WEEK 10: Expansion Microscopy Measurement & Imaging
This journal documents the experiment of performing protein-retension expansion microscopy (ProExM), wherein proteins in the sample are covalently linked to the gel before expansion. This variant of ExM is used to anchor fluorescent antibodies or fluorescent proteins directly to the gel. See the supplementary readings for more information about expansion microscopy and super-resolution imaging.
Protein-Retention Expansion Microscopy of Cells and Tissues Labeled Using Standard Fluorescent Proteins and Antibodies. Tillberg et al., Nature Biotechnology, 2016.
This topic is supported by David Sun Kong (MIT Media Lab), Evan Daugharty (ReadCoor), and Paul Reginato (MIT)
I. INTRO
Expansion microscopy (ExM) enables imaging of preservedspecimens with nanoscale precision on diffraction-limitedinstead of specialized super-resolution microscopes. ExM worksby physically separating fluorescent probes after anchoringthem to a swellable gel. The first ExM method did not resultin the retention of native proteins in the gel and relied oncustom-made reagents that are not widely available. Here wedescribe protein retention ExM (proExM), a variant of ExMin which proteins are anchored to the swellable gel, allowingthe use of conventional fluorescently labeled antibodies andstreptavidin, and fluorescent proteins. We validated anddemonstrated the utility of proExM for multicolor superresolution (~70 nm) imaging of cells and mammalian tissueson conventional microscopes. (Tillberg et al.)
II. PART 1
Materials
Stock-X monomer solution (prepared for you)*,**
10 mg/mL Acryloyl-X in anhydrous DMSO (prepared for you)*
PBS
10% ammonium persulfate (APS) (prepared for you)*
10% N,N,N′,N′-tetramethylethylenediamine (TEMED)(prepared for you)*
0.5% 4-hydroxy TEMPO (4HT) (prepared for you) *
1x digestion buffer (prepared for you)*,***
Proteinase K, 800 U/mL
MilliQ or double-distilled H2O
ice bucket
Microscope Slides (untreated)
coverslips (No. 1.5)
Small watercolor paintbrush
parafilm
4 mini binder clips
35 mm petri dish
10 cm petri dish
razor blade
1. treat a brain slice with acryloyl-X, which involves an overnight (or >6 hrs) incubation.
We prepared the brain slides with Acryloyl-X Treatment and incubated them in 37C.
Before we treated the brain slice with Acryloyl-X we check the brain slice on a microscope to get the situation before expansion.
III. PART 2
1. Gel-embedding and digestion
We used a paintbrush to gently transfer the brain slice into the activated monomer solution.
We rinse the brain slice with PBS to remove residual AcX, and then wash it twice for 3 minutes with PBS.
Incubate on ice for 30 mins, solidify for an hour and we put the slides into our digestion solution into a 35 mm petri dish. Then we wait overnight
IV. PART 3
1. Expansion and visualization
We carefully(brain slides very delicate!) pipet away the digestion solution and than wash the gel 3 times for 10 minutes in deionized water to get the expansion. Finally we removed the water and image the expanded brain slides on the microscope using the settings for GFP.
The neurons expanded a lot!
