The presented method enables visualization of fluorescently labeled cellular proteins with expansion microscopy leading to a resolution of 70 nm on a conventional microscope.
Disruption of the glomerular filter composed of the glomerular endothelium, glomerular basement membrane and podocytes, results in albuminuria. Podocyte foot processes contain actin bundles that bind to cytoskeletal adaptor proteins such as podocin. Those adaptor proteins, such as podocin, link the backbone of the glomerular slit diaphragm, such as nephrin, to the actin cytoskeleton. Studying the localization and function of these and other podocytic proteins is essential for the understanding of the glomerular filter's role in health and disease. The presented protocol enables the user to visualize actin, podocin, and nephrin in cells with super resolution imaging on a conventional microscope. First, cells are stained with a conventional immunofluorescence technique. All proteins within the sample are then covalently anchored to a swellable hydrogel. Through digestion with proteinase K, structural proteins are cleaved allowing isotropical swelling of the gel in the last step. Dialysis of the sample in water results in a 4-4.5-fold expansion of the sample and the sample can be imaged via a conventional fluorescence microscope, rendering a potential resolution of 70 nm.
Albuminuria is a surrogate parameter of cardiovascular risk and results from disruption of the glomerular filter1. The glomerular filter is composed of the fenestrated endothelium, the glomerular basement membrane and the slit diaphragm formed by podocytes. Primary and secondary foot processes of podocytes wrap around the capillary wall of the glomerulum2. The delicate structure of foot processes is maintained by cortical actin bundles which also serve as anchors for multiple slit diaphragm proteins and other adaptor proteins2. The slit diaphragm's backbone protein is called nephrin and interacts in a homophilic manner with nephrin molecules of opposing podocytes. Via diverse adaptor proteins, nephrin is linked to the actin cytoskeleton2,3. Mutations in the nephrin-encoding gene NPHS1 lead to nephrotic syndrome of the Finnish type4.
One of nephrin's interacting proteins is podocin, a hairpin-like protein of the stomatin family3. Podocin recruits nephrin to lipid rafts and links it to the actin cytoskeleton5. Podocin is encoded by the NPHS2 gene. Mutations in NPHS2 lead to steroid-resistant nephrotic syndrome6.
To visualize and co-localize actin adaptor proteins, immunofluorescence techniques may be used. Unfortunately, the diffraction barrier of the light limits the resolution of conventional fluorescence microscopes to 200-350 nm7. Novel microscopy techniques, e.g., stimulated emission depletion (STED)8, photo-activated localization microscopy (PALM)9, stochastic optical reconstruction microscopy (STORM or dSTORM) or ground state deletion microscopy followed by individual molecule return (GSDIM)9,10,11, enable a resolution up to approximately 10 nm. However, these super resolution techniques require highly expensive microscopes, well-trained personnel and are therefore not available in many laboratories.
Expansion microscopy (ExM) is a novel and simple technique that enables super resolution imaging with conventional microscopes and is potentially available to a large research community12. In protein retention expansion microscopy (proExM), the sample of interest (cells or tissue) is fixed and stained with fluorophores13. Proteins within the sample are then covalently anchored by a small molecule (6-((Acryloyl)amino)hexanoic acid, succinimidyl ester, AcX) into a swellable hydrogel13. Through enzymatic digestion with proteinase K (ProK), proteins and fluorophores maintain their relative position within the gel after expansion13. After swelling of the gel, the sample expands up to 4.5-fold (90-fold volumetric expansion) leading to an effective lateral resolution of approximately 60-70 nm (300 nm/4.5). Modifications of this technique can even allow for a 10-fold expansion (1,000-fold volumetric expansion), rendering a resolution of 20-30 nm on conventional microscopes14,15,16.
Glomerular structures of mouse and human kidneys have been visualized via ExM17. Within this paper, we present a detailed proExM protocol to visualize super resolution images of F-actin and the actin-adapter protein podocin within cells using a conventional fluorescence microscope.
1. Splitting and seeding of cells
2. Transfection of cells
3. Immunolabeling of cellular structures
4. Expansion microscopy
The concept and timing of this proExM protocol is depicted in Figure 1. On day 5, transfected cells are fixed and stained with fluorescent antibodies targeting the protein of interest (Figure 1A,B). On day 6, treatment with AcX leads to formation of amine groups on all proteins (including fluorophores) (Figure 1A,B)12. Upon polymerization of the hydrogel, these amine groups bind covalently to the hydrogel (day 6). After polymerization of the gel, homogenization (digestion) is performed with proteinase K resulting in the destruction of structural proteins of the cell (day 6, Figure 1A,B). Fluorescently labeled antibodies remain mostly preserved after digestion. Due to the disruption of structural proteins, water dialysis of the hydrogel results in isotropic expansion of the cell within the hydrogel on day 7 (Figure 1A,B). Imaging of the sample is performed with a conventional fluorescence microscope (Figure 1A). Data validation to determine the expansion factor and to exclude distortions should be performed (Figure 1A).
To perform expansion of the cell isotropically, the gelation step is essential. Figure 2 shows the lateral and top view of a gelation chamber. Glass cover slips build the spacers of the gelation chamber (Figure 2A1-3/C1-3). The cover glass with the fixed and stained cells is positioned with the cells upward onto a glass slide (Figure 2A4-C4). The lid of the gelation chamber is wrapped with parafilm and is closed bubble-free (Figure 2A5-C5).
This ExM protocol enables expansion of up to four-fold. To determine the expansion factor, it is essential to image cells before and after expansion (Figure 3A + B). Insufficient anchoring and homogenization may lead to distortions and ruptures of cells. Figure 4A + B shows representative examples of ruptured cells in different magnification images.
This method can be used to investigate the co-localization of F-actin and actin adaptor proteins, e.g., podocin and nephrin (Figure 5). Podocin is depicted in green while actin is labeled in blue (Figure 5). Nephrin is marked in green. White areas indicate co-localization.
Figure 1: Concept and timing of this ExM protocol. (A) In the "protocol" column, each step of the protocol is outlined. (A + B) After seeding and transfecting cells, immunofluorescent labeling is performed (Immunolabeling). (A + B) The small molecule AcX (red dot) binds to all proteins and anchors them to the hydrogel (Anchoring). (A + B) Via polymerization all proteins including fluorophores are covalently bound via AcX to the hydrogel (Polymerization). (A + B) Homogenization leads to digestion of structural matrix proteins. (A + B) Expansion is achieved by dialysis in water. (A) Imaging and validation of imaging finalizes the experiment. (A) The entire protocol requires 7 days (column "day") with many incubation steps (total time per day column "total time"), but actual bench time is much less as indicated in the respective column "bench time". Modified from14. Please click here to view a larger version of this figure.
Figure 2: Building the gelation chamber. Side view (A1) and top view (B1 + C1) of a glass slide with four #1.5 cover stripes. By adding a droplet of water between the glass slide and the cover slip stripes, the stripes will adhere to the glass slide (side view A2, top view B2, C2). Droplets of water on the #1.5 cover stripes lead to adhesion of #1.0 cover stripes laid on top of the #1.5 cover stripes (side view A3, top view B3, C3). The sample on the cover slip is placed in the middle of the rectangle using forceps. The gel is pipetted on top (side view A4, top view B4, C4). (A5) Side view and top view (B5 and C5) of the assembled gelation chamber including the closed lid which is built from a cover slip wrapped in parafilm. Please click here to view a larger version of this figure.
Figure 3: Cells before and after expansion. (A) Cells before expansion stained for actin. The box indicates in which area the expanded cells in Figure 3B lie. (B) Cells after expansion stained for actin in red and podocin in green. Podocin co-localizes with actin in the cell periphery. Scale bar = 5 µm, expansion factor = 2. Please click here to view a larger version of this figure.
Figure 4: Distortions and ruptures of cells. (A + B) Representative microscopic images of cos7 cells immuno-stained for actin (red). Cells were fixed, stained, anchored, digested and expanded. (A) Ruptures of cells. Arrows indicated ruptured areas. Scale bar = 5 µm, expansion factor = 4. (B) Ruptures and distortion of cells. White arrows indicated ruptured areas. Scale bar = 5 µm, expansion factor = 4. Please click here to view a larger version of this figure.
Figure 5: Podocin co-localizes with nephrin and actin. Cos7 cells immunofluorescently labeled for podocin, actin, and nephrin. (A) Cos7 cells stained for podocin (green), actin (blue), and nephrin (red) with ExM. Podocin co-localizes with actin and nephrin. Scale bar = 200 nm, expansion factor = 4. (B) Magnification of the indicated area in (A), Scale bar = 40 nm. Please click here to view a larger version of this figure.
Solutions for ExM | |||
Anchoring buffer | final concentration | ||
NaHCO3 | 150 mM | ||
Acryloyl-X, SE (AcX) | 0.1 mg/ml | ||
Monomer solution | Stock solution concentration g/100ml | amount (ml) | final concentration (g/100 ml) |
sodium acrylate | 38 | 2.25 | 8.6 |
acrylamide | 50 | 0.5 | 2.5 |
N,N’-Methylenebisacrylamide | 2 | 0.5 | 0.10 |
sodium chloride | 29.2 | 4 | 11.7 |
PBS | 10x | 1 | 1x |
water | 1.15 | ||
total | 9.4 | ||
Gelling solution | Stock solution concentration | amount (µl) | final concentration (mg/ml) |
monomer solution | NA | 190 | NA |
APS | 10% | 4 | 2 |
TEMED | 10% | 4 | 2 |
water | NA | 2 | NA |
total | 200 | ||
Digestion solution | Stock solution concentration | amount (µl) | final concentration |
Tris Cl, pH 8.0 | 1 M | 1000 | 50 mM |
EDTA pH 8.0 | 0.5 M | 40 | 1 mM |
Triton X-100 | 10% | 1000 | 0.5 % |
Guanidin HCL | 8M | 2000 | 0.8 M |
water | ad 20 ml | ||
proteinase K | 800 U/ml | 100 | 4 U/ml |
Table 1: Solutions for ExM.
The presented method enables the investigator to visualize cellular proteins, e.g., podocin, nephrin, and cytoskeletal components, e.g., F-actin. Within this protocol, transfected cos7 cells are used as a model to study interaction of slit diaphragm proteins with F-actin. Unfortunately, immortalized podocyte cell lines do not express sufficient endogenous amounts of slit diaphragm proteins19.
With this method, cellular proteins can be visualized with nanoscale resolution using a conventional fluorescence microscope. The most critical steps within the protocol are: 1) sufficient anchoring of protein amine groups to the hydrogel with AcX, 2) adequate polymerization of the hydrogel, 3) optimal timing for digestion and 4) selection of compatible fluorophores.
Anchoring of cellular proteins to the hydrogel is essential for this method in order to preserve the protein's position within the hydrogel during expansion. AcX is a small molecule that binds to amine groups of proteins within cells and tissues. AcX creates a carbon-carbon double band with proteins, enabling incorporation of the proteins into the hydrogel in the polymerization step20. AcX also integrates antibodies so that labeling with immunofluorescence antibodies can be performed before AcX treatment. Insufficient anchoring may lead to ruptures and distortions of cells. Due to modification of amine groups by fixatives, one needs to optimize the fixative or the time of fixation. In addition, insufficient storage or non-optimized anchoring conditions may result in ruptures and distortions. Based on our experience, AcX loses its optimal effect when used for more than 3-4 months.
Polymerization of the gel is temperature dependent. We, therefore, recommend keeping the polymerization solutions on ice before pipetting it into the gelation chamber. In addition, the handling time of the gelation step should be kept short (less than 5 min) in order to avoid premature gel formation. Thorough mixing of the polymerization solution prevents uneven polymerization. Air bubbles will affect the expansion process when touching the sample and can be prevented by adding more polymerization solution.
After incorporation of the cellular proteins within the hydrogel, the mechanical homogenization step (or digestion) is needed to ensure expansion. Different methods, e.g., heat and detergent or enzymatic digestion, exist and need to be customized to the investigated sample12,14,20. Within this protocol, the protease Proteinase K is used for enzymatic digestion. Proteinase K is applied at a dosage sufficient to destroy structural proteins whilst preserving most other proteins including fluorescent antibodies12. If digestion is incomplete, the sample expansion is insufficient. In addition, the sample can tear during the expansion process (Figure 3). If an inadequate sample expansion has occurred, water replacement is recommended. Alternatively, the time for the enzymatic digestion can be adjusted or a new aliquot of the Proteinase K opened.
If the sample is over-digested, fluorescence signals will be diminished. In this case, the digestion time should be reduced. In ExM in general, the fluorescence signal intensity per unit of volume is reduced due to the volumetric expansion of the sample14. Therefore, longer exposure times during imaging need to be considered.
It is essential to select ExM compatible fluorophores. Cyanine dyes are degraded during the polymerization step13. Fluorescence proteins based on bacteriophytochromes are also largely destroyed13. However, most GFP-like proteins will be preserved13. In addition, streptavidin can also be applied pre-expansion, labeling post-translational modifications such as S-nitrolysation via a small molecule tag13.
Phalloidin, a small labeling molecule to target the actin cytoskeleton, is not compatible with ExM21. To overcome insufficient anchoring of phalloidin, trivalent anchoring (TRITON) has been introduced21. This approach offers simultaneous targeting, labeling and grafting of biomolecules21.
This method can be modified to stain RNA molecules (ExFish)22. In iterative expansion microscopy (iExM) or X10 microscopy, the resolution of 60-70 nm can be extended to approximately 25 nm by applying a second swellable gel within the first expanded hydrogel or conducting a single expansion step using a different hydrogel15,16. Ultrastructure expansion microscopy (U-ExM) enables super resolution of proteins preserving their attribution to an ultrastructural element (e.g., mitochrondria, microtubules)23. A combination of ExFish (RNA and DNA) and proExM methods have previously been performed as well22,24. The presented protocol uses transfected cos7 cells as a model to investigate slit diaphragm proteins. We expect that other resident cultured kidney cells, e.g., HEK293T cells, can be similarly used for this protocol. Depending on the cell line, adjustments may need to be made for the different culturing and transfection conditions.
ExM enhances resolution of immuno-stained samples by about 4-fold reaching a lateral spatial resolution of 70 nm13. Compared to other super-resolution techniques, ExM is performed on a conventional fluorescence microscope13,14. Therefore, no expensive equipment or specifically trained personnel is necessary to conduct the ExM method14. Even though not all fluorophores are compatible with ExM, there are generally many available antibodies with optimized fluorophores with photo-physical properties needed for super resolution microscopy14. The main disadvantage of this method is that ExM is incompatible with live samples12,14.
In the future, improving the hydrogel's chemical composition may lead to even higher spatial resolution12. The combination of different protocols may also enable visualization of proteins, RNA, DNA, or lipids in complexes within the same sample with such high resolution12.
The authors have nothing to disclose.
The authors would like to thank Blanka Duvnjak and Nikola Kuhr for their excellent technical assistance.
Acrylamide >99% | Sigma-Aldrich | A3553-100G | |
6-((Acryloyl)amino)hexanoic acid, succinimidyl ester, Acryloyl-X, SE | invitrogen | A-20770 | store up to 4 months |
APS | Sigma-Aldrich | A3678-25G | |
Deckgläser (cover glasses) | Engelbrecht | K12432 | 24x32mm #1.0 |
Diamont cutter | VWR | 201-0392 | for cutting the cover slips |
Guanidine HCl | Sigma-Aldrich | G3272-100G | 8M Stock can be kept at RT |
Marten hair paintbrush | Leon Hardy | 3 (770) | |
"Menzel" Deckgläser (cover glasses) | Thermo Fischer | 15654786 | 24x24mm #1.5 |
N,N`-Methylenbisacrylamide | Sigma-Aldrich | M7256-25G | |
Objektträger UniMark | Marienfeld | 703010 | |
Proteinase K | New England Biolabs | P8107S | |
Sodium Acrylate | Sigma-Aldrich | 408220 | check purity |
Sodium Bicarbonate | Sigma-Aldrich | S5761 | |
Staining chamber | produced at the university's workshop | ||
TEMED | ROTH | 2367.1 | |
6-Well glass bottom plates | Cellvis | P06-1.5H-N | |
Antibodies | |||
Actin-ExM 546 | chrometra | non-available | 1:40 |
Anti Podocin produced in rabbit | Sigma | P-0372-200UL | 1:200 |
Donkey anti guinea-pig CF633 | Sigma | SAB4600129-50UL | 1:200 |
Goat anti rabbit 488 | Life Technologies | A11034 | 1:1000 |
Guinea pig anti nephrin | Origene | BP5030 | 1:100 |
Software | |||
FIJI | |||
Visiview | |||
microscope | |||
AXIO Observer Z1 | Zeiss | non-available |