The overall goal of this procedure is to measure a topology of cell adhesion on a substrate with nanometer precision. This is accomplished by first cultivating cells on transparent glass slides and incubating them with a fluorescent marker. Then the angular position of the microscope's adjustable mirror is calibrated and the position is stored in the mirror positioning program.
The next step is to record the fluorescence images upon irradiation at about 10 different angles. The final step is to calculate cell substrate distances with a precision of a few nanometers. Ultimately, results of topology of cell adhesion on substrate are used to show differences of adhesion between various cell lines, for example, cancer cells and less malignant cells.
The main advantage of this technique over existing methods like confocal microscopy, is that actual resolution is in the range of only a few nanometers. Though this method can provide insight into cell membranes. It can also be applied to other systems such as small vesicles or nanomaterials.
Demonstrating the experiment will be our postdoc Petra, who will prepare the cell samples and our engineer, Michael Wagner, who will run the experiment To prepare cells for variable angle, total internal reflection. Fluorescence microscopy seed individual cells at a density of 100 cells per square millimeter on a glass slide in culture medium supplemented with 10%fetal calf, serum, and antibiotics. Grow cells for 72 hours in an incubator at 37 degrees Celsius and 5%carbon dioxide.
Next, apply to the cells the fluorescent membrane, marker six D, decano two, dimethyl amino naphthalene, or lorane at a concentration of eight micromolar in culture, medium incubate for 60 minutes prior to fluorescence microscopy. Wash the cells with PBS pipette the buffer solution over the object slide to remove medium afterwards, wipe off the buffer solution from the backside of the object slide. An upright microscope such as a Zeiss Axio plan, equipped with a back illuminated and or exon E-M-C-C-D camera is used for variable angle.
T-I-R-F-M replace its condenser by a custom made illumination device with a hemi cylindrical glass prism optically coupled to the object slide. The laser beam is incident from a collated mono mode fiber, which after imaging by the glass prism and further optical components, again results in a parallel beam on the surface of the sample. Take care that the electrical field vector is polarized, perpendicular to the plane of incidents.
This position is marked on the output coupler of the fiber. Use an adjustable mirror located inside the condenser, which is imaged in the sample plane and illuminates the sample under a variable angle of incidents or object ray. The adjustable mirror may be driven by a step motor, which is controlled by software.
Each position corresponds to a well-defined angle of incidents as determined by go metric proceeded measurements. To calibrate the angular position of the adjustable mirror, use a fluorescent dye such as one millimolar flavin nucleotide, dissolved in water. To visualize the critical angle upon variation of the angle of incidents, use the critical angle of 61.3 degrees for a glass water transition as a fixed value, the angular position of the mirror at which the angle of incidents equals the critical angle is stored in the mirror positioning program.
Using the camera software, set the parameters of the E-M-C-C-D camera, including temperature gain, recording time, and shift speed. Use immersion oil to assure optical contact of the sample. With a glass prism, select an objective lens of appropriate magnification for the cells and moderate numeric aperture.
To visualize the object in the microscope, use an appropriate long pass or band pass filter for fluorescence detection. Record fluorescence images of identical samples upon variation of the angle of illumination. The angles of incidents should be the same or greater than the critical angle.
With the critical angle corresponding to 64.5 degrees for a cell glass interface, an angular range of 66 degrees to 74 degrees with steps of 0.5 degrees or one degree is recommended. To begin data analysis, use the lab view program to read the images recorded at various angles as well as the camera settings, the excitation wavelength, and the refractive indices. For glass, which is 1.52, and cells, which is 1.37, the settings are imported for proper evaluation of the data.
For each set of parameters, the penetration depth and the transmission factor are calculated automatically For the success of the procedure. It is important that images are carefully recorded and selected for analysis. Select images used for evaluation of cell substrate distances according to the equation.
For a membrane marker, individual images within homogenous illumination, such as shown in this example, may be excluded. Calculate images of cell substrate topology and select an appropriate color code for display during the evaluation. Profiles of individual pixels can be displayed as depicted in this figure.
These profiles may be used as an indicator for the quality of the fit. Finally, store the evaluation protocol. This figure depicts total internal reflection images of U2 51 mg glioblastoma cells with an activated TP 53 tumor suppressor gene.
Upon incubation with luan recorded at various angles of incidents, 66 degrees corresponding to a penetration depth of the evanescent electromagnetic field of about 140 nanometers, 69 degrees, corresponding to a penetration depth of 75 nanometers and 73 degrees corresponding to a penetration depth of 60 nanometers with decreasing penetration. Depth fluorescence decreases in intensity and originates from more superficial sites of the cells, such as focal contacts on their edges. Cell substrate distances are depicted in Panel D by a color code ranging from zero to 600 nanometers as seen here.
Cell substrate distances vary greatly over the cells between only a few nanometers represented by white to yellow and 250 to 300 nanometers represented by dark red. This indicates focal contacts and larger cell substrate distances are in close vicinity. A similar variation in cell substrate distances is calculated for U2 51 mg glioblastoma cells with an activated tumor suppressor gene P 10.
In contrast, cell substrate distances are rather constant for U2 51 mg control cells represented by yellow and wild type cells represented by orange to red After its development. This technique paved in the way for researchers in the field of cell substrate topology to explore tumor cells and the morphological changes upon laser therapy. After watching this video, you should have a good understanding of how to visualize surfaces with an actual resolution in the nanometer range at low light exposure, which is well tolerable to living organisms.
