The overall goal of this study is to use digital holographic microscopy or DHM to provide automated stain-free quantitative phase contrast imaging of intestinal inflammation colonic tissue samples for the multi-modal assessment of epithelial wound healing in vitro. This method can help answer key questions in the field of gastrointestinal inflammation and wound healing about the disease activity inflamed human and marine The main advantage of this technique is that it allows the detection and quantification of cell and tissue by physical parameters in a stain-free and objective manner. Demonstrating the procedures will be Fittest Ketelhut, a technician, from the Biomedical Technology Center.
Set up the cell culture, incubate four times ten to the fifth, Caco two cells per centimeters squared in 35 millimeter Petri dishes equipped with high culture inserts in RPMI medium supplemented with FBS and antibiotics at 37 degrees Celsius in 95%humidity and five percent carbon dioxide. After two days, treat the cells with the agent of interest and return the cultures to the incubator for an additional 24 hours. The next day, aspirate the supernatant and use tweezers to remove the inserts.
Wash the culture one to two times with one milliliter of HEPES buffered cell-cultured medium to remove any dead cells and any remaining cellular components from the wound area. Then, add two milliliters of fresh HEPES buffered medium and close the dish. Now place the Petri dish in the digital holographic microscope and select the 10 x objective.
Open the image acquisition software and select the bright field imaging mode. Set the heating chamber to 37 degrees Celsius and place the culture into the heating chamber. Next, move the viewing field until a suitable wound with single humongous cell layer and straight borders on both sides with no dead cells or serum can be visualized in the live monitoring window.
Capture a white light image of the initial wound area. Then, turn of the white light illumination and select the digital holographic microscopy mode. Switch on the laser illumination and select an exposure time of less than three milliseconds for the hologram recording.
If the holographic off-access interference fringes appear with an adequate contrast, capture a sample hologram and reconstruct a quantitative phase image with the reconstruction software to check the image quality. Then, start a time lapsed DHM observation of the wound stopping the recording at the appropriate endpoint and obtain a final image of the sample under white light illumination. For Acute Dextran Sodium Sulfate, or DSS colitis induction, provide three grams of DSS dissolved in 100 milliliters of autoclaved water to mice ablibidim.
After seven days, obtain seven micrometer thick sections of Swiss rolled colon samples from the control and treated animals. Immerse each section in 50 to 100 micro liters of PBS and cover the samples with individual glass cover slips. Confirm that the bottoms of the slides are free from dust and other contamination.
Then, place the first slide on the microscope stage and adjust the field of view until the tissue area of interest is visible. Bring the sample into sharp focus and capture a bright field image in the image acquisition software. Then, select the DHM imaging mode and switch to the laser light illumination.
Set an exposure time of under three milliseconds and confirm that the holographic off access interference fringes appear with an adequate contrast in the live imaging window. Then, capture a digital hologram of the tissue. Endoscopically the evidence of moderate to severe colitis includes colonic wall-thickening, alterations of the vascular pattern, fiber nedudations, and mucosal surface granularity.
Conventional histological evaluation of the colonic sections reveals a marked edema in the colonic wall of DSS treated versus the control animals predominately located in the submucosa. Corresponding quantitative DHM phase contrast images and related refractive index maps also, demonstrate that these tissue alterations with no visible differences in the densities of epithelial menstroma are especially apparent in the native unstained tissue samples. The refractive index is significantly reduced in the epithelium, submucosa, and stroma of the colitic mice compared to the healthy controls with a significant correlation between the relative loss of body weight and the refractive index in the experimental animals.
Here the representative continuous monitoring of a wound healing assay by digital holographic microscopy over 45 hours is shown. Allowing the calculations of the cell covered surface area, average cell thickness, and cellular dry mass over the course of the healing process. We have shown that DHM allows continuous monitoring of epithelial wound healing while simultaneously providing bio core information about cytokine induced cell alterations.
Moreover, we demonstrate the accurate assessment of histological damage in colitis models and human colonic tissue samples by DHM through measurement of refractive index changes. As DHM is based on the detection of the optical path lengths delay, absolute biophysical parameters, such as tissue density, dry mass, or cellular thickness, can be analyzed by stain-free quantitative phase contrast imaging. The culmination of DHM with common research microscopes and the robustness of the technique paves the way for routine applications of this method in cell biology and digital pathology.
After watching this video, you should have a good understanding of how to use digital holographic microscopy for the analysis of tissues of living cell cultures in the biomedical laboratory environment. In conclusion, DHM creates a valuable, novel, and quantitative tool for assessment of inflammation absolute parameters, as well as for the simplified quantification of epithelial voluntary in vitro with a high potential for translational diagnostic use.
