Expansion microscopy of zebrafish for neuroscience and

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in Fig 1 and SI Appendix Fig S1 A B and D and one of two larvae used for. A the analysis shown in SI Appendix Fig S1C were raised in Danieau s medium at. Optic tectum, the Max Planck Institute of Neurobiology These animal procedures conformed. to the institutional guidelines of the Max Planck Society and the local gov. ernment Regierung von Oberbayern Experimental protocols were approved. by Regierung von Oberbayern 55 2 1 54 2532 101 12 and 55 2 1 54 2532 31. pre expansion post expansion 2016 All larvae were raised on a standard 14 h light 10 h dark cycle at a. temperature of 28 C, Transgenic Fish Lines The genotypes of the larvae and embryos used to. RG2 generate each figure are detailed in SI Appendix SI Methods The transgenic. RG1 fish lines that were crossed to produce these larvae and embryos were all. RG1 previously described All larval brain images are from 6 d postfertiliza. tion larvae and all embryo images are from shield stage i e 6 h. postfertilization embryos, Immunohistochemistry Immunohistochemistry was performed following. standard previously published procedures 28 The exact protocol as well as. a detailed list of antibodies used is provided in SI Appendix SI Methods. Expansion Expansion was performed using the previously described proExM. protocol 2 SI Appendix SI Methods, Imaging Both pre and postexpansion brains and embryos were imaged on an. Andor spinning disk CSU X1 Yokogawa confocal system with a 40 1 15 N A. water immersion objective Nikon with the exception of some images in SI. Appendix Fig S1 The first and third images in SI Appendix Fig S1A and all. preexpansion images used to generate SI Appendix Fig S1C were acquired. using a Deltavision OMX Blaze GE Healthcare structured illumination micro. scope SIM with a 60 1 42 N A oil immersion objective Olympus These brains. were immersed in SlowFade Diamond Antifade mounting medium Invitrogen. endfeet for refractive index matching and suppression of bleaching The first and third. images in SI Appendix Fig S1B and all preexpansion images used to generate SI. C C Appendix Fig S1D were acquired using a Leica TCS SP8 STED microscope with a. 100 1 4 N A oil immersion objective These brains were immersed in SlowFade. Gold Antifade mounting medium Invitrogen for refractive index matching and. suppression of bleaching The second and fourth images in SI Appendix Fig S1B. and all postexpansion images used to generate SI Appendix Fig S1D were ac. quired using a Leica TCS SP8 confocal microscope with a 40 1 1 N A water. immersion objective Images of the samples were also obtained with a 10. SIN 0 45 N A air objective and used to aid in the comparison of pre and. postexpansion data and computation of expansion factors Details of ex. citation and emission collection are provided in SI Appendix SI Methods. For embryos postexpansion imaging was also performed via a Nikon Ti E. D D epifluorescence microscope with a 4 0 13 N A air objective to allow. capturing of the entire sample for computation of expansion factors For. brains expansion factors were computed by measuring the size of specific. anatomical features e g the axon cap pre vs postexpansion and taking. the ratio of the respective sizes For embryos the diameter of the embryo. pre vs postexpansion was compared Scale bars on postexpansion images. reflect these expansion factor computations For the expanded embryos. imaged in SI Appendix Fig S10 pre and postexpansion images were. taken from very different angles thus an exact expansion factor could. not be computed For SI Appendix Fig S10 an expansion factor of 4. similar to the expansion factors computed for other embryos 3 8 and. E1 E 4 1 was estimated for the purpose of drawing scale bars. Intensity AU,Intensity AU, Image Processing Each figure panel constitutes a single plane from a z stack.
where the area of interest was cropped out of the field of view using Fiji 29. or a maximal intensity projection as indicated in the figure legends The. brightness and contrast of individual channels were adjusted in ImageJ NIH. after cropping the area of interest The STED preexpansion images shown in. Distance pixel Distance pixel SI Appendix Fig S1B first and third panels and used in SI Appendix Fig. S1D were deconvolved using Huygens Scientific Volume Imaging Tracing. Fig 1 ExM helps resolve the morphology of fine cellular processes A of cellular processes shown in SI Appendix Fig S2 was performed using. Schematic of the larval zebrafish brain showing the imaged area red rect Imaris This tracing algorithm is intensity based First start and end points. angle within the left optic tectum B and B Maximum intensity projections. of part of the tectum highlighted in red in A of a 6 d postfertilization larval. zebrafish sparsely expressing membrane bound EGFP and stained for GFP. preexpansion B and postexpansion B showing radial glial cells two of of extratectal fibers preexpansion D and postexpansion D from the re. which are labeled RG1 and RG2 and projection fiber bundles arrowheads gions highlighted by arrowheads in B and B respectively is shown E and E. C and C Single confocal slices show projections of cell RG1 preexpansion Intensity plots along the orange line in D and D respectively AU arbitrary. C and postexpansion C Endfeet processes of this cell wrap around the units Scale bars B 10 m B 10 m physical size postexpansion 35 m C. cell body of a superficial interneuron SIN 113 arrow D and D A bundle and D 5 m C and D 5 m 17 5 m. E10800 www pnas org cgi doi 10 1073 pnas 1706281114 Freifeld et al. A B post expansion,Midbrain nIV isl1 nuclei,Hindbrain 6 7FRHcrtR. projections isl1 GFP,6 7FRHcrtR kaede,6 7FRHcrtR kaede isl1 GFP overlay. I II III IV,synaptotagmin,I II III IV,post expansion. synaptotagmin,V VI VII VIII, Fig 2 ExM analysis of synaptic connections A Schematic of larval zebrafish brain showing nIII and nIV nuclei labeled by Tg isl1 GFP rw0 GFP yellow and. neural projections labeled by Tg 6 7FRhcrtR Gal4VP16 Tg UAS Kaede Kaede magenta The rectangular area is imaged in B B Maximal intensity pro. jection of an 33 m thick volume corresponding to the rectangular area shown in A The fish is 6 d postfertilization dpf and is stained with anti GFP. yellow anti Kaede magenta and anti pan MAGUK not shown C GFP labeled cells yellow and Kaede labeled projections magenta in the nIII. nucleus C I IV and I IV Two nearby planes one in each row from an expanded 6 dpf brain stained with anti GFP yellow anti Kaede magenta and. anti synaptotagmin2b cyan Arrows point to Kaede expressing and synaptotagmin2b stained varicosities and terminals next to GFP labeled neuropil IV. and cell bodies IV Arrowheads point to a cluster of synaptotagmin2b unlabeled by Kaede next to a GFP labeled cell IV and a Kaede labeled. synaptotagmin2b stained varicosity next to a GFP negative cell IV C V VIII Single plane from a brain stained with anti pan MAGUK cyan Arrows. point to Kaede labeled varicosities and terminals next to GFP labeled cells and neuropil exhibiting colocalized MAGUK puncta Arrowheads point to a. MAGUK punctum on a GFP negative cell opposed to a Kaede labeled terminal Top arrowhead and to MAGUK puncta on GFP labeled cell bodies and. neuropil in the absence of nearby Kaede labeled projections Bottom two arrowheads Scale bars B 10 m 38 m C I IV and I IV 5 m 23 m C V VIII. are detected and then these points are connected with traces following the of expansion at the nanoscale was validated by comparison of. image intensity Fig 2B is a maximal intensity projection of four stacks ac postexpansion confocal images with superresolution structured. quired separately and stitched together using Fiji s pairwise stitching plug in illumination microscopy SIM images of the same samples. 30 The data shown in Fig 3 B E and SI Appendix Figs S5 and S6 were preexpansion cell culture mouse brain and pancreas slices 2. cropped from stacks following illumination correction using CIDRE 31 and. and human breast biopsy tissue slices 32 Interestingly ExM. deconvolution using Huygens Since the illumination model is dictated by. has rapidly become trusted enough that it has also been used. the microscope optics a single illumination model was learned using CIDRE. by pooling the datasets together and then this same model was used for the. without such validation sometimes even in novel species or tis. correction of both pre and postexpansion datasets After the application of sue types 33 36 38 While the widespread validation and trust in. illumination correction a dataset specific threshold was manually set ExM bodes well for the use of the technology we here sought to. according to the characteristic background noise level Both pre and post pursue validation nonetheless. expansion datasets were then deconvolved using the exact same procedure We first validated the isotropy of expansion of larval zebrafish. and parameters brains at the nanoscale using the previously developed method. ology for validation by comparison with a classical superresolution. NEUROSCIENCE, Measurement Error Quantification Errors were quantified using the same method over small regions that could be imaged by both methods.
procedures as previously described 2 32 with a few exceptions SI Ap Thus we imaged larval zebrafish brains preexpansion with two. pendix SI Methods different types of superresolution microscopes SIM and a stimu. lated emission depletion STED microscope and found these. Results images to be nearly identical to postexpansion images of the same. Validation of ExM of Zebrafish The proExM protocol has been regions in the same brains SI Appendix Fig S1 A and B We do. previously successfully used in a variety of applications and note however that depths of field vary in pre and post. tissue types including cell culture as well as mouse brain slices expansion images due to expansion occurring in the axial di. 2 33 mouse lung spleen and pancreas slices 2 isolated mension 1 as well as in lateral directions and for the images. mouse mitochondria 34 planaria 35 the central nervous shown here also due to the use of different microscopes and. system and the germarium tip of ovaries in Drosophila 36 objectives thus sample features sometimes appear in one. 37 and human tissue specimens prepared in a variety of dif image but not the other This difference in appearance is further. ferent manners 32 38 In many of these tissue types the isotropy aggravated by higher tissue scattering preexpansion compared. Freifeld et al PNAS Published online November 21 2017 E10801. spiral fiber neurons,Hindbrain M cells,B spiral fiber neurons Merge. pre expansion,synaptotagmin,glycine receptors,post expansion. synaptotagmin,glycine receptors,D pre expansion post expansion. 1 2 3 4 5 6 7, Fig 3 Expansion enables the resolving of synaptic heterogeneity and structure in intrasynaptic protein distributions A Schematic of a larval zebrafish. brain showing the M cells blue and spiral fiber neurons magenta The rectangle illustrates the region focused in on in B D consisting of the axon cap and a. part of the M cell body B Preexpansion images of the axon cap area showing spiral fiber neurons magenta wrapping around the M cell axon initial. segment the unlabeled tube passing through these fibers better visualized as a black stripe in C as well as synaptotagmin2b Top cyan and glycine. receptors Bottom cyan C Same as in B but postexpansion Note The synaptotagmin2b axon cap shown Top is not from the same brain as in B Top. Top Left and Right Arrows point to a Kaede labeled varicosity bearing synaptotagmin2b at a low density Top Left and Right Arrowheads point to a Kaede. negative varicosity bearing dense synaptotagmin2b staining Center Arrowheads point to varicosities in spiral fiber neuron projections forming the M cell. axon cap D Maximal intensity projection of the M cell body and axon initial segment area showing the distribution of glycine receptors cyan preexpansion. Left vs postexpansion Right Note This is the same axon cap as shown in B and C Bottom Boxes highlight seven examples of ring shaped clusters zoomed. in on in E E Seven examples of ring shaped clusters of various sizes present on the M cell body 1 6 and axon 7 Scale bars B Top and Bottom 5 m C. Top 5 m 23 m C Bottom 5 m 20 m D Left 5 m D Right 5 m 20 m E 1 m 4 m. with the optically clear 1 postexpansion samples causing re ones remaining after image registration result in pre and post. duced signal to noise ratios that may obscure some features in expansion images representing overlapping yet distinct optical. preexpansion samples Finally orientation differences even sections in the samples. E10802 www pnas org cgi doi 10 1073 pnas 1706281114 Freifeld et al. Nevertheless a quantitative comparison of the data shows to GFP positive cells in preexpansion data from this pan. that in length measurements performed using postexpansion MAGUK stained brain we identified 12 example terminals where. images the root mean square rms of measurement errors rel the data suggested more than one GFP positive cell as a putative. ative to the lengths measured was comparable to the results postsynaptic target SI Appendix Fig S4 Top However examining. obtained in previously published ExM papers In particular the the same regions in the same brain postexpansion we found that. rms of measurement errors was no larger than 5 for pre MAGUK puncta were often present only on a subset of these pu. expansion images acquired with SIM and 2 for preexpansi. roscience and developmental biology using the zebrafish model Regarding neuroscience studies ExM enables the trac ing of cellular processes in the zebrafish brain as well as the imaging of synapses and their biomolecular content and or ganization Regarding development ExM enablesthe resolving of nuclear compartments particularly nuclear invaginations and channels and helps relate such

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