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www(npl(co(uk HighCSpeed Structured Illumination Microscopy in Thick Samples lydia(zajiczek@npl(co(uk 6 National Physical LaboratoryY .nalytical Science 'ivisionY Eampton RoadY TeddingtonY MiddlesexY TW66 °LWY UX Lydia Zajiczek b F Kevin O2Holleran A and Michael Shaw b 4 "ambridge .dvanced /maging "entreY University of "ambridgeY .natomy 7uildingY "ambridgeY "74 8'YY UX Widefield WLR-S/M OS-S/M Weighted Linear Reconstruction Polarization Rotation: θ:64°° θ:[°° λ : 5ww nm λ : z[6 nm λ : [8w nm -.zimuthal polarization maximizes contrast of illumination pattern [6] -"o-rotate polarization of illumination light with SLM pattern via Pockels cell and QWPY also accounts for rotation from SLM θ:°° L 4 Microscope 7ody L 8 Pockels "ell Quarterwave Plate fQWPB L 6 "ollimator Laser engine coupled into PM fiber Spatial Light Modulator WSLMR: Spatial Filter: L 5 L z M HighCSpeed SIM System -Located at Jourier plane of SLM -7locks zeroth order and higher harmonics of JT of SLM patternsY allows ±6st ordersY 8 orientations -Resulting interference in focal plane of microscope is sinusoidal -"an be modified to match polarization and intensity of zeroth and first orders for 8 beam S/M -Three spatial orientations of illumination gives near-isotropic resolution enhancement PatternsQ widefield WLR-S/M SR-S/M WLR-S/M 8'-S/M -Structured illumination allows for extraction of high-spatial frequency information from a sample that is outside the optical transfer function fOTJB support of a widefield microscope due to frequency mixing between the illumination pattern and normally unobservable features in the sample [4] -Reconstruction separates information contained in different passbands and shifts it back to its true location in Jourier spaceY out-of-focus light generates image artefacts not related to real structure -Out-of-focus light can be attenuated by maximizing axial extent of OTJ support [8] which is done by multiplying frequency passbands with weighting functions fsee belowB -Requires overlap of information passbands fmissing conesB which typically means limiting spatial frequency of illumination pattern and reducing absolute improvement in spatial resolution Figure: OTJ support ftopB for 4' S/M with spatial frequency of illumination pattern equal to half the incoherent cutoff frequency of the system and weighting functions fbottomB applied to passbands to attenuate out-of-focus lightO Red color is zeroth-order passbandY blue is first-order passbandO Current Issues with Structured Illumination Microscopy WSIMR S/M has emerged as one of the most important super-resolution microscopy techniques in recent yearsO /ts primary drawbacks are the number of images required to reconstruct a super-resolution image and the adverse impacts of out-of-focus lightY limiting its ability to capture dynamic processes and restricting the types of samples that can be imagedO The use of 8' S/M is one method of addressing the issue of out-of- focus light at the expense of increased light exposure and imaging timeO We present a system capable of acquiring single-plane 4' S/M images in excess of 65 Ez for brightY photostable samplesO /n additionY we describe a weighted linear reconstruction algorithm that attenuates out-of-focus light present in single-plane images of optically thick samples requiring less images per plane than 8' S/MO Summary -Main problems with S/MQ difficulty imaging dynamic processes and artefacts from out-of-focus light -Eigh-speed S/M system presented here can exceed imaging speeds of 65 Ez for suitable samples -WLR reconstruction method attenuates out-of-focus light present in optically thick samples fup to z°-]° μm in diameterB without need to image multiple planes -4' WLR-S/M reconstruction requires overlap between 0missing cones0 of 6st order passband and widefield passband fusually means sacrificing improvement in spatial resolutionB -"urrent system and reconstruction method can potentially be modified to implement 8-beam S/M for improvement in axial resolution as well References [6] XO OIEolleranY MO ShawY OptO LettOY 8] f4°64B 5[°8-5[°zO [4] MO9OLO 9ustafsson etO alOY 7iophysO %OY k5 f4°°wB 5kz]-5k]°O [8] XO OIEolleranY MO ShawY 7iomedO OptO GxpressY z f4°65B 4zw°-4zk° OTF Support 4N.=λ 8N.=λ Passband Weighting 6 ° k z k r OS-S/M WLR-S/M Widefield Left: "ut through of widefield fupper rightB and WLR-S/M flower leftB images of alpha tubulin fredB and G-cadherin fgreenB in epidermis of Drosophilia embryoO "ontrast enhanced °O°z2 for clarityO Right: Single plane S/M images of alpha-tubulin fdashed box in lower left imageB with different reconstruction techniquesO "ontrast enhanced 62 for clarityO .rtefacts present in the SR-S/M reconstructed image due to out-of-focus light have been removed in the WLR-S/M imageO The 8'-S/M image was obtained with a commercial system for comparisonO 8'- S/M should have the highest signal-to-noise ratio of all techniques presented here but this requires imaging the entire volume of the sample without any movementO This particular commerical reconstruction algorithm was not as effective in suppressing out-of-focus lightO Above: Tail region of C. elegans animal imaged via diffraction-limited widefield microscopeY conventional 4' fSRB S/MY non-linear optically sectioned fOSB S/M and weighted linear reconstruction fWLRB S/M Opposite: cross-section of above images showing reduction of out-of-focus light present in SR- and OS-S/M Bottom: reconstructed images of cluster of 'il labelled structures inside pharynx of C. elegans animal Widefield OS-S/M SR-S/M WLR-S/M A μm Acknowledgements The authors gratefully acknowledge Muna Glmi from University "ollege London for preparation of "O elegans samplesY %uan Manuel 9omez from the 7rown Lab at the 9urdon /nstituteY University of "ambridge for preparation and imaging of 'rosophilia embryos and Nicola Lawrence from the 9urdon /nstituteY University of "ambridge for helpful discussions about the OMX microscope systemO Xevin OIEolleran is supported by the /saac Newton Trust and the MR" Next 9eneration Optical Microscopy /nitiativeO This work was partially funded by the UXIs National Measurement SystemO A μm A μm θ:°° θ:[°° θ:64°°
