We present a detailed protocol for Epon post-embedding correlative light and electron microscopy using a fluorescent protein called mScarlet. This method can maintain the fluorescence and the ultrastructure simultaneously. This technique is amenable to a wide variety of biological applications.
Correlative light and electron microscopy (CLEM) is a comprehensive microscopy that combines the localization information provided by fluorescence microscopy (FM) and the context of cellular ultrastructure acquired by electron microscopy (EM). CLEM is a trade-off between fluorescence and ultrastructure, and usually, ultrastructure compromises fluorescence. Compared with other hydrophilic embedding resins, such as glycidyl methacrylate, HM20, or K4M, Epon is superior in ultrastructure preservation and sectioning properties. Previously, we had demonstrated that mEosEM can survive osmium tetroxide fixation and Epon embedding. Using mEosEM, we achieved, for the first time, Epon post embedding CLEM, which maintains the fluorescence and the ultrastructure simultaneously. Here, we provide step-by-step details about the EM sample preparation, the FM imaging, the EM imaging, and the image alignment. We also improve the procedures for identifying the same cell imaged by FM imaging during the EM imaging and detail the registration between the FM and EM images. We believe one can easily achieve Epon post embedding correlative light and electron microscopy following this new protocol in traditional EM facilities.
Fluorescence microscopy (FM) can be used to obtain the localization and distribution of the target protein. However, the context that surrounds the target protein is lost, which is crucial for investigating the target protein thoroughly. Electron microscopy (EM) has the highest imaging resolution, providing several subcellular details. Nevertheless, EM lacks target labeling. By accurately merging the fluorescence image taken by FM with the gray image acquired by EM, correlative light and electron microscopy (CLEM) can combine the information obtained by these two imaging modes1,2,3,4.
CLEM is a trade-off between fluorescence and the ultrastructure1. Because of the limitations of current fluorescent proteins and the traditional EM sample preparation procedures, especially the use of osmic acid (OsO4) and hydrophobic resins such as Epon, the ultrastructure always compromises fluorescence5. OsO4 is an indispensable reagent in EM sample preparation, which is used to improve the contrast of EM images. Compared with other embedding resins, Epon is superior in ultrastructure preservation and sectioning properties5. However, no fluorescent proteins can retain the fluorescence signal after the treatment of OsO4 and Epon embedding6. To overcome the limitations of fluorescent proteins, pre embedding CLEM was developed, in which FM imaging is done before EM sample preparation6. However, the drawback of pre embedding CLEM is the imprecise registration between the FM and the EM images5.
On the contrary, post embedding CLEM method performs the FM imaging after the EM sample preparation, the registration accuracy of which can reach 6-7 nm5,6. To retain the fluorescence of fluorescent proteins, very low concentrations of OsO4 (0.001%)3 or the high-pressure frozen (HPF) and freeze substitution (FS) EM preparation methods4,7 have been used at the expense of compromised ultrastructure or the contrast of the EM image. The development of mEos4b greatly promotes the progress of post embedding CLEM, although glycidyl methacrylate is used as the embedding resin5. With the development of mEosEM, which can survive the OsO4 staining and Epon embedding, Epon post embedding super-resolution CLEM was achieved for the first time, maintaining the fluorescence and ultrastructure simultaneously6. After mEosEM, several fluorescent proteins that can survive the OsO4 staining and the Epon embedding were developed8,9,10,11. This greatly promotes the development of CLEM.
There are three key aspects to Epon post-embedding CLEM. The first is the fluorescent protein, which should maintain the fluorescent signal after EM sample preparation. According to our experience, mScarlet is superior to other reported fluorescent proteins. The second is how to find the same cell imaged by FM imaging in EM imaging. To solve this problem, we improve the procedure for this step so that one can readily find the targeted cell. The last is the method to align the FM image with the EM image. Here, we detail the registration between the FM and the EM images. In this protocol, we express mScarlet in VGLUT2 neurons and demonstrate that mScarlet can target secondary lysosomes using Epon post-embedding CLEM. We provide step-by-step details for Epon post-embedding CLEM, without compromising the fluorescence and the ultrastructure.
Animal husbandry and experiments were approved by the Institutional Animal Care and Use Committee of Fujian Medical University Medical Center. The step-by-step workflow of the current protocol is shown in Figure 1.
1. Sample preparation
2. EM sample preparation
3. Coating of the coverglass with gold nanoparticles
4. Ultrathin sectioning
5. Light microscopy imaging
6. Preparation for EM imaging
NOTE: The workflow is shown in Figure 3B.
7. Image analysis
8. EM imaging
9. Registration of the FM image with the EM image
Previous reports demonstrated that mScarlet can target the lysosome15. In this protocol, AAV expressing mScarlet (rAAV-hSyn-DIO-mScarlet-WPRE-pA) was injected into the M1 (ML: ±1.2 AP: +1.3 DV: -1.5) of Vglut2-ires-cre mouse brain using stereotaxic instruments. Following the protocol described above, the final correlated image is shown in Figure 4A. The FM image can be accurately aligned with the EM image using gold nanoparticles (the green dots) as fiducial markers. As shown in the EM image (Figure 4C,F), mScarlet targeted the lysosome, and the contents inside lysosomes are heterogeneous, which are the typical characteristics of the secondary lysosome.
Figure 1: Schematic overview of correlative light and electron microscopy. (A) Mouse brain preparation. Adeno-associated viruses were injected into the mouse brain. After 30 days, the fluorescent regions of mouse brain slices were cut into small blocks for EM sample preparation. (B) EM sample preparation. (C) Ultrathin section using a diamond knife and the schematic diagram of the coverglass. (D) FM imaging and TEM imaging. Abbreviations: EM = electron microscopy; FM = fluorescence microscopy; TEM = transmission electron microscopy. Please click here to view a larger version of this figure.
Figure 2: Coating coverglasses with gold nanoparticles. (A) Cover the coverglass with pioloform by centrifugation. (B) Incubate the coverglass surface with poly-L-lysine. (C) Incubate the coverglass surface with the diluted gold nanoparticles. (D) Schematic diagram of the coated coverglass. Please click here to view a larger version of this figure.
Figure 3: The schedule of preparations for FM imaging and EM imaging. (A) The workflow of the preparations for FM imaging consists of five steps: (i) scooping the ultrathin section, (ii) Air drying, (iii) placing a second coverglass after adding the buffer, (iv) fixing both coverglasses in the chamber, and FM imaging. (B) The workflow of the preparations for EM imaging also consists of five steps: (i) separating the two coverglasses, (ii) floating the ultrathin section, (iii) scooping up the ultrathin section, (iv) moving the ultrathin section onto the slot grid, and (v) EM imaging. Abbreviations: EM = electron microscopy; FM = fluorescence microscopy; TEM = transmission electron microscopy. Please click here to view a larger version of this figure.
Figure 4: Representative CLEM image of mScarlet. (A) The CLEM image of the whole neuron. (B,E) The CLEM images of the inset. (C,F) The EM images of the inset. (D,G) The fluorescence images of the inset. Green dots indicate gold nanoparticles. Scale bar = 5 µm (A), 1 µm (inset images). Abbreviations: EM = electron microscopy; FM = fluorescence microscopy; CLEM = correlative light and electron microscopy. Please click here to view a larger version of this figure.
The protocol presented here is a versatile imaging method, which can combine the localization information of the target protein from light microscopy (LM) and the context surrounding the target protein from electron microscopy (EM)6. With the limitations of current fluorescent proteins, the widely used method is pre embedding correlative light and electron microscopy (CLEM), which means the LM imaging is done before the EM sample preparation. Almost all existing fluorescent proteins can be examined in pre embedding CLEM. However, because of the inevitable distortions and shrinkages, accurate alignment of the final image is impossible6. Therefore, the information provided by pre embedding CLEM is to check the ultrastructures of the same cell imaged by LM in EM imaging.
The method provided by this study is post-embedding CLEM, in which LM imaging is done after EM sample preparation. The shrinkage caused by the chemical fixation in EM sample preparation does not affect the final image's accurate alignment. Furthermore, because both LM imaging and EM imaging are done on the same section and with the help of gold nanoparticles, the final image alignment can be very accurate. Post-embedding CLEM requires that the fluorescent proteins should retain a fluorescent signal after conventional EM sample preparation. Previously, we had reported the first fluorescent protein called mEosEM, which can retain fluorescent signals using Epon as embedding resin8. Compared with other resins as embedding resins, Epon has superior ultrastructure preservation and sectioning properties. Following mEosEM, other resistant Epon-embedding fluorescent proteins had been reported, such as mKate211,16, mCherry210,17, mWasabi10,18, CoGFPv010,19, mEosEM-E8, mScarlet8,20, mScarlet-I8,20, mScarlet-H8,20, and HfYFP9.
According to our experience, mScarlet is superior to other fluorescent proteins. Fixative solution can affect the fluorescence of fluorescent proteins. In general, the fixation proceeding speed of paraformaldehyde (PFA) is much slower than that of glutaraldehyde (GA); conversely, PFA penetrates the sample more quickly than the larger GA. A mixture of PFA and GA provides a balance between fixing the sample quickly enough that its quality is maintained but slowly enough that sample damage such as oxidation does not occur. The higher GA concentration will produce higher autofluorescence. From our experience, the fluorescence of mScarlet in resin block can be detected 6 months after polymerization. But we recommend to do CLEM imaging immediately after polymerization.
Another key factor in post embedding CLEM is how to register the LM image with the EM image accurately. Based on previously reported methods6,21, we made some modifications to the protocol. The first is how to find the same cell imaged by LM in EM imaging. We took different FOVs to stitch the navigation map of the whole ultrathin section using DIC and fluorescence imaging mode. Using the navigation map, the cells of interest could be easily identified. Another improvement is accurate image alignment. Previously, we used the fluorescent signal in fluorescence imaging mode and a high electron contrast signal in the EM imaging mode of the gold nanoparticles as the fiducial alignment markers6. However, when using mScarlet, the fluorescent signal of mScarlet was much higher than the fluorescent signal of the gold nanoparticles and it was difficult to detect the fluorescent signal of gold nanoparticles. To solve this problem, we used the brightfield signal of the gold nanoparticles for registration instead of the fluorescent signal. Following this modified protocol, it was easy to perform post-embedding CLEM.
However, there are some limitations of the current protocol, which should be taken into consideration before using it. Although it works well in the overexpression system, the fluorescence is quite faint if the target proteins are under their native promoter22. Another limitation is that although mScarlet is the best fluorescent protein (according to our experience) that can retain enough fluorescence signals after EM sample preparation and works well in mammalian cells, it will be a problem when using mScarlet as a fluorescent tag in neurons, which can lead to the mistaken location of the target proteins15. In these cases, we suggest using oScarlet15, a mutation of mScarlet, which works well in neurons.
The potential applications of this modified protocol can be divided into two categories. The first is to zoom into the subcellular level, which means studying the target protein in the subcellular context. In our previously published reports, we used post-embedding CLEM to examine the formation of cytoplasmic virion assembly compartments (cVACs) during infection by a γ-herpesvirus23. In future applications, post embedding CLEM can be combined with electron tomography to obtain the 3D distribution of the target proteins and solve the 3D structure natively24. The second type of potential application is to zoom out to the cellular level, which can be used to draw and analyze neural circuits. Due to its high-resolution capability, EM has become an effective means of mapping fine brain connections. However, EM cannot accurately provide the identities of neurons, which limits the in-depth analysis of the neuronal circuit. Epon post-embedding CLEM has the potential to bring the identities of neurons into the neuronal circuit without compromising the ultrastructures.
The authors have nothing to disclose.
This project was supported by the National Natural Science Foundation of China (32201235 to Zhifei Fu), the Natural Science Foundation of Fujian Province, China (2022J01287 to Zhifei Fu), the Research Foundation for Advanced Talents at Fujian Medical University, China (XRCZX2021013 to Zhifei Fu), the Finance Special Science Foundation of Fujian Province, China (22SCZZX002 to Zhifei Fu), Foundation of NHC Key Laboratory of Technical Evaluation of Fertility Regulation for Non-human Primate, and Fujian Maternity and Child Health Hospital (2022-NHP-04 to Zhifei Fu). We thank Linying Zhou, Minxia Wu, Xi Lin, and Yan Hu at the Public Technology Service Center, Fujian Medical University for support with EM sample preparation and EM imaging.
0.2 M Phosphate Buffer (PB) | NaH2PO4 · 2H2O+Na2HPO4 · 12H2O | ||
0.2 M Tris-Cl (pH 8.5) | Shanghai yuanye Bio-Technology | R26284 | |
25% Glutaraldehyde (GA) | Alfa Aesar | A17876 | Hazardous chemical |
Abbelight 3D | Nanolnsights | ||
Acetone | SCR | 10000418 | |
Ammonium hydroxide | J&K Scientific | 335213 | |
BioPhotometer D30 | eppendorf | ||
Cleaning buffer of cover glasses | 50 mL Ammonium hydroxide, 50 mL Hydrogen peroxide, 250 mL H2O | ||
Coverglass | Warner | 64-0715 | |
DABCO | Sigma | 290734 | Hazardous chemical |
DDSA | SPI company | GS02827 | Hazardous chemical |
Desktop centrifuge | WIGGENS | MINICEN 10E | |
Diamond knife | DiATOME | MX6353 | |
DMP-30 | SPI company | GS02823 | Hazardous chemical |
DNA transfection reagent | Thermo Fisher | 2696953 | Lipofectamine 3000 Transfection Kit |
Epon 812 | SPI company | GS02659 | Hazardous chemical |
Ethanol | SCR | 10009218 | |
Fiji image J | National Institutes of Health | ||
Fixative solution | 4% PFA+0.25% GA+0.02 M PB | ||
Formvar | Sigma | 9823 | |
Glycerol | SCR | 10010618 | |
Gold nanoparticles | Corpuscular | 790120-010 | |
Gradient resin | Acetone to resin 3:1, 1:1, 1:3 | ||
Hydrofluoric acid | SCR | 10011118 | |
Hydrogen peroxide | SCR | 10011218 | |
ICY (https://icy.bioimageanalysis.org/about/) | Easy CLEMv0 Plugin | ||
Imaging chamber | Thermo Fisher | A7816 | |
Large gelatin capsules | Electron Microscopy Sciences | 70117 | |
Mounting buffer | Mowiol 4-88, Glycerol, 0.2 M Tris-Cl (pH 8.5), DABCO | ||
Mowiol 4-88 | Sigma | 9002-89-5 | |
Na2HPO4 ž12H2O | SCR | 10020318 | |
NaH2PO4 ž2H2O | SCR | 20040718 | |
NMA | SPI company | GS02828 | Hazardous chemical |
Oligonucleotide primers | Takara Biomedical Technology (Beijing) | Three oligonucleotides primers were used to detect Vglut2-ires-Cre and wild-type simultaneously. The primers 5,-ATCGACCGGTAATGCAGGCAA-3, and 5,-CGGTACCACCAAATCTTACGG-3, aimed to detect Vglut2-ires-Cre. The primers 5,-CGGTACCACCAAATCTTACGG-3, and 5,-CATGGTCTGTTTTGAATTCAG-3, aimed to detect wild-type. | |
Oscillating microtome | Leica | VT1000S | |
Osmium tetroxide | SCR | L01210302 | Hazardous chemical |
OsO4 solution | 1% Osmium tetroxide+1.5% K4Fe (CN)6·3H2O | ||
Parafilm | Amcor | PM-996 | |
Paraformaldehyde (PFA) | SCR | 80096618 | Hazardous chemical |
Perfusion buffer | 4% PFA+0.1 M PB | ||
Pioloform | Sigma | 63148-65-2 | Hazardous chemical |
Poly-L-lysine | Sigma | 25986-63-0 | |
Potassium ferrocyanide (K4Fe (CN)6·3H2O) | SCR | 10016818 | |
Scalpel blades | Merck | S2771 | |
Scalpel handles | Merck | S2896-1EA | |
Stereomicroscope | OLYMPUS | MVX10 | |
Transgenic mice | The Jackson Laboratory | Vglut2-ires-Cre mice (strain: 129S6/SvEvTac) were housed in standard conditions (25 °C, a 12 h light/dark cycle, with water and food given ad libitum. Male and Female mice were used at 2–3 months old, weight range 20-30 g. | |
Transmission electron microscope (TEM) | FEI | TECNAL G2 | |
UA solution (2% UA) | Aqueous solution | ||
Ultramicrotome | Leica | LEICA EM UC6 | |
Uranyl acetate (UA) | TED PELLA | 19481 | Hazardous chemical |