FAQ’s: Glass Bottom Dishes

A general procedure for their use follows.

1. Maintain sterility: Open dishes in a sterile environment (e.g. laminar flow hood).
2. Pre-equilibrate dishes: Incubate the dishes with culture medium. Pipet 2-3 ml of medium into the 35 mm dishes or 3-4 ml into the 50 mm dishes and incubate at 37° C for 15 minutes.
3. Add cell suspension to microwell: Remove the culture medium by aspiration and plate cells onto the glass surface. Pipet 250 µl of the cell suspension (cells suspended in culture medium) into the 10 mm diameter microwells, 500 µl of cell suspension into the 14-mm microwells, or 1 ml of cell suspension into the 20-mm wells. Incubate the dishes for 1 hour at 37° C.
4. Add additional medium: After 1 hour, gently fill the remainder of the dish with medium. Add 2-3 ml to the 35 mm dishes or 3-4 ml for the 50 mm dishes.

Note: After the initial one hour period to allow cells to attach to the glass surface, it is important to fill the dish to normal levels in order to minimize the effects of evaporation and to avoid inducing changes in osmolarity.

Note: Since almost all microscope objectives are optimized for use with No. 1.5 coverslips, the No. 1.5 coverslip thickness will produce the highest quality images.

Note: If the exact glass bottom dish part number is unspecified in a literature article, it is safe to assume that uncoated dishes were used (e.g. Part #’s: P35G-1.5-14-C, P50G-1.5-30-F, etc.)

1. Under sterile conditions, pipette 250 µl of 1 N HCl onto non-coated 10-mm glass bottom dishes (e.g. Part #’s: P35G-x-10-C or P50G-x-10-F); use 500 µl or 1 ml of 1 N HCl for the 14-mm and 20-mm glass bottom culture dish, respectively (e.g. Part #’s: P35G-x-14-C or P35G-x-20-C).
2. After 15 minutes, decant the HCl and rinse the dish 3x with phosphate buffered saline (PBS) and 2x with ultrapure H2O.
3.  Apply the coating to the dishes.
4. Add a similar volume of the medium in which you will plate your cells to pre-equilibrate the glass surface. Incubate the medium in glass bottom dishes for 15 min at 37°C. Remove the medium and then plate your cells.

To try a sample of non-coated glass bottom dishes, go to our free sample page.

If necessary (e.g. for long term storage purposes), the coverslip can be removed using the following procedure:

1. Order Part # PDCF OS 30 (Fluid for removal of coverslips from glass bottom dishes)
2. Invert the cover of the dish.
3. Pipette 1.0 ml of fluid into the inverted cover.
4. Place the bottom of the dish onto the cover. Make sure that the liquid in the inverted cover is touching the bottom of the coverslip.
5. Allow the dish to sit in the fluid for 45 minutes at room temperature.
6. Dry the bottom of the coverslip with an absorbent paper towel.
7. Place the dish on a clean surface. Using forceps, press down on the inner edge of the coverslip to separate the coverslip from the dish.

Note: If the above procedure is followed, the PDCF OS 30 fluid will not contact the cells and will not disrupt cells on the coverslip or the staining thereof. Coverslips can be removed without breakage.

In order to approximate physiological conditions, the temperature of the medium contained within the glass bottom dishes can be controlled by using a microscope stage heater and an appropriate stage adapter.

For use with the P35G dishes (Corning 35 mm dishes): Culture dish heaters (part#: DH-35), microscope stage adapters (part#: SA-microscope type), heater controller (part#: TC324-B), and connecter cable (part#: CC-28) are available from Warner Instrument Corporation. Information is available online at: https://www.warneronline.com/

The gridded coverslips allow one to refer to specific cells and follow them over time. For instance, individual cells can be microinjected, returned to the incubator, and observed at multiple time points since each cell can be identified with a unique alpha-numeric coordinate in the dish. Glass bottom dishes containing gridded Bellco Glass coverslips are available. Standard gridded dishes are designated as part #’s: P35G-1.5-14-CGRD and P50G-1.5-14-FGRD.

Grid size: The grid on part #’s P35G-1.5-14-CGRD and P50G-1.5-14-FGRD consists of 520 unique alphanumeric squares. Each square measures 600 μm x 600 μm. The line thickness is 20 μm.
Visualization of the grid: The grid should be readily visible using a 10X brightfield objective. After locating the cell of interest, simply switch to a higher magnification or fluorescence objective. The grid will not be visible using higher magnification or fluorescence objectives.
I can’t see the grid: A confluent monolayer of cells will typically mask the grid making it difficult or impossible to visualize. If your cells are confluent, you can utilize Part # P35G-1.5-14-CGRD-D which positions the grid on the outside of the dish where it is unaffected by the cells growing on the coverslip. To use this dish, find a cell of interest and then focus down to the bottom side of the coverslip to get its coordinates.

Gridded coverslips (18 mm x 18 mm, No. 1.5 thickness, non-sterile, part# PCS-1.5-1818-GRD) are available for purchase.

1. Incident ultraviolet rays with wavelengths longer than 320 nm do not cause fluorescence.
2. Mercury lines at 334 and 365 nm do not create auto-fluorescence. (Note: For mercury illumination, filter out the mercury lines with wavelengths shorter than 313 nm to obtain best possible results.)
3. Refractive index (@ 20°C): nd= 1.5230 tolerance ± 0.0015
4. Abbe number V = 55.

For super-high resolution microscopy techniques, we offer glass bottom dishes with high tolerance No. 1.5 coverslips designated in part #s as -0.170 (e.g. part #: P35G-0.170-14-C).

The actual thickness of the glass coverslips depends on the Coverslip No./part #, as follows:

MatTek uses the highest quality, borosilicate German glass coverslips in our glass bottom dishes. The coverslip properties are as follows:

1. Highest hydrolytic resistance (hydrolytic class 1).
2. Excellent resistance to chemicals.
3. Emission of alkali approximately 15 to 24 µg Na2O/g glass.
4. Excellent properties for fluorescent microscopy.
5. Incident ultraviolet rays with wavelengths longer than 320 nm do not cause fluorescence.
6. Mercury lines at 334 and 365 nm do not create auto-fluorescence.(Note: For mercury illumination, filter out the mercury lines with wavelengths shorter than 313 nm
   to obtain best possible results.)
7. Refractive index (@ 20°C): nD ** = 1.5230 tolerance ± 0.0015.
8. Abbe number V = 55.

The depth of the micro-wells in the glass bottom dishes depends on the type of dish and the thickness of the dish bottom as follows:

1. Corning 35 mm: 0.70-0.75 mm
2. Falcon 50 mm: 1.00-1.10 mm
3. Falcon 60 mm: 1.15-1.20 mm
4. Falcon 100 mm: 0.90-1.00 mm*
5. Falcon 6-well plate: 1.45-1.55 mm
6. Falcon 12-well plate: 1.45-1.55 mm
7. Falcon 24-well plate: 1.10-1.20 mm
8. Falcon 96-well plate: 1.05-1.25 mm

The body of the glass bottom dishes and multi-well plates is made from polystyrene. Therefore, they have limited compatibility with organic solvents. Please see the chemical compatibility table.

The P35G-0.170-14-C dishes will improve picture quality (see Figure below) versus the P35G-1.5-14-C dishes for any high numerical aperture objective used in confocal, fluorescence, GSDIM, dSTORM, PALM total internal reflection (TIRF), and other high numerical aperture objectives. For example, quantitative measurements using P35G-0.170-14-C dishes gave z-resolution of +/- 9.5% while z-resolution in the P35G-1.5-14-C dishes gave z-resolution of +/- 17.3% (n=5).

A
B

Figure: Improved Z-axis resolution – Effect of high tolerance glass coverslips (in P35G-0.170-14-C glass bottom dishes) on imaging of sub-resolution beads using:
A) P35G-0.170-14-C and B) P35G-1.5-14-C glass bottom dishes.
5 µl of FITC labeled 175nm PS-Speck sub-resolution beads were added to well of the glass bottom dishes and allowed to dry. After drying, 200 ul of water were added and the beads were imaged using a Zeiss LSM510 confocal microscope equipped with an Olympus UPLSAPO 60x (NA=1.2) water immersion objective.

Figures and measurements courtesy of Teemu Ihalainen, Ph. D., University of Jyvaskyla, Finland (2008).