This protocol describes the necessary steps to obtain subcellular protein localization results on zebrafish retina by correlating super-resolution light microscopy and scanning electron microscopy images.
We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.
Methods to determine the subcellular localization of proteins and their relationship to different compartments of the cell are essential tools to understand their functions and possible interactions. Super-resolution microscopy in combination with electron microscopy provides such information1. Ground state depletion microscopy followed by individual molecule return (GSDIM) is a super-resolution microscopy technology compatible with a wide range of organic and genetically encoded fluorophores2 and achieves a lateral resolution up to 20 nm3. The incorporation of methods with higher resolution than standard diffraction-limited microscopy improves the accuracy of the correlation4,5,6. In order to achieve the best correlation of protein expression with a specific subcellular compartment and to reduce the volume of uncertainty7 the use of the same ultrathin section for light and electron microscopy is recommended. Among the different sectioning methods, Tokuyasu cryo-section protocol does not require dehydration or resin embedding and, in addition, preserves the antigenicity of many epitopes and provides good tissue ultrastructure8. Several methods have demonstrated the applicability of these sections in correlative light and electron microscopy (CLEM)4,5,9,10.
The zebrafish retina is a valuable model to study visual development and human disease mechanisms given its highly conserved structure and function across vertebrates. In particular, retinal photoreceptors display the same architecture as mammalian photoreceptors, with a basal synapse, an apico-basally elongated nucleus, clustering of mitochondria in the more apical inner segment and an outer segment composed of membrane disks in the most apical position11. Protein localization to the diverse cellular compartments is conserved between zebrafish and human, allowing investigation of the biological function of human disease-relevant proteins12,13.
Here we present a protocol to prepare larval zebrafish retina samples to resolve the localization of the mitochondrial outer membrane protein Tom20 by correlative super-resolution light and electron microscopy. The method is based on collecting cryo-sections on silicon wafers and obtaining contrast by topographical information produced after application of a thin layer of platinum. These steps are clear technical improvements in terms of ease of use, reproducibility, and time to complete experiments. We have recently demonstrated the applicability of the method to detect nuclear pores and mitochondrial proteins in mouse tissue14.
All experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the local authorities.
1. Preparation of Ultrathin Sections on Silicon Wafers
2. Immunolabelling
3. Super-resolution Microscopy
4. Platinum Shadowing
5. Scanning Electron Microscopy
6. Alignment of Light and Electron Microscopy Images
The expression of the protein Tom20, a subunit of the translocase mitochondrial outer membrane complex20, was determined, in thin sections of larval zebrafish retina, by super-resolution light microscopy (Figure 1) and this information was complemented with the topographical signal obtained by scanning electron microscopy after platinum shadowing of the same sections. These correlative data confirm the localization of a protein in association with a particular compartment, the outer mitochondrial membrane and, in addition provide information about the relation of the protein with other organelles of the cell.
Figure 1: CLEM on zebrafish retina. A. Low magnification widefield image of a 5 dpf zebrafish retinal section, nuclei stained with DAPI (cyan). B. Scanning electron microscopy of the same area. C. Higher magnification widefield image of frame in B. Nuclei stained with DAPI (cyan) and Tom20 mitochondrial staining appears in red. D. Widefield image of same section at higher magnification. The pattern of Tom20 expression is at the clusters of mitochondria. E. Expression of Tom20 (red dots) detected by GSDIM microscopy. Nuclei stained with DAPI (cyan). F. Same section as E combining correlative super-resolution and scanning electron microscopy. Tom20 staining (red dots) appears at the mitochondrial cluster (M) at the outer membranes of mitochondria. Fluorescence DAPI signal in the nuclei (N) corresponds with the topography of the SEM image. G. High magnification image of frame in F. The scanning electron microscopy image provides context to the GSDIM image (red dots). Mitochondrial cristae are clearly visible and the Tom20 staining is localized to the outer membranes of mitochondria. The membranes of the outer segment the photoreceptors (OS) are clearly resolved. Image pixel size 5 nm. Scale bars: A, B, and C: 10 µm; D: 2 µm; E and F: 1 µm and G: 0.2 µm. Please click here to view a larger version of this figure.
Supplementary Figure 1: Alignment of light and electron microscopy images. A. Screenshot from TrackEM2 interface with SEM image and numbered landmarks (yellow) along different nuclei. B. Screenshot from TrackEM2 interface with fluorescence image and numbered landmarks (yellow) along different DAPI stained nuclei. To change layer transparency the sliders on the left upper part of the menu can be used. Scale bar: 1 µm. Please click here to download this file.
This method combines super-resolved protein localization with context information to determine the precise position of proteins in an organelle. We demonstrate here the completion of the experiment to visualize the expression of Tom20 in the outer membrane of mitochondria, and its relation to other organelles like nuclei or outer segments of the photoreceptor in the larval zebrafish retina.
Tokuyasu cryo-sectioning requires some training to acquire well-preserved sections. However, this is a method employed in many laboratories with demonstrated success21. Transfer of the sections to the silicon wafer is very simple and no special considerations are needed. The use of glycerol in the imaging buffer is a very critical step to avoid drying of the sections. For super-resolution imaging the best results are obtained when the wafer is very close to the glass bottom of the Petri dish. The silicone stripes help maintaining the wafer in this position. Special care has to be taken while removing the stripes in order to avoid damaging the sections.
The thickness of the cryo-sections, around 100 nm, thinner than the optical resolution in the Z-dimension, additionally facilitates the accuracy of this correlative method as the super-resolution microscopy signal is coming only from this thin layer and the scanning electron microscope signal is displaying the topography of the sample. This method could also be combined with multicolor imaging. However, special care must be taken that the sample can be imaged under same conditions (e.g. imaging buffer) and cross talk should be prevented.
One limitation of the method is its two dimensional approach, since only a limited number of serial sections (about 3 to 7 per wafer) can be collected. Thus, projects dealing with volume analysis would not be ideal. However, it is a method that can be applied to detect protein expression in any type of tissue by simple sectioning of the sample. We provide a method without use of classical contrast agents like uranyl acetate or lead citrate. The contrast by platinum shadowing provides very informative topographical contrast, but in some cases membranes are difficult to resolve. This may pose problems for projects that need, for example, to determine the expression of proteins in small vesicles.
Our protocol, based on Tokuyasu cryo-sections, uses standard equipment to obtain correlative results from super-resolution and scanning electron microscopes. The collection of sections on silicon wafers and the use of platinum for contrast are simple steps to provide stability and reproducibility to the sample preparation.
The authors have nothing to disclose.
Funding ION and RGB: Swiss National Science Foundation Ambizione-SCORE grant PZ00P3 142404/1 and PZ00P3 163979.
Paraformaldehyde | Sigma-Aldrich | #158127 | |
Glutaraldehyde EM Grade | EMS, USA | #16220 | |
Cacodylate | Merck | #8.20670 | |
Tricaine | Sigma-Aldrich | #886-86-2 | |
Agarose, peqGOLD Universal | VWR International GmbH | 35-1020 | |
Flat embedding molds | BEEM Flat | ||
Local food brand gelatin | Dr.Oetker | Extra Gold | |
Sucrose | Merck | #1.07687 | |
Methylcellulose | Sigma | #M-6385 | |
Glycine | Sigma | #G-7126 | |
Gelatin type B | Sigma | #G-6650 | |
BSA | Applichem | #A6588.0050 | |
Silicon wafer | Si-Mat Silicon Materials | Type: P/Boron; Orientation <111> ON; Growth method: CZ; Resistivity:1-30 ohm/cm; Surface: polished; Laser cut at 7 x7 mm | |
Cryo-pin | Baltic Preparation | #16701950 | |
Wired loop "Perfect loop" | |||
Silicone stripes | Picodent | Twinsil 22 | |
Glucose | Sigma-Aldrich | #G8270 | |
glucoseoxidase | Sigma-Aldrich | #G7141 | |
catalase | Sigma-Aldrich | #C40 | |
beta-Mercaptoethylamine hydrochloride | Sigma-Aldrich | #M6500 | |
anti-Tom20 | Santa Cruz Biotechnology | #sc – 11415 | |
AlexaFluor 647 AffiniPure F(ab')2 fragment donkey anti rabbit IgG | Jackson Immuno-Research | #711-606-152 | |
DAPI | ROCHE, Switzerland | #10236276001 | |
Glycerol solution | Sigma-Aldrich | #49782 | |
Glass bottom petri dish | Ibidi, Germany | u-Dish 35mm, high glass bottom, #81158 | |
SEM aluminium stub | Agar Scientific | #G301F | |
Conducting carbon cement Leit-C | Plano, Germany | AG3300 | |
Name | Company | Catalog Number | コメント |
Instruments | |||
Diamond Knife (Cryo Immuno) | Diatome | DCIMM3520 | |
Cryo-ultramicrotome | Leica Microsystems | Leica EM FCS | |
Widefield-TIRF microscope GSD Leica SR GSD 3D | Leica Microsystems | ||
160x 1.43 TIRF objective | Leica Microsystems | 11523048 | |
SEM – Zeiss Supra 50 VP | Zeiss | Supra 50 VP | |
FIB-SEM – Zeiss Auriga 40 | Zeiss | Auriga 40 |