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Microscopy and Optics M Laboratory Report
Experiment No 2: Resolution and Contrast
Aims and Objectives
The aims and objectives of the specific experiment in resolution and contrast were to gain a good understanding of the Köhler Illumination process and the phase contrast technique and their importance in Microscopy, to obtain knowledge about optical resolution and magnification, to learn how to choose the right microscope settings and technique for each type of sample and lastly to obtain useful experience and the necessary confidence in using an optical microscope in the laboratory. Following a specific protocol and gaining the aforementioned knowledge, we were able to obtain clear images of various types of samples and, consequently, measure their features.
Experimental Equipment and Method
The equipment used for this experiment was the following:
- The microscope used was the Motic AE 30/31 (Motic Incorporations Ltd), which is an inverted microscope. The specific microscope has 4x, 10x, 20x and 40x objective lenses and all of them were used during the course of the experiment.
- The camera used was the ProgRes® CF (Jenoptik Germany), a CCD camera and the program used for the image acquisition was the ProgRes® CapturePro 184.108.40.206.
- The image analysis program used was ImageJ, an open source Java image processing program by NIH Image (1). It should be noted that in order for this specific program to be used, the images should be saved in .tiff format.
- The samples for this experiment were a scale bar, a microfabricated polycarbonate square and a cell sample.
The experimental procedure began with the familiarization with the Motic AE 30/31, by reading the microscope manual, in order to learn its parts and specifications. The switching on of the microscope followed and the first operation (Köhler Illumination) took place.
For the Köhler Illumination a specific list of steps were followed. Firstly, the phase annular diaphragm was set in position O (center position). Secondly, the FOV diaphragm was fully opened, the aperture diaphragm was in the open position and the specimen was brought into focus. Then the FOV diaphragm was closed to its minimum setting, the image of the field diaphragm was formed on the surface of the specimen using the condenser focus knob and finally the center of the field diaphragm image was adjusted to fit the center of the FOV using the condenser centering screws. After opening the diaphragm, the microscope was corrected and ready for normal observation.
An image was acquired before the Köhler Illumination process and one after the process.
It should also be noted that this process took place every time the objective lens was changed for every objective lens (4x, 10x, 20x and 40x).
For the second part of the experiment, the combinations of objectives and phase contrast, all four objective lenses were used and for each lens three pictures were taken: one with the phase annulus diaphragm in the 10/20 position, one in the empty diaphragm position and one in the 40 position. A total of 12 pictures were taken.
The same process took place for the cell sample, but in this stage only the 20x and 40x objectives were used. A total of 6 pictures were taken.
The final step was to obtain an image of a sample consisting of a scale. A different image was taken for each objective. A total of 4 images were taken.
The images were acquired clicking the “Capture” button and they were saved in a folder created by the team.
The scale images were used as a reference in order to add a scale bar to the rest of the images in ImageJ. A scale was defined by the relationship between the pixels and the distance. The process was the following:
- ImageJ -> File -> Open -> Analyze -> Set Scale
In Set Scale option, the known distance of the smallest scale interval (20 μm) was imported to the program, the unit chosen was micrometers and the Global box was ticked. The Global choice enabled the scaling of a group of images taken using the same objective lens.
This process took part for all 4 scale images in order for all the images to be scaled correctly.
Results and Discussion
The results of the aforementioned experimental procedure are presented below in the form of scaled images.
Figure 1: Image with 4x objective lens and closed field of view diaphragm. From left to right: The image before the Köhler Illumination process and the condenser away from the proper position and after performing the Köhler Illumination operation with the condenser in the correct position
Figure 2: Image with 4x objective lens and open field of view diaphragm. From left to right: The image before the Köhler Illumination process and the condenser away from the proper position and after performing the Köhler Illumination operation with the condenser in the correct position
Figure 3: Image with 10x objective lens and open field of view diaphragm. From left to right: The phase annulus diaphragm in the 10/20 setting, in the empty diaphragm setting and in the 40 setting
Figure 4: Image with 20x objective lens and open field of view diaphragm. From left to right: The phase annulus diaphragm in the 10/20 setting, in the empty diaphragm setting and in the 40 setting
Figure 5: Image of a cell sample with 20x objective lens and open field of view diaphragm. From left to right: The phase annulus diaphragm in the 10/20 setting, in the empty diaphragm setting and in the 40 setting
Figure 6: Image of a cell sample with 40x objective lens and open field of view diaphragm. From left to right: The phase annulus diaphragm in the 10/20 setting, in the empty diaphragm setting and in the 40 setting
The images of a sample consisting of a scale that were used to scale the rest of the images are shown below.
Figure 7: Image of the scale sample with 4x objective lens and open field of view diaphragm
Figure 8: Image of the scale sample with 10x objective lens and open field of view diaphragm
Figure 9: Image of the scale sample with 20x objective lens and open field of view diaphragm
Figure 10: Image of the scale sample with 40x objective lens and open field of view diaphragm
The Köhler Illumination process that was the first process that took place in this experiment is the basic operation for bright field optical microscopy. This process took place in order for each specimen to be properly illuminated and the images acquired to be of high quality, good resolution and contrast. Throughout the process both the amount and area of the sample that was illuminated were adjusted resulting in the high quality images shown in Figure 1 and 2 (2).In Figure 1 it is also apparent that in the image on the left the condenser is moved from the position of Köhler Illumination, resulting in a less focused and clearly less sharpened image in comparison to the image on the right. This image would be unsuitable for collecting necessary information.
As far as the phase contrast techniques are concerned, their function is to impart contrast to unstained biological material by transforming differences in phase caused by differences in the refractive index between cellular components into differences in amplitude of light which can be observed (3). In practice, the task that had to be accomplished was to specifically match each objective to its corresponding phase plate (i.e. 20x objective with the 10/20 phase annulus diaphragm) in order for the microscope to be aligned to superimpose illuminating rays passing through the annulus onto the objective phase ring. The result was high contrast images (4). It is obvious from Figures 3 through 6 that the best images were those with the correct match of objective lens and phase plate.
Comparing the images in Figures from 3 to 4 and 5 to 6 it is clear that the change of the objective lens affect the resolution. From theory it is known that the numerical aperture determines the resolving power of the objective and in general the higher the magnification the higher the numerical aperture. As a result it is expected that the images taken with the 40x objective will have a higher resolution than those taken with the 20x objective (Fig. 5,6) and those taken with the 20x objective will have a higher resolution than those taken with the 10x objective (Fig. 3,4). The acquired images verify this claim. It should however be noted that in higher magnifications an image may appear not so sharp but it would still be resolved to the maximum ability of the particular objective.
This laboratory experiment in resolution and contrast fulfilled its objectives which were to understand the definitions of optical resolution and magnification, to understand the importance of the Köhler Illumination and the phase contrast technique, to be able to choose the right settings and technique for various different types of samples and to obtain experience in use of optical microscopy.
However it could be useful if the lab hours were extended. This extension would give more time to the students to familiarize with the instruments and it would give them more time to understand and correct potential mistakes in the course of the experiment.
The knowledge I gained from this laboratory exercise was on the main definitions and techniques that define optical microscopy and I obtained necessary hands on experience that will be useful for the future.
- https://imagej.nih.gov/ij/features.html. [Online]
- Class Handout-Lecture 3.
- Ruzin. Plant Microtechnique and Microscopy. 1999.
- Nikon, Phase Contrast Configuration. https://www.microscopyu.com/techniques/phase-contrast/phase-contrast-microscope-configuration. [Online]
 Charged-coupled device
 Field of View
 In ImageJ the micrometer unit is imported as um
 Class Handout-Lecture 3
 Ruzin, Plant Microtechnique and Microscopy, 1999
 Nikon, Phase Microscope Configuration
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