Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

Summary

The CLEM technique has been adapted to analyze ultrastructural morphology of membranes, organelles, and subcellular structures affected by microinjected molecules. This method combines the powerful techniques of micromanipulation/microinjection, confocal fluorescent microscopy, and electron microscopy to allow millimeter to multi-nanometer resolution. This technique is amenable to a wide variety of applications.

Abstract

The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our understanding of cellular regulation. Imaging techniques such as fluorescent and confocal microscopy make ascertaining the position of a fluorescently tagged small molecule relatively straightforward, however the resolution of very small structures is limited ¹.

On the other hand, electron microscopy has revealed details of subcellular morphology at very high resolution, but its static nature makes it difficult to measure highly dynamic processes with precision ^2,3. Thus, the combination of light microscopy with electron microscopy of the same sample, termed Correlative Light and Electron Microscopy (CLEM) ^4,5, affords the dual advantages of ultrafast fluorescent imaging with the high-resolution of electron microscopy ⁶. This powerful technique has been implemented to study many aspects of cell biology ^5,7. Since its inception, this procedure has increased our ability to distinguish subcellular architectures and morphologies at high resolution.

Here, we present a streamlined method for performing rapid microinjection followed by CLEM (Fig. 1). The microinjection CLEM procedure can be used to introduce specific quantities of small molecules and/or proteins directly into the eukaryotic cell cytoplasm and study the effects from millimeter to multi-nanometer resolution (Fig. 2). The technique is based on microinjecting cells grown on laser etched glass gridded coverslips affixed to the bottom of live cell dishes and imaging with both confocal fluorescent and electron microscopy. Localization of the cell(s) of interest is facilitated by the grid pattern, which is easily transferred, along with the cells of interest, to the Epon resin used for immobilization of samples and sectioning prior to electron microscopy analysis (Fig. 3). Overlay of fluorescent and EM images allows the user to determine the subcellular localization as well as any morphological and/or ultrastructural changes induced by the microinjected molecule of interest (Fig. 4). This technique is amenable to time points ranging from ≤5 s up to several hours, depending on the nature of the microinjected sample.

Protocol

1. Mammalian Cell Culture

1. Normal Rat Kidney (NRK), immortalized cervical cancer (HeLa), or other appropriate mammalian cell lines are cultured at 37°C, 5% CO₂, on photoetched glass gridded coverslips affixed to live cell dishes (see Table 1 for any reagent or apparatus used in this protocol) in DMEM + 10% FBS and 1% Pen/Strep. Precautions should be taken to maintain a sterile environment when working with cultured mammalian cells.
2. Depending on your experimental time-line (0-5 hours vs 1-2 days post injection) the confluency of cells should be adjusted to ~50% or ~25%, respectivel....

Discussion

The method presented here enables the direct delivery of purified proteins, nucleic acids, or small molecules to the eukaryotic cytoplasm and affords ultra high-resolution analysis through the correlation of fluorescent and electron microscopy. The use of this method is simple yet robust and can be performed in-house with many existing core facilities and/or appropriately equipped electron microscopy labs. The technique is also amenable to live-cell, allowing the user to monitor the dynamics of fluorescently tagged molec.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Phot–tched Coverslip and Live-cell Plate	MatTek Corp.	P35G-2-14-CGRD
Texas Red	Invitrogen	D3329
Cascade Blue	Invitrogen	D7132
Rhodamine Labeling Kit	Thermo Fisher Scientific, Inc.	53002
0.22 μm centrifugal filtration unit	EMD Millipore	UFC30GV00
Borosilicate Glass Pipettes	Sutter Instrument Co.	BF100-50-10
Micropipette Puller	Sutter Instrument Co.	Model P-97	Use program #4 after performing ramp test
FemtoJet Microinjection System	Eppendorf	Injectman NI2
Coverslip Removal Fluid	MatTek Corp.	PDCF OS 30
Technai Transmission Electron Microscope	FEI	Technai G2 BioTWIN
Ultramicrotombe	Leica Microsystems	EM UC6