Light Microscope

• Syed Ibrahim

Introduction

The development of the microscope has been vital to much scientific advancement in biology (Kriss & Kriss 1998). Microscopes allow humans to see objects that would otherwise be unseen by the naked eye. The light microscope uses a series of three lenses to magnify an object. The condenser lens align and focus the light from the illumination source through the stage, onto the specimen. (Murphy, 2001) After passing through the specimen, the light goes to the objective lens which collect diffracted light and magnify the image of the specimen, typically 4X, 10X, 40X, or 100X (Murphy, 2001). The light finally reaches the ocular lens. The ocular lens also focus and magnify the image, but this is typically 10X or 15X (Murphy, 2001). After passing through the ocular lens, the light reaches the observer’s eyes.

Microscopes do not just magnify the image of an object, but also increase its resolution (Heidcamp et al., 2014). Magnification is the increase in the dimensions of an image, while resolution is the ability to distinguish two components of the image (Alberts et al., 2008). In other words, the magnification is the size of the image while the resolution is the clarity or quality of the image (Heidcamp et al., 2014). There is no limit of magnification because the size of an image can be increased indefinitely, but there is a limit of resolution because of the properties of light (Alberts et al., 2008). Due to diffraction, the limit of resolution for light microscopes is close to half the wavelength of light divided by the numerical aperture. (Hell, 2007). The numerical aperture is a measure of the number of light rays collected by the objective lens of a microscope, and it is dependent on the refractive index and the sine of half of the cone angle (Heidcamp et al., 2014). These can be combined to give the following equation (Heidcamp et al., 2014):

Where:

= wavelength of light

= refractive index

= half of the cone angle

Based on the above equation, decreasing the wavelength of light, increasing the refractive index, or increasing the cone angle will decrease the limit of resolution, thus increasing the resolution of an image. The smallest limit of resolution of a light microscope is 0.2μm (Alberts et al., 2008).

Microscopes can be used to examine microorganisms. In this lab Spirogyra, Paramecium and Saccharomyces cerevisiae were examined. Spirogyra are filamentous algae that are typically 10µm-100µm wide and their filaments may be a few centimeters long (Parmentier, 1999). Spirogyra are often found in freshwater are distinguishable by their spiral chloroplasts (Fathima et al., 2007). Paramecium are unicellular protists with cilia that are typically found in aquatic habitats and are usually 100µm-3500µm (Morgan, 1999; Wichterman, 1986). Saccharomyces cerevisiae (yeasts) are unicellular fungi that are typically 3µm-6µm in size (Schneiter, 2004). Since the naked eye’s limit of resolution is 100µm, these organisms are too small to be observed by the human eye alone (Heidcamp et al., 2014). Light microscopy was used to increase magnification and resolution so that the individual organism as well as their internal structures may be clearly observed.

The purpose of this lab was to use a bright field microscope to determine the scale of each objective, to examine Spirogyra, Paramecium, wild-type yeasts and fab1Δ mutant yeasts under a microscope, as well as to learn the essentials of micropipetting.

Results

Part A: Lab 1 Report Sheets

Please refer to attached sheets.

Part B: Answers to Assigned Questions

1. When the dimensions for the letter “e” using 4X, 10X or the naked eye were compared in Exercise 1.2, they were all approximately the same, as seen below. Using the light microscope gave more precise dimensions as compared to the naked eye. When comparing the different magnifications of the light microscope, they had percentage differences of 4% and 8% in the length and width respectively. Overall, it makes sense that all three measurements gave roughly the same dimensions as they were all measuring the same specimen.

Dimensions of the letter “e”

Naked Eye:

Light Microscope (4X):

Light Microscope (10X):

Percentage difference between 4X and 10X

2. Based on the observations from Exercise 1.3, it was apparent that Spirogyra have cell walls while Paramecium do not. As well, Paramecium have cilia while Spirogyra do not.
3. After pipetting as required for Exercise 1.4, a minute amount of water remained in the Eppendorf tube, and there was no air gap in the tip of the pipette. This means that slightly more than 50μL of water was pipetted into the Eppendorf tube. For this reason we practiced again, and this time no liquid remained. For future labs, we must ensure that we are extra attentive to ensure we pipette the correct amount of liquid.
4. During Exercise 1.5, it was observed that fab1Δ mutant yeasts appeared to have a thicker cell membrane than the wild-type yeasts. This thicker cell membrane may have been an enlarged vacuole within the cell that was pressing up against the cell membrane.

Part C: Research

There are many types of light microscopes, including bright-field microscopes, dark-field microscopes and phase-contrast microscopes (Alberts et al., 2008). Phase contrast microscopes rely on the phase-shifting of light as it passes through parts of the specimen of different relative thickness and density (Zernike, 1942).

Search Engine: Web of Science

Search Terms: phase contrast microscopic [filtered by date from 1900 to 1950]

Reference: Zernike, F. (1942). Phase contrast, a new method for the microscopic observation of transparent objects.Physica,9(7), 686-698.

After researching, a microscope was found with the following specifications and price (Cole-Parmer, 2014):

Microscope: Phase Contrast Microscope with Digital Camera (3 megapixels), Binocular, 115 VAC, 60 Hz

Distributer: Cole-Parmer

Model Number: RK-48925-04

Approximate Price: $2,932.46CND/EACH

Search Engine: Google

Search Terms: Phase Contrast Microscope with Digital Camera

Reference: Cole-Parmer. (2014). Phase Contrast Microscope with Digital Camera.Cole-Parmer. Retrieved September 15, 2014, from http://www.coleparmer.ca/Product/Phase_Contrast_Microscope_with_Digital_Camera_Binocular_115_VAC_60_Hz/RK-48925-04

References

Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2008).Molecular Biology of the Cell(5th ed.). New York: Garland Science.

Cole-Parmer. (2014). Phase Contrast Microscope with Digital Camera.Cole-Parmer. Retrieved September 15, 2014, from http://www.coleparmer.ca/Product/Phase_Contrast_Microscope_with_Digital_Camera_Binocular_115_VAC_60_Hz/RK-48925-04

Fathima, M., Shantha, N., & Rajagovindan, N. (2007).Botany(Revised ed.). Chennai: Tamil Nadu Textbook Corporation.

Heidcamp, W., Antonescu, C., Botelho, R., & Victorio-Walz, L. (2014).Laboratory Manual: Cell Biology – BLG311(Fall 2014 ed.). Toronto: Ryerson University.

Hell, S. W. (2007). Far-Field Optical Nanoscopy.Science,316(5828), 1153-1158.

Kriss, T. C., & Kriss, V. M. (1998). History of the Operating Microscope: From Magnifying Glass to Microneurosurgery. Neurosurgery,42(4), 899-907.

Morgan, M. (1999). Paramecium. Microscopy-UK. Retrieved September 15, 2014, from http://www.microscopy-uk.org.uk/index.html?http://www.microscopy-uk.org.uk/ponddip/paramecium.html

Murphy, D. B. (2001).Fundamentals of light microscopy and electronic imaging. New York: Wiley-Liss.

Parmentier, J. (1999). Spirogyra. Microscopy-UK. Retrieved September 15, 2014, from http://www.microscopy-uk.org.uk/index.html?http://www.microscopy-uk.org.uk/ponddip/spirogyra.html

Schneiter, R. (2004).Genetics, Molecular and Cell Biology of Yeast. Fribourg : University of Fribourg Switzerland.

Wichterman, R. (1986).The Biology of Paramecium(2nd ed.). New York: Plenum Press.

Zernike, F. (1942). Phase contrast, a new method for the microscopic observation of transparent objects.Physica,9(7), 686-698.