Structured illumination microscopy: artefact analysis and reduction utilizing a parameter optimization approach

Schaefer, Lutz H.; Schuster, Dietwald; Schaffer, J.

doi:10.1111/j.0022-2720.2004.01411.x

Cited by 126 publications

(96 citation statements)

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“…Any inaccuracy in grid movement will result in residual grid lines visible in the sectioned image. A more general approach to calculation of the optical section was proposed by Schaefer et al [12] and later also by Tkaczyk et al [13]. Their approach has the advantage of working with arbitrary phase shifts.…”

Section: Structured Illumination For Optical Sectioningmentioning

confidence: 99%

“…Furthermore, this general approach allows the use of more than three images. In this case, a least squares approach can be used to produce results with greater accuracy and improved SNR [12].…”

Section: Structured Illumination For Optical Sectioningmentioning

confidence: 99%

“…Finally, all commercial instruments use rectangular grating patterns instead of the ideal sinusoidal ones, which also is a reason for artifacts in the final image. Schaefer and colleagues modeled all 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 F o r P e e r R e v i e w these sources of potential artifacts and included a parameter-optimization approach to achieve efficient artifact removal [12] which was incorporated into the Zeiss ApoTome software.…”

Section: Structured Illumination For Optical Sectioningmentioning

confidence: 99%

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Structure brings clarity: Structured illumination microscopy in cell biology

Langhorst

Schaffer

Goetze

2009

Biotechnology Journal

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International audienceBiological samples are three dimensional and therefore optical sectioning is mandatory for microscopic images to precisely show the localization or function of structures within biological samples. Today, researchers can choose from a variety of methods to obtain optical sections. This article focuses on structured illumination microscopy, which is a group of techniques utilizing a combination of optics and mathematics to obtain optical sections: A structure is imaged onto the sample by optical means and the additional information thereby encoded in the image is used to calculate an optical section from several acquired images. Different methods of structured illumination microscopy (mainly grid projection and aperture correlation) are discussed from a practical point of view, concentrating on advantages, limitations and future prospects of these techniques and their use in cell biology. Structured illumination can also be used to obtain superresolution information if structures of higher frequency are projected onto the sample. This promising approach to superresolution microscopy is also briefly discussed from a user's perspective

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Section: Structured Illumination For Optical Sectioningmentioning

confidence: 99%

Section: Structured Illumination For Optical Sectioningmentioning

confidence: 99%

Section: Structured Illumination For Optical Sectioningmentioning

confidence: 99%

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Structure brings clarity: Structured illumination microscopy in cell biology

Langhorst

Schaffer

Goetze

2009

Biotechnology Journal

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“…7 The drawback of SIM lies in image artifacts that result from imprecise instrumentation and sample bleaching. 8,9 Moreover, to adapt a standard wide-field fluorescence microscope for SIM, an additional illumination add-on module is required.In this letter, we present a generic wide-field optical sectioning scheme, photobleaching imprinting microscopy (PIM). Compared to DM and SIM, wide-field PIM is easy to implement-it does not require knowledge of the system's point-spread-function (PSF) or require an extra illumination module.…”

mentioning

confidence: 99%

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confidence: 99%

Optical sectioning by wide-field photobleaching imprinting microscopy

Li¹,

Gao²,

Liu³

et al. 2013

Appl. Phys. Lett.

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We present a generic wide-field optical sectioning scheme, photobleaching imprinting microscopy (PIM), for depth-resolved cross-sectional fluorescence imaging. Wide-field PIM works by extracting a nonlinear component that depends on the excitation fluence as a result of photobleaching-induced fluorescence decay. Since no specific fluorescent dyes or illumination modules are required, wide-field PIM is easy to implement on a standard microscope. Moreover, wide-field PIM is superior to deconvolution microscopy in removing background fluorescence, yielding a six-fold improvement in image contrast. V C 2013 AIP Publishing LLC.[http://dx.doi.org/10.1063/1.4827535] Three-dimensional fluorescence microscopy is an indispensable tool in studying cell and tissue biology. 1 However, because of out-of-focus light, conventional wide-field fluorescence microscopy generally does not have sectioning capabilities. To achieve depth-resolved wide-field fluorescence imaging, two major strategies are commonly used. One strategy, referred to as deconvolution microscopy (DM), computationally determines how much out-of-focus light is expected for the optics in use and then seeks to redistribute this light to its points of origin in the sample. 2 However, the reduction of out-of-focus light by DM is effective only for specimens in which the ratio of background fluorescence to the in-focus signal is no greater than $20:1. 3 Additionally, the contrast improvement by DM is achieved at the expense of a decreased signal-to-noise ratio and may also introduce structural artifacts. 4 The second strategy, referred to as structured illumination microscopy (SIM), projects a grid excitation pattern onto the sample and captures three phase-shifted fluorescence images. 5,6 The sectioned image is then calculated by a demodulation algorithm. 7 The drawback of SIM lies in image artifacts that result from imprecise instrumentation and sample bleaching. 8,9 Moreover, to adapt a standard wide-field fluorescence microscope for SIM, an additional illumination add-on module is required.In this letter, we present a generic wide-field optical sectioning scheme, photobleaching imprinting microscopy (PIM). Compared to DM and SIM, wide-field PIM is easy to implement-it does not require knowledge of the system's point-spread-function (PSF) or require an extra illumination module. A depth-resolved image can be simply derived from time-lapse imaging of photobleaching-induced fluorescent decay. The operating principle of wide-field PIM is illustrated in Fig. 1. Upon one-photon excitation, the light intensity measured by a wide-field microscope is the integration of the fluorescence emitted over all depths Iðx; yÞ ¼ C ð l a ðx; y; zÞFðx; y; zÞ Ã PSF z ðx; yÞdz;where C is a constant, l a is the absorption coefficient of the fluorophore, F is the excitation fluence distribution, PSF z is the point-spread-function at depth z, and the operator Ã represents 2D convolution. In fluorescence microscopy, photobleaching occurs when the excited electrons are trapped in a relative...

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