Candida albicans biofilms like many biological specimens are heavily translucent to opaque in their native state. In these protocols, we show that using clarification methods, the internal structure of fixed intact biofilms can be imaged by optical sectioning microscopy. With simple, inexpensive, and relatively fast processing steps, fixed intact biofilms can be made transparent for 3D imaging by confocal scanning fluorescence microscopy.

Demonstrating this procedure today will be Katie Lagree, a post-doctoral researcher here, and myself. To begin biofilm growth in a 12-well plate, prepare the plate as described in the text protocol. Place the plate on a 60 RPM orbital mixer in a humidified 37 degrees Celsius air incubator for 90 minutes to allow time for cell adhesion.

After 90 minutes, remove the medium and unattached cells and wash with sterile medium or PBS. Transfer the inoculated substrata into another multi-well plate containing pre-warmed filamentation medium. Return the plate to the 37 degrees Celsius humidified ambient air incubator and grow the biofilms up to 48 hours with 60 RPM orbital mixing for aeration.

Retrieve the 48-hour plate from the incubator and remove the culture medium from each specimen. Replace the medium with PBS and incubate for several minutes to dilute away serum proteins. After removing the PBS, refill the wells with fixative.

Sufficient fixative volume should be added to each dish to immerse all biofilm growth including any on the inner sides of the dish. Set the covered dish on a slow orbital mixer for 20 minutes. After removing the fixative as described in the text protocol, prepare for staining by draining away the PBS and quickly refilling the wells with a solution containing PBS and the requisite stain.

Do not allow the biofilm to drain or dry for more than a few seconds. Add the stain to the dish. The needed quantity of stain depends on the biofilm mass.

Then set the covered dish on a slow orbital mixer overnight and protect from light with foil or a piece of black paper. Using tweezers, transfer the fixed stained biofilm from PBS to five milliliters of 50/50 PBS-methanol in a 20 milliliter glass vial with the biofilm facing up. Manually mix with orbital motion at one minute intervals for five minutes.

Remove the solvent to a waste bottle being cautious to avoid contact between the pipette and biofilm or the biofilm and vial. Gently refill with three milliliters of neat methanol. Manually mix with orbital motion at one minute intervals for three minutes.

Now remove the solvent to a waste bottle and immediately refill with five milliliters of methanol. After manually mixing with orbital motion at one minute intervals for five to 10 minutes, remove the solvent to the waste bottle. Immediately refill with three milliliters of methanol.

Biofilms often look more opaque at this point than their initial appearance. Remove the solvent to the waste bottle and immediately refill the vial with five milliliters of 50/50 methanol-methyl salicylate. Manually mix with orbital motion at one minute intervals for five to 10 minutes.

The biofilm should be semi-transparent in this mixed solvent. After removing the solvent to a waste bottle, gently refill the vial with three milliliters of neat methyl salicylate. Manually mix with orbital motion at one minute intervals for three minutes.

Repeat the process again with five milliliters of neat methyl salicylate, manually mixing at one minute intervals for five to 10 minutes. At this point, the biofilm should be transparent. Remove the solvent to the waste bottle and immediately refill the vial with three milliliters of neat methyl salicylate.

The processed biofilm is stable in this solvent. If using an inverted microscope, use a solvent-proof dish that has a coverglass bottom and prepare spacers for support of the inverted specimen. Here, spacers are 13 millimeter silicone rings that are pre-soaked in methyl salicylate for one hour to minimize focus drift due to swelling.

For a biofilm on a medical grade silicone square, invert the square and set it on a spacer in a pool of methyl salicylate in the dish. Make sure to avoid any bubbles below the specimen. Mount the dish firmly on the stage of the microscope and make oil immersed contact with the objective.

Adjust the amount of methyl salicylate in the dish so that the meniscus holds the specimen firmly down on the spacer by surface tension. Place a droplet of methyl salicylate on top of the inverted square substratum to reduce light scatter from the matte finish. Then cover the dish with a glass plate to limit evaporation.

Wait several minutes for the specimen to settle onto the spacers prior to imaging. Now, perform confocal microscopy to image the specimen as described in the text protocol. To process the images, open ImageJ or Fiji.

Check to make sure the program has sufficient assigned memory for the data to be processed. Processing a two gigabyte dataset requires at least eight gigabytes of dedicated RAM for a smooth operation. Open the serial plane image file to be processed.

If the computer has insufficient RAM to process the entire stack, substacks may be processed and then reassembled into a single stack. Convert the data to 32-bit format to enable floating point arithmetic operations using image menu, type, 32-bit. This will double the file size.

Subtract the smooth background to increase the contrast of in-focus features. Process the entire stack by navigating to process menu, subtract background. Adjust the rolling ball radius if necessary.

The ball radius should be significantly larger than the largest dimension of the in-focus features to be preserved. To set all negative pixels to zero, add each image to its absolute value. First, duplicate the stack using image menu, duplicate, duplicate stack.

Then take the absolute value of the duplicated data using process menu, math, absolute value. Add the two stacks by navigating to process menu, image calculator, add. To axially reslice the image stack, use image menu, stacks, reslice.

Then select output spacing 1.0, start at top and avoid interpolation. The focus axis is now the vertical axis in the stack. The Y-dimension of the resliced stack equals the number of focus planes.

To make a side view projection of some or all of the stack, navigate to image menu, stacks, Z project. Set start slice and stop slice to include some or all of the side view planes. Select max intensity for best results.

Correct the axial scaling of the side view projection by navigating to image menu, scale. The projection must be corrected for the unequal image plane pixelation and focus increment using the axial scale factor. Set the X-scale to one and set the Y-scale to the axial scale factor.

Select bicubic interpolation. Select create new window. Adjust brightness and contrast.

Then convert to eight-bit format for display. Alternatively, apply a color lookup table and save as RGB or eight-bit color. Using serially miscible solvents of increasing refractive index, the maximum in clarity can be quickly estimated.

This is refined by conventional phase contrast microscopy to find the point of minimum contrast. One biofilm was serially exchanged between xylene and a narrow set of reference liquids in that range. After each exchange, the same field was relocated and viewed through a coverglass.

With increasing index, contrast reversal is first seen when n equals 1.530 for the cell wall, followed by cytoplasm when n equals 1.535. Deep imaging of mutant and wild type C.albicans biofilms is shown here. The wild type biofilm grew to a thickness of 502 microns as seen in the side view projection of the 538 plane confocal image stack.

Axial projections from apex to base show the variation in cell types and different strata of the biofilm. This example shows characteristic wild type structure. The mutant biofilm grew to a thickness of 500 microns as seen in the side view projection of the 556 plane image stack.

This mutant known to undergo filamentous growth in the absence of inducing stressors produced a dramatically different architecture. As seen in both the side view and axial projections, many branching cells give rise to radial outgrowths. This is a high refractive index deep imaging protocol.

Ideally, the refractive index of the specimen and the refractive index of the immersion oil should match. That is why in this case, we use an oil immersion objective. Most of our studies have been structural imaging of biofilms using cell wall markers, but any fluorescence method that is used with fixed specimens should work including immunofluorescence, fluorescence in situ hybridization, and reporter gene expression.

This particular organism, Candida albicans, is commensal in healthy humans, but is also an opportunistic pathogen that can cause mucosal or systemic yeast infections. At some institutions, BSL II containment methods and protocols are required. Additionally, formaldehyde and glutaraldehyde fixatives are volatile and hazardous.

They are known carcinogens. Make up dilutions of fixatives in a fume hood. Keep dishes containing diluted fixatives covered and do not put containers containing these fixatives in a biosafety cabinet or in a cell culture incubator.

Summary

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