒌 ℎ 𝒌 + 𝒆𝑖𝜃𝒏1
2𝑆 𝒌 − 𝒌𝑔 ℎ 𝒌 + 𝒆−𝑖𝜃𝒏
1
2𝑆 𝒌 + 𝒌𝑔 ℎ 𝒌
𝐶 0 𝒌 𝐶 1 𝒌 𝐶 −1 𝒌 Components:
Images:
Matrix notation:
𝑬 𝒌 =
𝐸 1 𝒌
𝐸 2 𝒌
𝐸 3 𝒌
Vector of images: 𝑪 𝒌 =
𝐶 −1 𝒌
𝐶 0 𝒌
𝐶 1 𝒌
Vector of components:
![Page 119: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/119.jpg)
In Fourier space
𝐸 𝑛 𝒌 = 𝐼 𝑛 ⊗ 𝑆 ℎ 𝒌
𝐼 𝑛 𝒌 = 𝛿 𝒌 +1
2𝒆𝑖𝜃𝒏 𝛿 𝒌 − 𝒌𝑔 +
1
2𝒆−𝑖𝜃𝒏 𝛿 𝒌 + 𝒌𝑔
𝐸 𝑛 𝒌 = 𝑆 𝒌 ℎ 𝒌 + 𝒆𝑖𝜃𝒏1
2𝑆 𝒌 − 𝒌𝑔 ℎ 𝒌 + 𝒆−𝑖𝜃𝒏
1
2𝑆 𝒌 + 𝒌𝑔 ℎ 𝒌
𝐶 0 𝒌 𝐶 1 𝒌 𝐶 −1 𝒌 Components:
Images:
Matrix notation:
𝑬 𝒌 =
𝐸 1 𝒌
𝐸 2 𝒌
𝐸 3 𝒌
Vector of images: 𝑪 𝒌 =
𝐶 −1 𝒌
𝐶 0 𝒌
𝐶 1 𝒌
Vector of components:
𝑬 𝒌 = 𝑴 𝑪 𝒌
𝑴 =𝑒−𝑖𝜃1 1 𝑒𝑖𝜃1
𝑒−𝑖𝜃2 1 𝑒𝑖𝜃2
𝑒−𝑖𝜃3 1 𝑒𝑖𝜃3
Component mixing matrix:
![Page 120: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/120.jpg)
Image reconstruction in SIM
![Page 121: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/121.jpg)
Image reconstruction in SIM
a. Component separation
b. Component recombination
![Page 122: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/122.jpg)
𝑬 𝒌 =
𝐸 1 𝒌
𝐸 2 𝒌
𝐸 3 𝒌
Vector of images: 𝑪 𝒌 =
𝐶 −1 𝒌
𝐶 0 𝒌
𝐶 1 𝒌
Vector of components:
𝑴 =𝑒−𝑖𝜃1 1 𝑒𝑖𝜃1
𝑒−𝑖𝜃2 1 𝑒𝑖𝜃2
𝑒−𝑖𝜃3 1 𝑒𝑖𝜃3
Component mixing matrix:
𝑬 𝒌 = 𝑴 𝑪 𝒌
Component separation
![Page 123: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/123.jpg)
𝑬 𝒌 =
𝐸 1 𝒌
𝐸 2 𝒌
𝐸 3 𝒌
Vector of images: 𝑪 𝒌 =
𝐶 −1 𝒌
𝐶 0 𝒌
𝐶 1 𝒌
Vector of components:
𝑴 =𝑒−𝑖𝜃1 1 𝑒𝑖𝜃1
𝑒−𝑖𝜃2 1 𝑒𝑖𝜃2
𝑒−𝑖𝜃3 1 𝑒𝑖𝜃3
Component mixing matrix:
𝑬 𝒌 = 𝑴 𝑪 𝒌
invert equation
𝑪 𝒌 = 𝑴 −𝟏 𝑬 𝒌
Component separation
![Page 124: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/124.jpg)
𝑬 𝒌 =
𝐸 1 𝒌
𝐸 2 𝒌
𝐸 3 𝒌
Vector of images: 𝑪 𝒌 =
𝐶 −1 𝒌
𝐶 0 𝒌
𝐶 1 𝒌
Vector of components:
𝑴 =𝑒−𝑖𝜃1 1 𝑒𝑖𝜃1
𝑒−𝑖𝜃2 1 𝑒𝑖𝜃2
𝑒−𝑖𝜃3 1 𝑒𝑖𝜃3
Component mixing matrix:
𝑬 𝒌 = 𝑴 𝑪 𝒌
invert equation
𝑪 𝒌 = 𝑴 −𝟏 𝑬 𝒌
𝐶 𝑚 𝒌 = 𝑴 𝒎𝒏−𝟏
𝟑
𝒏=𝟏
𝐸 𝑛 𝒌 Extracts the components from the recorded images.
Component separation
![Page 125: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/125.jpg)
Component recombination
𝑪 𝒌 =
𝐶 −1 𝒌
𝐶 0 𝒌
𝐶 1 𝒌
=
1
2𝑆 𝒌 + 𝒌𝑔 ℎ 𝒌
𝑆 𝒌 ℎ 𝒌1
2𝑆 𝒌 − 𝒌𝑔 ℎ 𝒌
Vector of components:
![Page 126: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/126.jpg)
Component recombination
𝑪 𝒌 =
𝐶 −1 𝒌
𝐶 0 𝒌
𝐶 1 𝒌
=
1
2𝑆 𝒌 + 𝒌𝑔 ℎ 𝒌
𝑆 𝒌 ℎ 𝒌1
2𝑆 𝒌 − 𝒌𝑔 ℎ 𝒌
Vector of components:
First step: shift components back to their true frequencies.
![Page 127: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/127.jpg)
Component recombination
𝑪 𝒌 =
𝐶 −1 𝒌
𝐶 0 𝒌
𝐶 1 𝒌
=
1
2𝑆 𝒌 + 𝒌𝑔 ℎ 𝒌
𝑆 𝒌 ℎ 𝒌1
2𝑆 𝒌 − 𝒌𝑔 ℎ 𝒌
Vector of components:
First step: shift components back to their true frequencies.
𝐶 −1 𝒌 − 𝒌𝑔 =1
2𝑆 𝒌 ℎ 𝒌 − 𝒌𝑔
𝐶 0 𝒌 = 𝑆 𝒌 ℎ 𝒌
𝐶 1 𝒌 + 𝒌𝑔 =1
2𝑆 𝒌 ℎ 𝒌 + 𝒌𝑔
![Page 128: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/128.jpg)
Component recombination
𝑪 𝒌 =
𝐶 −1 𝒌
𝐶 0 𝒌
𝐶 1 𝒌
=
1
2𝑆 𝒌 + 𝒌𝑔 ℎ 𝒌
𝑆 𝒌 ℎ 𝒌1
2𝑆 𝒌 − 𝒌𝑔 ℎ 𝒌
Vector of components:
First step: shift components back to their true frequencies.
𝐶 −1 𝒌 − 𝒌𝑔 =1
2𝑆 𝒌 ℎ 𝒌 − 𝒌𝑔
𝐶 0 𝒌 = 𝑆 𝒌 ℎ 𝒌
𝐶 1 𝒌 + 𝒌𝑔 =1
2𝑆 𝒌 ℎ 𝒌 + 𝒌𝑔
![Page 129: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/129.jpg)
Component recombination
𝑪 𝒌 =
𝐶 −1 𝒌
𝐶 0 𝒌
𝐶 1 𝒌
=
1
2𝑆 𝒌 + 𝒌𝑔 ℎ 𝒌
𝑆 𝒌 ℎ 𝒌1
2𝑆 𝒌 − 𝒌𝑔 ℎ 𝒌
Vector of components:
First step: shift components back to their true frequencies.
𝐶 −1 𝒌 − 𝒌𝑔 =1
2𝑆 𝒌 ℎ 𝒌 − 𝒌𝑔
𝐶 0 𝒌 = 𝑆 𝒌 ℎ 𝒌
𝐶 1 𝒌 + 𝒌𝑔 =1
2𝑆 𝒌 ℎ 𝒌 + 𝒌𝑔
Second step: recombine components.
(e.g. simple averaging)
Final image: 𝐹 𝒌 =
1
3𝐶 −1 𝒌 − 𝒌𝑔 + 𝐶 0 𝒌 + 𝐶 1 𝒌 + 𝒌𝑔
= 𝑆 𝒌1
3
1
2ℎ 𝒌 − 𝒌𝑔 + ℎ 𝒌 +
1
2ℎ 𝒌 + 𝒌𝑔
![Page 130: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/130.jpg)
Component recombination
𝑪 𝒌 =
𝐶 −1 𝒌
𝐶 0 𝒌
𝐶 1 𝒌
=
1
2𝑆 𝒌 + 𝒌𝑔 ℎ 𝒌
𝑆 𝒌 ℎ 𝒌1
2𝑆 𝒌 − 𝒌𝑔 ℎ 𝒌
Vector of components:
First step: shift components back to their true frequencies.
𝐶 −1 𝒌 − 𝒌𝑔 =1
2𝑆 𝒌 ℎ 𝒌 − 𝒌𝑔
𝐶 0 𝒌 = 𝑆 𝒌 ℎ 𝒌
𝐶 1 𝒌 + 𝒌𝑔 =1
2𝑆 𝒌 ℎ 𝒌 + 𝒌𝑔
Second step: recombine components.
(e.g. simple averaging)
Final image: 𝐹 𝒌 =
1
3𝐶 −1 𝒌 − 𝒌𝑔 + 𝐶 0 𝒌 + 𝐶 1 𝒌 + 𝒌𝑔
= 𝑆 𝒌1
3
1
2ℎ 𝒌 − 𝒌𝑔 + ℎ 𝒌 +
1
2ℎ 𝒌 + 𝒌𝑔
Effective SIM OTF
![Page 131: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/131.jpg)
4.
Weighted averaging in Fourier space (not covered in lecture)
![Page 132: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/132.jpg)
For signal-to-noise reasons, a simple averaging of components is not ideal!
Component recombination
𝐶 −1 𝒌 − 𝒌𝑔
𝐶 0 𝒌
𝐶 1 𝒌 + 𝒌𝑔
ℎ
𝒌
𝐹 𝒌 =1
3𝐶 −1 𝒌 − 𝒌𝑔 + 𝐶 0 𝒌 + 𝐶 1 𝒌 + 𝒌𝑔
= 𝑆 𝒌1
3
1
2ℎ 𝒌 − 𝒌𝑔 + ℎ 𝒌 +
1
2ℎ 𝒌 + 𝒌𝑔
Example: For high frequencies, only one component will contribute
information, but all components will contribute to noise!
![Page 133: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/133.jpg)
Solution: Weighted averaging!
Component recombination
𝐹 𝒌 =𝑤 −1 𝒌 𝐶 −1 𝒌 − 𝒌𝑔 + 𝑤 0 𝒌 𝐶 0 𝒌 + 𝑤 1 𝒌 𝐶 1 𝒌 + 𝒌𝑔
𝑤 −1 𝒌 + 𝑤 0 𝒌 + 𝑤 1 𝒌
= 𝑆 𝒌𝑤 −1 𝒌
12 ℎ 𝒌 − 𝒌𝑔 + 𝑤 0 𝒌 ℎ 𝒌 + 𝑤 1 𝒌
12 ℎ 𝒌 + 𝒌𝑔
𝑤 −1 𝒌 + 𝑤 0 𝒌 + 𝑤 1 𝒌
![Page 134: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/134.jpg)
Solution: Weighted averaging!
Component recombination
𝐹 𝒌 =𝑤 −1 𝒌 𝐶 −1 𝒌 − 𝒌𝑔 + 𝑤 0 𝒌 𝐶 0 𝒌 + 𝑤 1 𝒌 𝐶 1 𝒌 + 𝒌𝑔
𝑤 −1 𝒌 + 𝑤 0 𝒌 + 𝑤 1 𝒌
= 𝑆 𝒌𝑤 −1 𝒌
12 ℎ 𝒌 − 𝒌𝑔 + 𝑤 0 𝒌 ℎ 𝒌 + 𝑤 1 𝒌
12 ℎ 𝒌 + 𝒌𝑔
𝑤 −1 𝒌 + 𝑤 0 𝒌 + 𝑤 1 𝒌
𝑤 −1 𝒌 =1
2ℎ 𝒌 − 𝒌𝑔
𝑤 0 𝒌 = ℎ 𝒌
𝑤 1 𝒌 =1
2ℎ 𝒌 + 𝒌𝑔
With weights:
![Page 135: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/135.jpg)
Solution: Weighted averaging!
Component recombination
𝐹 𝒌 =
12 ℎ 𝒌 − 𝒌𝑔 𝐶 −1 𝒌 − 𝒌𝑔 + ℎ 𝒌 𝐶 0 𝒌 +
12 ℎ 𝒌 + 𝒌𝑔 𝐶 1 𝒌 + 𝒌𝑔
12 ℎ 𝒌 − 𝒌𝑔 + ℎ 𝒌 +
12 ℎ 𝒌 + 𝒌𝑔
= 𝑆 𝒌
14 ℎ 2 𝒌 − 𝒌𝑔 + ℎ 2 𝒌 +
14 ℎ 2 𝒌 + 𝒌𝑔
12 ℎ 𝒌 − 𝒌𝑔 + ℎ 𝒌 +
12 ℎ 𝒌 + 𝒌𝑔
![Page 136: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/136.jpg)
Solution: Weighted averaging!
Component recombination
𝐹 𝒌 =
12 ℎ 𝒌 − 𝒌𝑔 𝐶 −1 𝒌 − 𝒌𝑔 + ℎ 𝒌 𝐶 0 𝒌 +
12 ℎ 𝒌 + 𝒌𝑔 𝐶 1 𝒌 + 𝒌𝑔
12 ℎ 𝒌 − 𝒌𝑔 + ℎ 𝒌 +
12 ℎ 𝒌 + 𝒌𝑔
= 𝑆 𝒌
14 ℎ 2 𝒌 − 𝒌𝑔 + ℎ 2 𝒌 +
14 ℎ 2 𝒌 + 𝒌𝑔
12 ℎ 𝒌 − 𝒌𝑔 + ℎ 𝒌 +
12 ℎ 𝒌 + 𝒌𝑔
Effective SIM OTF
![Page 137: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/137.jpg)
Other things to consider
a. Separate and recombine components for several pattern orientations at once.
b. Wiener filter final reconstructed Fourier image.
![Page 138: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/138.jpg)
SIM is available in the ZEISS Elyra S.1 & PS.1
ZEISS Elyra S.1 & PS.1 Example Images
![Page 139: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/139.jpg)
SIM is available in the ZEISS Elyra S.1 & PS.1
ZEISS Elyra S.1 & PS.1 Example Images
![Page 140: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/140.jpg)
Shigella sp. Actin (yellow) and Chromatin (cyan) Images by: • Volker Brinkmann,
MPI for Infection Biology • Stephan Kuppig
Carl Zeiss Microscopy
![Page 141: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/141.jpg)
Shigella sp. Actin (yellow) and Chromatin (cyan) Images by: • Volker Brinkmann,
MPI for Infection Biology • Stephan Kuppig
Carl Zeiss Microscopy
![Page 142: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/142.jpg)
Shigella sp. Actin (yellow) and Chromatin (cyan) Images by: • Volker Brinkmann,
MPI for Infection Biology • Stephan Kuppig
Carl Zeiss Microscopy
![Page 143: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/143.jpg)
Shigella sp. Actin (yellow) and Chromatin (cyan) Images by: • Volker Brinkmann,
MPI for Infection Biology • Stephan Kuppig
Carl Zeiss Microscopy
![Page 144: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/144.jpg)
Shigella sp. Actin (yellow) and Chromatin (cyan) Images by: • Volker Brinkmann,
MPI for Infection Biology • Stephan Kuppig
Carl Zeiss Microscopy
![Page 145: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/145.jpg)
Shigella sp. Actin (yellow) and Chromatin (cyan) Images by: • Volker Brinkmann,
MPI for Infection Biology • Stephan Kuppig
Carl Zeiss Microscopy
![Page 146: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/146.jpg)
Non-linear Structured Illumination
Microscopy
Images: Mats Gustafsson
![Page 147: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/147.jpg)
Structured Illumination Microscopy How it works
Actin Filaments
488nm, > 510 nm
24 lp/mm = 88% of frequency limit
Plan-Apochromat 100x/1.4 oil iris
0.5 µm
We need powerful reconstruction algorithms! Otherwise we will get reconstruction artefacts.
![Page 148: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/148.jpg)
Structured Illumination Microscopy How it works
Actin Filaments
488nm, > 510 nm
24 lp/mm = 88% of frequency limit
Plan-Apochromat 100x/1.4 oil iris
0.5 µm 0.5 µm
We need powerful reconstruction algorithms! Otherwise we will get reconstruction artefacts.
![Page 149: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/149.jpg)
0.5 µm
Structured Illumination Microscopy How it works
We need powerful reconstruction algorithms! Otherwise we will get reconstruction artefacts.
Actin Filaments
488nm, > 510 nm
24 lp/mm = 88% of frequency limit
Plan-Apochromat 100x/1.4 oil iris
0.5 µm
![Page 150: Structured Illumination Microscopy - Nanoimagingnanoimaging.de/.../07/Microscopy20190614.pdf · Microscopy lecture 14. June 2019 Structured Illumination Microscopy (plus a little](https://reader033.fdocuments.in/reader033/viewer/2022060212/607c8f3319d09e0abf3dc4bd/html5/thumbnails/150.jpg)
Thank you for your attention!