Our lab is interested in understanding how intracellular pH dynamics modulate cellular behaviors. We are interested in behaviors that contribute to cancer progression because we know that intracellular pH is increased in most cancers. We do a lot of microscopy and develop protocols to quantify data from these images.
We're learning more about cellular pathways and processes that are regulated by pH. Most cancer cell lines have a higher intracellular pH and many show increased levels of autophagy, but we did not know this was connected. We recently showed that higher intracellular pH in genetically normal cells is sufficient to increase autophagy.
Our lab is one of only a few labs studying how intracellular pH or PHI regulates cell behaviors in a whole animal model. We have found that increased PHI promotes increased cell proliferation, invasive cell migration, and tissue disorganization. Most recently, we found that higher PHI promotes autophagy, which is a form of cellular cannibalism.
We are addressing the gap created by the high cost and complexity of preparing samples for SEM, which limits its accessibility for many labs. At San Jose State, we lack the resources for SEM. So we developed a more accessible and affordable protocol allowing us to capture the high resolution images without the need for expensive equipment.
This protocol combines traditional techniques used in entomology and offers a cost effective and user-friendly alternative to scanning electron microscopy. It can be easily adapted to capture high resolution images of different parts of the fly or different specimens. To begin, select the desired drosophila strain and place it in a vial containing fly food.
Incubate the vial at 25 degrees Celsius until the flies have grown and the adults are close. Next, anesthetize the adult flies with carbon dioxide and place them on a carbon dioxide pad. Prepare a feather fly sorter by trimming a goose feather to fit the tapered end of a one milliliter serological pipette.
Sort the flies with a feather and select the individuals with straight wings. Next, add one milliliter of 70%ethanol into a 1.7 milliliter micro centrifuge tube. Transfer the selected flies into the tube and place the tube on ice.
Using a specialized point punch. Cut small triangular points from 65 pound archival card stock. Then use Dumont number five fine tip forceps to bend the tip of each point to a 90 degree angle.
Remove the flies from the micro centrifuge tube using Dumont number five fine tip forceps. Gently blot the flies with lint-free tissue to remove excess ethanol. Place each fly on its left side on an index card under a dissecting microscope.
Using a transfer pipette, mix one to two drops of hide glue with one to two drops of deionized water on an index card. Pick up a prepared card point at the broad end with forceps and dab the bent tip into the glue water mix to apply a small amount of glue. Apply the bent tip of the card point to the anterior side of the fly's right abdomen around abdominal segments two and three.
Before the glue dries, adjust the fly so that its anterior posterior axis is perpendicular to the bent tip of the card point. Insert a number three mounting pin into the wide end of the card point, and secure it to an insect pinning block. Label each pin or row of pins with the corresponding genotype.
To acquire high resolution photographs of point mounted drosophila eyes turn on the stacking imaging system. Then set up a DSLR camera body with a 70 to 200 millimeter telephoto lens attached to a 20x Apo microscope objective via a 77 millimeter lens adapter. Ensure the specimen is illuminated with a flash through a diffuser.
Control the Z positioning of the camera using a StackShot controller and macro rail. Position the point mounted fly on the universal stage gimbal with the eye facing the lens. Adjust the head position, carefully using forceps for optimal alignment.
Tether the camera to a laptop and adjust the acquisition settings. Set the magnification to 20x, shutter speed, 1/200 of a second, aperture f/2.8 and ISO 400. Then, set the location for saving the resulting image stack to the desired file folder.
Now, adjust the focus stack settings on the StackShot control unit in auto distance mode. Set the step size to five micrometers and calculate the number of steps by setting the start and stop positions of the focus stack. View the specimen in live view mode with the camera in auto shoot mode.
Move the rail so that the closest part of the specimen is in focus. Then, move to the furthest feature of interest and adjust the focus. Return the camera back to manual shoot mode and start the image acquisition from the StackShot control unit.
After imaging, open the acquired image files in the referenced focus stacking software. Select Stack, then align and stack all or Pmax to generate a stacked image. Then click File and save output image to save the final stacked image as a tif file.
After imaging pin-mounted drosophila eyes, select the stacked image for analysis where the eye is centered and aligned with adequate lighting and minimal peripheral blurring. Open the 500 micrometers scale bar image in Fiji software. Measure the length of the scale bar using the straight line tool.
Click Analyze and Measure to record the pixel distance, which corresponds to 500 micrometers. Then, calculate pixels per micron and use this to convert pixel measurements into micrometer measurements. Open the stacked image file in Fiji.
Select magnifying glass from the toolbar to enlarge the area of focus. Fill the screen with the eye and surrounding head cuticle. Next, from the toolbar, select the freehand select tool and outline the retinal area following the outermost row of ommatidia.
To remove part of the selection, hold down the option button and select the pixels to remove. To add to the selection, hold down the option and shift buttons and select the pixels to add. To calculate the area, select Analyze and Measure.
A new window will display the area, mean, minimum and maximum parameters. Copy and paste the data into a spreadsheet for documentation and conversion from pixels to micrometer measurements. Over-expression of DNhe2 in GMR-DNhe2 flies resulted in significantly smaller adult eyes compared to wild type adult flies.
GMR-DNhe2 flies showed a 41%reduction in eye area compared to wild type flies and a 48%reduction compared to GMRGAL4 heterozygous flies. Eye size was restored in GMR-DNhe2 flies heterozygous for Atg1 allele 3, bringing it to 129.9 square micrometers from 74.28 square micrometers.
