Image Scanning Microscopy Research Articles

Optical photon reassignment with increased axial resolution by structured illumination

Fluorescent microscopy methods linked to the reassignment principle as image scanning microscopy (ISM), re-scan confocal (RSC), optical photon reassignment (OPRA) and instant structured illumination microscopy (iSIM) have the potential to replace confocal microscopy as the standard microscopy technique. Photon reassignment methods are known to link the most important properties in biological imaging as resolution, sensitivity, imaging speed and combinability with fluorophores in an elegant way. On the example of OPRA, we show how this method could be easily extended to the third dimension. If OPRA is used in combination with a structured illumination pattern the sectioning ability can be improved while maintaining the very high signal intensity. We present a detailed analysis about the imaging properties of OPRA in three dimensions and show experimental results on biological samples.

Interpretation of the optical transfer function: Significance for image scanning microscopy.

The optical transfer function (OTF) is widely used to compare the performance of different optical systems. Conventionally, the OTF is normalized to unity for zero spatial frequency, but in some cases it is better to consider the unnormalized OTF, which gives the absolute value of the image signal. Examples are in confocal microscopy and image scanning microscopy, where the signal level increases with pinhole or array size. Comparison of the respective unnormalized OTFs gives useful insight into their relative performance. The significance of other properties of the general OTF is discussed.

Deconvolution approach for 3D scanning microscopy with helical phase engineering.

RESCH (refocusing after scanning using helical phase engineering) microscopy is a scanning technique using engineered point spread functions which provides volumetric information. We present a strategy for processing the collected raw data with a multi-view maximum likelihood deconvolution algorithm, which inherently comprises the resolution gain of pixel-reassignment microscopy. The method, which we term MD-RESCH (for multi-view deconvolved RESCH), achieves in our current implementation a 20% resolution advantage along all three axes compared to RESCH and confocal microscopy. Along the axial direction, the resolution is comparable to that of image scanning microscopy. However, because the method inherently reconstructs a volume from a single 2D scan, a significantly higher optical sectioning becomes directly visible to the user, which would otherwise require collecting multiple 2D scans taken at a series of axial positions. Further, we introduce the use of a single-helical detection PSF to obtain an increased post-acquisition refocusing range. We present data from numerical simulations as well as experiments to confirm the validity of our approach.

Superconcentration of light: circumventing the classical limit to achievable irradiance.

Concentration of light is limited by a fundamental physical principle, which ensures that étendue, the product of area and solid angle, can never decrease in an optical system. In microscopy, many superresolving methods, which can overcome the classical resolution limit, have recently emerged. We propose, and demonstrate experimentally, that it is also possible to circumvent the classical light concentration limit. Actually, most superresolution methods exhibit a common drawback: with respect to the total number of emitted photons, they are less efficient than standard widefield microscopy. Most methods "shave"' the point spread function (PSF) by discarding the disturbing signal from its edge. We show, that in contrast to PSF-shaving, methods related to reassignment microscopy (image scanning microscopy, optical photon reassignment, rescan confocal, instant structured illumination microscopy) concentrate all detected photons in their superresolving images and thereby increase the detected signal per sample area compared to widefield microsopy. We term this behavior superconcentration, as it breaks the classical light concentration limit.

Boost Your Microscope by Exploring New Dimensions

Due to diffraction of light, the conventional fluorescent microscope is a system with a band-limited information spatial channel. Every super-resolved fluorescent microscope circumvents this limitation by avoiding the simultaneous signaling of adjacent (< diffraction limit distance) fluorophores recording them sequentially in time. Notably, the confocal microscope also agrees to this perspective. Thus, the current super-resolved systems encode extra spatial information in the temporal channel.Here we discuss if other channels (dimensions) can be used to further improve the resolution of a fluorescence microscope. In particular, we describe time-gated STED microscopy and image-scanning microscopy (ISM) as examples of exploring extra dimensions, respectively the photon arrival time and the spatial image plane dimensions.Confocal scanning microscopy (CSM) can theoretically surpass the diffraction limit by a factor of √2. Practically, this improvement is sacrificed to obtain a good signal-to-noise ratio (SNR). Image scanning microscopy (ISM) solved this limitation by substituting the single point detector with a 2D array of detector. We showed that ISM can be straightforwardly implemented by using a quadrant detector. This implementation offers resolution close to the CSM theoretical value, improves the SNR by factor of 1.5 with respect to the CSM counterpart, and may be implemented without losing the optical sectioning capability and the system versatility.Time-gated detection increases the spatial resolution of a gated continuous-wave-(CW-) STED microscope, with the drawback of reducing the SNR. Thus, in sub-optimal conditions, such as a low-photons budget regime, the SNR reduction can cancel-out the expected gain in resolution. We developed a method which does not discard photons, but instead sorts all the photons in different time-gates according to their arrival time and recombines them through a multi-image deconvolution. Our results show that the SNR of the restored image improves, thereby improving the effective resolution.

Image scanning microscopy with a quadrant detector.

Confocal scanning microscopy (CSM) is the most widely used modern optical microscopy technique. Theoretically, it allows the diffraction barrier to be surpassed by a factor of 2, but practically this improvement is sacrificed to obtain a good signal-to-noise ratio (SNR). Image scanning microscopy (ISM) solves this limitation but, in the current implementations, the system complexity is increased and the versatility of CSM is reduced. Here we show that ISM can be straightforwardly implemented by substituting the single point detector of a confocal microscope with a quadrant detector of the same size, thus using a small number of detector elements. This implementation offers resolution close to the CSM theoretical value and improves the SNR by a factor of 1.5 with respect to the CSM counterpart without losing the optical sectioning capability and the system versatility.

Post-processing strategies in image scanning microscopy

Image scanning microscopy (ISM) coupled with pixel reassignment offers a resolution improvement of √2 over standard widefield imaging. By scanning point-wise across the specimen and capturing an image of the fluorescent signal generated at each scan position, additional information about specimen structure is recorded and the highest accessible spatial frequency is doubled. Pixel reassignment can be achieved optically in real time or computationally a posteriori and is frequently combined with the use of a physical or digital pinhole to reject out of focus light. Here, we simulate an ISM dataset using a test image and apply standard and non-standard processing methods to address problems typically encountered in computational pixel reassignment and pinholing. We demonstrate that the predicted improvement in resolution is achieved by applying standard pixel reassignment to a simulated dataset and explore the effect of realistic displacements between the reference and true excitation positions. By identifying the position of the detected fluorescence maximum using localisation software and centring the digital pinhole on this co-ordinate before scaling around translated excitation positions, we can recover signal that would otherwise be degraded by the use of a pinhole aligned to an inaccurate excitation reference. This strategy is demonstrated using experimental data from a multiphoton ISM instrument. Finally we investigate the effect that imaging through tissue has on the positions of excitation foci at depth and observe a global scaling with respect to the applied reference grid. Using simulated and experimental data we explore the impact of a globally scaled reference on the ISM image and, by pinholing around the detected maxima, recover the signal across the whole field of view.

A joint Richardson—Lucy deconvolution algorithm for the reconstruction of multifocal structured illumination microscopy data

We demonstrate the reconstruction of images obtained by multifocal structured illumination microscopy, MSIM, using a joint Richardson–Lucy, jRL-MSIM, deconvolution algorithm, which is based on an underlying widefield image-formation model. The method is efficient in the suppression of out-of-focus light and greatly improves image contrast and resolution. Furthermore, it is particularly well suited for the processing of noise corrupted data. The principle is verified on simulated as well as experimental data and a comparison of the jRL-MSIM approach with the standard reconstruction procedure, which is based on image scanning microscopy, ISM, is made. Our algorithm is efficient and freely available in a user friendly software package.

Richardson–Lucy Deconvolution as a General Tool for Combining Images with Complementary Strengths

We use Richardson-Lucy (RL) deconvolution to combine multiple images of a simulated object into a single image in the context of modern fluorescence microscopy techniques. RL deconvolution can merge images with very different point-spread functions, such as in multiview light-sheet microscopes,1, 2 while preserving the best resolution information present in each image. We show that RL deconvolution is also easily applied to merge high-resolution, high-noise images with low-resolution, low-noise images, relevant when complementing conventional microscopy with localization microscopy. We also use RL deconvolution to merge images produced by different simulated illumination patterns, relevant to structured illumination microscopy (SIM)3, 4 and image scanning microscopy (ISM). The quality of our ISM reconstructions is at least as good as reconstructions using standard inversion algorithms for ISM data, but our method follows a simpler recipe that requires no mathematical insight. Finally, we apply RL deconvolution to merge a series of ten images with varying signal and resolution levels. This combination is relevant to gated stimulated-emission depletion (STED) microscopy, and shows that merges of high-quality images are possible even in cases for which a non-iterative inversion algorithm is unknown.

Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy

We demonstrate how a conventional confocal spinning-disk (CSD) microscope can be converted into a doubly resolving image scanning microscopy (ISM) system without changing any part of its optical or mechanical elements. Making use of the intrinsic properties of a CSD microscope, we illuminate stroboscopically, generating an array of excitation foci that are moved across the sample by varying the phase between stroboscopic excitation and rotation of the spinning disk. ISM then generates an image with nearly doubled resolution. Using conventional fluorophores, we have imaged single nuclear pore complexes in the nuclear membrane and aggregates of GFP-conjugated Tau protein in three dimensions. Multicolor ISM was shown on cytoskeletal-associated structural proteins and on 3D four-color images including MitoTracker and Hoechst staining. The simple adaptation of conventional CSD equipment allows superresolution investigations of a broad variety of cell biological questions.

Superresolution by image scanning microscopy using pixel reassignment

The effect of detector array size on resolution and signal collection efficiency of image scanning microscopy based on pixel reassignment is studied. It is shown how the method can also be employed if there is a Stokes shift in fluorescence emission wavelength. With no Stokes shift, the width of the point spread function can be sharpened by a factor of 1.53, and its peak intensity increased by a factor of 1.84.

Image Scanning Microscopy

A new microscopy technique is introduced, image scanning microscopy (ISM), which combines conventional confocal-laser scanning microscopy with fast wide-field CCD detection. The technique allows for doubling the lateral optical resolution in fluorescence imaging. The physical principle behind ISM is similar to structured illumination microscopy, by combining the resolving power of confocal-laser scanning microscopy with that of a wide-field imaging microscopy. This Letter describes the theoretical foundation and experimental realization of ISM.