Lab 15: Confocal Microscopy BioE 331-Integrated Bioelectronics Lab Peter Sords

April 19, 2012

Objective/Scope: The purpose of this experiment was to learn the basic concepts, operation principles, and visualization techniques of optical and confocal microscopes. In order to achieve this, it will be necessary to understand the theory behind how microscopes and optics work, as well as the limits of resolution, magnification, optical contrasts, refractive indices, and fluorescence imaging of modern optical microscopy. For a simple lens and a compound microscope, the geometrical optics are displayed in Figure 1.

Figure 1: Convex lens optics

Fluorescence is the emission of light by a substance that has absorbed light or other EM radiation of a different wavelength. The emitted light has a longer wavelength, and hence lower energy, than the absorbed radiation most often. Using fluorescence, we can track different particles or biological molecules that fluoresce at particular wavelengths. Confocal microscopy is utilized to increase optical resolution and contrast by using point illumination and a spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane. Using computer software in conjunction with this technique, we are able to develop 3D images from the obtained images. Confocal microscopy aims to eliminate out-of-focus images, and hence the resolution is much better than that of wide-field microscopes. Experiment Description: The first part of this experiment dealt with measuring the size of particles using their innate autofluorescent properties. In order to do this, we placed fluorescent 1-micron polystyrene (PS) spheres under magnification, and determined the pixel size of each particle. This was done for 5 unique polystyrene particles using 80m and 800m pinhole sizes to compare and contrast the resulting fluorescent images resolution. The freeware program Image J was used to accurately analyze the intensity profile across the particle images. Afterwards, were supposed to observe a sample of biological cells pre-labeled with fluorescent proteins, but due to a lack of sample, had to postpone this part of the experiment.

Measurement Data / Results:

Figure 2: PS1 80 micron

Figure 3: PS1 800 micron

Figure 4: PS2 80 micron

Figure 5: PS2 800 micron

Figure 6: PS3 80 micron

Figure 7: PS3 800 micron

Figure 8: PS4 80 micron

Figure 9: PS4 800 micron

Figure 10: PS5 80 micron

Figure 11: PS5 800 micron

Discussion: In our week 1 experiment, we observed that the particle size was 11.3453 microns when using the 100X oil, the particle size was 10.3627 when using the 60X oil, and 7.8947 microns using 10X. A trend that can be noticed from this data is that the larger the magnification is, the larger the apparent particle size. In our week 2 experiment, we used fluorescence to measure the size of cells. We first used a green leaf sample, and determined the size of the cell was approximately 1 x 1.3 microns. We searched for t1 and t2, which were the widest portions of the width and the height, respectively. The t1 and t2 were subjective, being that there was no set dimension to measure, but rather we had to estimate which axis was the longest; therefore, some human error needs to be accounted for. We also examined a red leaf under green light, and found the dimensions to be comparable, but slightly smaller; about 1 x 1 micron. We made sure that there were no air bubbles under each slide to ensure best possible results. In the week 3 experiment, we used a confocal microscope to obtain multiple images of the infratstructure of the cell. With these images, we performed a depth scan of the cell to determine the

width and the thickness of the cell. In part I (the pinhole), the importance of the size of the pinhole was evident. The 600-micron pinhole offers higher resolution than the 100-micron pinhole. This was apparent in both the images of the cells and of the microspheres. Conclusion: Over the course of these labs, we used two different microscopes and multiple techniques to determine the sizes of cells and form 3D images of them. Some potential problems we obtained with the labs were the processes necessary to obtain image size. For example, the light intensity vs. pixel size graphs were not extremely accurate, and were subject to human error. Using these graphs, there could have been error in determining the minimum light intensity, error in centering the line on the image at the region of smallest light intensity, etc. Also, for the week 1 experiment, we obtained different values for the particle size as we changed the objective. Therefore, error in week 1 was a function of magnification and resolution. In the week 3 experiment dealing with confocal microscopy, due to the quantity of settings and software factors we were not accustomed to, there could have been a great deal of error. It was very difficult to focus the images using this type of microscopy, and it is possible we did not obtain the best possible representation of the cell that we could have. Also, long exposure times were necessary for the confocal microscopy because although this microscopy technique offers higher resolution, it comes at the price of signal intensity.