High precision wavefront control in point spread function engineering for single emitter localization

Siemons, Marijn; Hulleman, Christiaan N.; Thorsen, Rasmus Ø.; Smith, Carlas; Stallinga, Sjoerd

doi:10.1364/oe.26.008397

Cited by 62 publications

(46 citation statements)

References 51 publications

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“…A much more challenging problem is simultaneously imaging more types of emitters, where splitting the imaging channels multiple times is more cumbersome and expensive. With the standard PSF, our initial approach showed significantly-reduced effectiveness relative to the two-color classifier, described in further detail later; therefore, we employed PSF engineering, where a wavelength-dependent aberration is added to help uniquify each color [18,30,31]. Phasemodulating elements, placed in the back focal plane of the objective, can be designed to engineer PSFs ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Multicolor localization microscopy and point-spread-function engineering by deep learning

Park

Shoman

et al. 2019

Opt. Express

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Deep learning has become an extremely effective tool for image classification and image restoration problems. Here, we apply deep learning to microscopy and demonstrate how neural networks can exploit the chromatic dependence of the point-spread function to classify the colors of single emitters imaged on a grayscale camera. While existing localization microscopy methods for spectral classification require additional optical elements in the emission path, e.g., spectral filters, prisms, or phase masks, our neural net correctly identifies static and mobile emitters with high efficiency using a standard, unmodified singlechannel configuration. Furthermore, we show how deep learning can be used to design new phase-modulating elements that, when implemented into the imaging path, result in further improved color differentiation between species, including simultaneously differentiating four species in a single image.

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Section: Resultsmentioning

confidence: 99%

Multicolor localization microscopy and point-spread-function engineering by deep learning

Park

Shoman

et al. 2019

Opt. Express

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“…An important addition to the fitting model that we make is to take into account calibrated aberrations as done previously (52) and extended further to incorporate the field-dependence of aberrations (56). We improve upon this treatment by modeling the field dependence of the aberration coefficients using the so-called Nodal Aberration Theory (NAT) instead of 2D polynomials of arbitrary order.…”

Section: Methodsmentioning

confidence: 99%

Simultaneous orientation and 3D localization microscopy with a Vortex point spread function

Hulleman

Thorsen

Stallinga

et al. 2020

Preprint

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We have developed an engineered Point Spread Function (PSF) to enable the simultaneous estimation of dipole orientation, 3D position and degree of rotational constraint of single-molecule emitters from a single 2D focal plane. Besides giving access to orientation information, the Vortex PSF along with the vectorial PSF fitter avoids localization bias common in localization microscopy for fixed dipole emitters. We demonstrate this technique on reorienting single-molecules and using binding-activated localization microscopy on DNA intercalators, corroborating perpendicular azimuthal angles to the DNA axis for in-plane emitters but find a non-uniform distribution as a function of the polar angle. The Vortex PSF is realized by an affordable glass phase mask and has a compact footprint that can easily be combined with localization microscopy techniques on rotationally constrained emitters.

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“…The pattern function g is chosen such that light is uniformly distributed over the first 9 diffraction orders (−4 th to +4 th order). The third term will introduce a defocus, but for nonzero ì σ 0 it also introduces a lateral shift [27]. The measurement is repeated using a range of 41 values of B spaced with ∆B = 90 mλ.…”

Section: Alignment Of the Spatial Light Modulatormentioning

confidence: 99%

“…The major challenge in realizing this new scanning concept lies in the area of PSF engineering, in which many approaches targeting the imaging light path, often in the context of single-molecule imaging [23][24][25][26][27][28], as well as the illumination light path, in particular light sheet based techniques [29,30], have been proposed. In the current case the single laser beam must be transformed into a set of parallel scan lines projected at different depths inside the sample.…”

Section: Introductionmentioning

confidence: 99%

Multi-line fluorescence scanning microscope for multi-focal imaging with unlimited field of view

Graaff¹,

Leenders²,

Boyaval³

et al. 2019

Biomed. Opt. Express

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Confocal scanning microscopy is the de facto standard modality for fluorescence imaging. Point scanning, however, leads to a limited throughput and makes the technique unsuitable for fast multi-focal scanning over large areas. We propose an architecture for multifocal fluorescence imaging that is scalable to large area imaging. The design is based on the concept of line scanning with continuous 'push broom' scanning. Instead of a line sensor, we use an area sensor that is tilted with respect to the optical axis to acquire image data from multiple depths inside the sample simultaneously. A multi-line illumination where the lines span a plane conjugate to the tilted sensor is created by means of a diffractive optics design, implemented on a spatial light modulator. In particular, we describe a design that uses higher order astigmatism to generate focal lines of substantially constant peak intensity along the lines. The proposed method is suitable for fast 3D image acquisition with unlimited field of view, it requires no moving components except for the sample scanning stage, and provides intrinsic alignment of the simultaneously scanned focal slices. As proof of concept, we have scanned 9 focal slices simultaneously over an area of 36 mm 2 at 0.29 µm pixel size in object space. The projected ultimate throughput that can be realized with the proposed architecture is in excess of 100 Mpixel/s.

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High precision wavefront control in point spread function engineering for single emitter localization

Cited by 62 publications

References 51 publications

Multicolor localization microscopy and point-spread-function engineering by deep learning

Multicolor localization microscopy and point-spread-function engineering by deep learning

Simultaneous orientation and 3D localization microscopy with a Vortex point spread function

Multi-line fluorescence scanning microscope for multi-focal imaging with unlimited field of view

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