cell seeding and transfection on electron microscopy (em) grids for cryotomography

Vorbereitung von elektronenmikroskopischen Gittern für die in situ zelluläre Kryotomographie

In situ cellular cryotomography is a powerful technique allowing for the study of biologically-relevant structures in cells without chemical fixation. By attaching cells to EM grids and plunge-freezing the grids in a cryogen, objects of interest are frozen in their natural cellular contexts without the formation of crystalline ice from intracellular water1,2. Both chemical fixation and crystalline ice formation can disrupt the structures of relevant molecules, such as proteins and lipids, reducing the biological accuracy of images obtained using these techniques3,4. In tomography, grids are imaged at incremental angles using electron microscopy, and these images are then used to construct three-dimensional representations of the target region imaged5. In situ cryotomography can be used alongside other microscopy techniques for integrative and correlative imaging, such as cryofluorescence imaging, soft x-ray tomography, and cryoFIB/SEM (cryogenic Focused Ion Beam/Scanning Electron Microscopy)6,7,8,9,10,11. Integration of multiple techniques allows for more information to be obtained about a structure or process than any single microscopy technique could achieve.
Despite all of the benefits of in situ cellular cryotomography, sample preparation can prove to be challenging for a variety of reasons. Due to their fragility, forceful manipulation of electron microscopy grids can lead to damage, with the thin carbon layer in particular being delicate and prone to tearing, reducing the imageable area of the grids. Electron microscopy grids are also difficult to manipulate due to their small size and are prone to becoming detached from the surface of the wells or microslide used to grow cells. Manipulation of the grids within the wells or microslides can prove difficult due to the geometry of these. Improper preparation of the grids (e.g., allowing them to float) can lead to low cell density and reducing the number of potential imaging areas, especially when cells are not prone to attach to the grids themselves. For direct cellular cryotomography, cells must&#160;spread very thin, which can be disrupted for many reasons, including improper temperatures or rough handling of the grids.
Through a variety of optimizations, the techniques presented in this article are meant to handle these most common pitfalls which arise during the preparation of electron microscopy grids for cryotomography. The use of 5/15 angled tweezers allows for the manipulation of grids within well plates or microslides. A fibronectin solution applied to both sides of the grids prior to plating makes floating grids less likely, which is beneficial in ensuring that grids have adequate cell density and that the grids are less likely to be damaged due to manipulation. By keeping the grids incubated at 37&#176;C until just before plunge freezing, we also ensure that the cells are kept in a comfortable environment to prevent the cells from retracting their thin edges. Blotting the grids from the back side also prevents damage to the cells from mechanical force. Altogether, these measures increase the success rate of sample preparation for in situ cellular cryotomography studies, increasing the accessibility of this imaging approach.

Autor im Rampenlicht: Optimierung der Gittervorbereitung für eine verbesserte Kryoelektronentomographie 

Probenvorbereitung für die In-situ-Kryotomographie von Säugetierzellen

The scope of this research is to develop a methodology for sample preparation that is both accessible and reliably reproducible. By achieving this, the power of direct cellular cryo-electron tomography can be used to answer a wide variety of biological questions. The challenges of grid preparation are extensive.Cells do not readily attach to grids, and manipulation can result in damage to the carbon layer, resulting in the loss of imageable grid areas. Additionally, the prevalence of thin cellular protrusions is reduced without attention to proper sample preparation conditions. Current protocols typically require specialized equipment to solve the major bottlenecks of seeding, such as micro patterning and 3D printed holders for cell attachment and manipulation, respectively.This protocol provides solutions with greater accessibility, while still maintaining flexibility for these specialized tools to be used for downstream applications.

Watch this Scientific Journal Video about Cell Seeding and Transfection on Electron Microscopy EM Grids for Cryotomography at JoVE.com

Cell Seeding and Transfection on Electron Microscopy (EM) Grids for Cryotomography  

Zellaussaat und Transfektion auf elektronenmikroskopischen (EM) Gittern für die Kryotomographie

Um mit der Zellaussaat zu beginnen, zählen Sie die Zellen, nachdem Sie sie mit mechanischen oder enzymatischen Methoden abgetrennt haben. Geben Sie mit einer Mikropipette die berechnete Anzahl von Zellen pro Well in die Sechs-Well-Platte, die vorgefertigte Elektronenmikroskopie-Gitter enthält, und vermeiden Sie dabei Blasen. Stellen Sie sicher, dass 1,5 bis 2,0 Milliliter Zellsuspension pro Vertiefung beibehalten werden.Für die Transfektion von HIV-Molekülklonen werden die Zellen 16 bis 24 Stunden lang bei 37 Grad Celsius inkubiert. Geben Sie 50 Mikroliter serumfreies DMEM und ein Mikrogramm DNA in ein sauberes Mikrozentrifugenröhrchen. Verdünnen Sie jeweils drei Mikroliter Transfektionsreagenz in serumfreiem DMEM in ein separates, sauberes Mikrozentrifugenröhrchen.Homogenisieren Sie die Mischung separat mit einer Mikropipette. Anschließend wird die Transfektionsreagenzmischung per Mikropipette zur DNA-Mischung gegeben und 15 Minuten bei Raumtemperatur inkubiert. Danach geben Sie 100 Mikroliter des Transfektionsreagenzes und der DNA-Mischung tropfenweise in jede Vertiefung direkt auf die Gitter.Ersetzen Sie das Wachstumsmedium 16 bis 24 Stunden nach der Transfektion. 24 Stunden nach der Co-Transfektionsphase zeigten Mikroskopie und Fluoreszenzmikroskopie, dass alle Gitter nur minimale Risse in der Kohlenstoffschicht aufwiesen. Zellen sowohl auf den simulierten Gittern als auch auf den co-transfizierten Gittern enthielten lebensfähige Zellen in mehreren Gitterquadraten.

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Research

JoVE Journal

Biochemistry

Cell Seeding and Transfection on Electron Microscopy (EM) Grids for Cryotomography

423 Aufrufe.  University of Minnesota. Um mit der Zellaussaat zu beginnen, zählen Sie die Zellen, nachdem Sie sie mit mechanischen oder enzymatischen Methoden abgetrennt haben. Geben Sie mit einer Mikropipette die berechnete Anzahl von Zellen pro Well in die Sechs-Well-Platte, die vorgefertigte Elektronenmikroskopie-Gitter enthält, und vermeiden Sie dabei Blasen. Stellen Sie sicher, dass 1,5 bis 2,0 Milliliter Zellsuspension pro Vertiefung beibehalten werden.Für die Transfektion von HIV-Molekülklonen werden die Zellen 1...

Serie: Cell Seeding and Transfection on Electron Microscopy (EM) Grids for Cryotomography  

Sample Preparation for In Situ Cryotomography of Mammalian Cells

In situ cellular cryotomography is a powerful technique for studying complex objects in their native frozen-hydrated cellular context, making it highly relevant to cellular biology and virology. The potential of combining cryotomography with other microscopy modalities makes it a perfect technique for integrative and correlative imaging. However, sample preparation for in situ cellular tomography is not straightforward, as cells do not readily attach and stretch over the electron microscopy grid. Additionally, the grids themselves are fragile and can break if handled too forcefully, resulting in the loss of imageable areas. The geometry of tissue culture dishes can also pose a challenge when manipulating the grids with tweezers. Here, we describe the tips and tricks to overcome these (and other) challenges and prepare good-quality samples for in situ cellular cryotomography and correlative imaging of adherent mammalian cells. With continued advances in cryomicroscopy technology, this technique holds enormous promise for advancing our understanding of complex biological systems.

This method provides an accessible and flexible protocol for the preparation of electron microscopy (EM) grids for in situ cellular cryotomography and correlative light and electron microscopy (CLEM).

In situ cellular cryotomography is a powerful technique for studying complex objects in their native frozen-hydrated cellular context, making it highly ...

Forschung

Biochemie

Preparing Electron Microscopy (EM) Grids for Cellular Cryotomography

To begin preparing the electron microscopy grids for cellular cryotomography, place the grid, loaded with perforated carbon support film in a glow discharge device with the carbon side of the grid facing up. Then, glow discharge the grid at 15 milliamperes, under 0.39 millibar atmospheric pressure for 45 seconds. Once done, place the grid in a clean container, lined with filter paper.Next, transfer the grid along with fibronectin, phosphate buffered saline or PBS, plates and tweezers to a biosafety cabinet. Using a micro pipette, cast two generously sized dots of bovine fibronectin onto a sterile surface like a six well plate lid. Ensure the dots are large enough to envelop the entire grid.Then, obtain five by 15 tweezers and treat both sides of the grid with the fibronectin adherence solution. Once treated with bovine fibronectin, the grid will become sticky. Gently touch the non-carbon surface of the grid to the bottom of an empty well in a six well plate and release the tweezers to attach the grid to the new surface.Next, incubate the grid for 30 minutes in the fibronectin adherence solution. To prevent the grids from drying, add one to two drops of adherence solution on top of the grids, using a micro pipette every 15 minutes. Following the incubation, remove the excess adherence solution with a micro pipette.Wash the grids by adding drops of PBS directly on top of the grids, using the micro pipette two to three times. Sterilize the grids under UV light in a biosafety cabinet for one hour. Position the grids as close to the UV as possible for maximum sterilization.Prevent the grids from drying during sterilization by micro pipetting one to two drops of PBS on top of the grid every 10 minutes.

Vorbereitung von Elektronenmikroskopie-Gittern (EM) für die zelluläre Kryotomographie

To begin cell seeding, count the cells after detaching them using mechanical or enzymatic methods. Using a micro pipette, add the calculated number of cells per well in the six well plate containing pre-prepared electron microscopy grids while avoiding bubbles. Ensure to maintain 1.5 to 2.0 milliliters of cell suspension per well.For the transfection of HIV molecular clones, incubate the cells for 16 to 24 hours at 37 degrees Celsius. Add 50 microliters of serum free DMEM and one microgram of DNA to a clean micro centrifuge tube. For each, well dilute three microliters of transfection reagent in serum free DMEM into a separate clean micro centrifuge tube.Homogenize the mixture separately using a micro pipette. Then add the transfection reagent mixture to the DNA mixture via micro pipette and incubate for 15 minutes at room temperature. After that, add 100 microliters of the transfection reagent and DNA mixture drop wise into each well directly on top of the grids.Replace the growth medium 16 to 24 hours after transfection. 24 hours following the co-transfection phase liked microscopy and fluorescent like microscopy, showed that all the grids had minimal tearing in the carbon layer. Cells on both the mock grids and the co-transfected grids contained viable cells in multiple grid squares.

Plunge Freezing of Electron Microscopy (EM) Grids for Cryotomography

Before plunge freezing the electron microscopy grids containing the cells, inspect the grids under an inverted optical microscope. Record the cell densities on each grid to adjust blotting times during plunge freezing. Warm two to three milliliters of phosphate buffered saline or PBS in an empty plate at 37 degrees Celsius.For gold fiducials preparation, pipette 50 microliters of 10 nanometer colloidal gold bead solution into a clean 1.5 milliliter tube. Add two microliters of bovine serum albumin to it and homogenized by micro pipetting repeatedly. Centrifuge the tube at 15, 000 to 20, 000 G for 15 minutes.Carefully remove the supernatant without disturbing the pellet using a micro pipette. Re-suspend the pellet in 50 microliters of PBS and centrifuge the tube again, as previously demonstrated. Aspirate four microliters of the pellet using a 10 microliter tip and transfer it to a clean 1.5 milliliter tube.Set up the environmental chamber at 95%humidity and 30 degrees Celsius. Using five by 15 tweezers, select a grid and wash it with PBS. Transfer the washed grid to plunging tweezers.Once the grid is secured, remove the five by 15 tweezers and slide the clamp on the plunging tweezers. Insert the grid into the plunge freezer while keeping the clamp secure. Add one microliter of gold fiducials to the backside of the grid.Then add two to three microliters of PBS to the carbon side of the grid. Utilize automated blotting to blot the backside of the grid before plunge freezing into liquid ethane. A cryo correlative light and electron microscopy image of transfected U2OS cells revealed the presence of HIV producing cells.An electron cryo tomography image showed multiple HIV particles budding from the plasma membrane of U2OS cells.