Visualization and Microscopy

The Microscopic Examination of Samples Can Be Performed in Three Modes:

Viewing from below the plate (through transparent PET or CM membranes)
Millicell devices using PET or CM membrane have been designed to allow visualization of cells from below using an inverted microscope. For viewing live cells, microscopic observations can be made through the receiver or plastic plate containing media. In order to focus on the cells, the microscope objective (typically 5–20X) must have an appropriate working distance. (For objective specifications, visit the websites listed in the Microscope Objective Information section.) Fixed cells that do not require to be visualized in media can be viewed directly without the receiver plate. However, care should be taken not to contaminate the objective with liquid residue (media, mounting fluid) on the membrane.
Viewing from above the plate (Millicell 6-well inserts, Millicell-24 cell culture insert plates)
Some cell culture platforms can allow the cells to be viewed in a conventional microscope directly from above using low magnification. Cells can be visualized through the lid to maintain sterility or with the lid removed for fixed cells or when maintenance of sterility is not required. Working distances of the objective must be longer when reading from above compared to when reading from below. If using immunofluorescence, it is recommended to use a mounting fluid that contains an anti-fade additive to prevent photobleaching.
Visualizing membranes on microscope slides (for higher magnification or withobjectives with short working distances [less than 2 mm])
The membrane can be removed from each well for microscopic evaluation. This allows for higher magnification examination and storage of the slides for future use.

For visualizing from above the membrane, typically 5–20X objectives are used that have at least a 13.59 mm (A) or a 18.03 mm (B) working distance when viewing without or with the lid, respectively. For visualizing from below the membrane, 5–20X objectives are used that have at least a 2 mm (C) working distance.

To prepare membranes on microscope slide

Remove the membrane from the well using a sharp scalpel to make a small incision in the edge of the membrane. Carefully cut along the inside of the well wall for approximately one quarter of the well diameter. Using forceps (Millipore cat. no. XX62 000 06), carefully hold the membrane while continuing to cut around the well diameter to remove membrane. Alternatively, a cork borer may be used to remove the membrane.

Note: Use care to prevent membrane from curling.
Place the membrane disk, cells facing up, onto a microscope slide.
Add 50 µL mounting fluid to the membrane disk and allow it to wet out in order to prevent bubbles under the disk.
Slowly lower a cover slip onto the membrane at an angle to allow air bubbles to be removed.

Microscope Objective Information

Information regarding microscope objective magnification power and working distances can be obtained from individual optical dealers or from the microscope vendors:

Nikon Instruments: http://www.nikoninstruments.com
Olympus Corporation: http://www.olympusamerica.com
Carl Zeiss: http://www.zeiss.com

Note It is assumed that users of this procedure will be knowledgeable in TEM procedures.

Materials and Reagents

Millicell Cell Culture Inserts
Phosphate Buffered Solution (PBS)
Glutaraldehyde
Osmium tetroxide
Sodium cacodylate
Sucrose, reagent grade
Calcium chloride
Lead nitrate
Sodium citrate/Sodium hydroxide
Uranyl acetate
Ethanol
Flat embedding trays
Embedding Resin (i.e. EPON812)
Fine forceps — Millipore cat. no. XX66 000 06
Diamond Knife
Cork Borer
Durapore Filter Disk — Millipore

Sample Pictures

A. Processing/Cell Preparation

Note: Steps 1–5 should be done on an intact Millicell cell culture insert or plate well.

Wash cells briefly (2 times for 5 minutes each) at room temperature with phosphate buffered solution without fixative.
Fix cells in 2% glutaraldehyde in 100 mm sodium cacodylate buffer, pH 7.5, at room temperature from 15 minutes to 2 hours.

Note: At this point, cells can be stored in the above buffer with 7 g sucrose/100 mL buffer at 4°C.
Fix cells in 1% osmium tetroxide in either 100 mm sodium cacodylate or suitable phosphate buffer.
Dehydrate cells in the following concentrations of ethanol:

Ethanol Concentration Kit (%)	Time (minutes)
30	15
50	15
70	15
95	15
100	3 x 15

Note: Dehydration of Millicell-HA units should be performed in a metal pan that will be used as the embedding tray due to the tendency of the cellulosic membranes to be less rigid during the dehydration process. Attempts to transfer the membranes during these steps could lead to mechanical damage to the cells.

For infiltration, EPON812, an EDPON substitution, or LX112 is suitable for both devices (do not use Spurr’s). The following is a general infiltration scheme:

Ethanol Concentration Kit (%)/Tray (% Plastic)	Time (minutes)
75/25	30 on a shaker
50/50	30 on a shaker
0/100	30 each/3x on a shaker
0/100	Overnight

Note: It is not necessary to use any other agent, such as propylene oxide, with plastic. Propylene oxide will dissolve the cellulosic filters. In addition, the standard inversion/rotation of specimens used in these steps is not advised since either (1) damage to the cell layer or (2) stretching of the cellulosic filter may occur. Mild shaking on a gel shaker apparatus is sufficient for successful infiltration.

Note: Before the next step the membrane must be detached from the surrounding plastic ring. Sometimes this will occur without manipulation since the EPON may loosen the membrane-to-ring bond. If this does not occur, use a sharp scalpel or a cork borer and cut the membrane. It may also help to cut the membrane over a 47 mm filter support disk. Under no circumstances should the membrane be left attached to the ring during polymerization.

Transfer to fresh plastic and polymerize at 68°C overnight.

B. Sectioning Notes

Nitrocellulose (HA), polycarbonate (PC) and polyethylene terepthalate (PET) membrane: These membranes can be sectioned in any plane without difficulty.
CM (Biopore) membrane: The Biopore membrane must be processed in one of two ways based on the final thickness of the section.

Sections of 90 nanometers (nm) or thicker – for standard transmission electron microscopy In sections of 90 nm or thicker, the cells will adhere to the Biopore membrane. Membranes should be oriented with the axis of the membrane/cells perpendicular to the knife-edge. This orientation will allow sections of cells to be made without passing them over the knife that has been previously used to section the underlying Teflon®. Some wrinkling of the section occurs because after it is sectioned, the plastic may expand slightly but the Biopore membrane will not. However, this problem is minimal and minor wrinkles resolve under the heat of the electron beam.

Note: Best sectioning of the CM membrane is achieved with a diamond knife.
Sections of less than 90 nm — for exceedingly thin sections. In sections of less than 90 nm, the cells will be separated from the membrane after sectioning because they will not adhere to the Biopore membrane. Sections of this size must be mounted on a Formvar® (polyvinyl formal)-coated grid to prevent edges from curling during mounting. The separation of the cell layer from the Teflon does not damage the membrane surface of the cell membrane. Therefore, studies examining the basal surface of cells can be accomplished using sections of less than 90 nm.

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