Brief History
Optical microscopy, as discussed in the introduction of the previous lab, uses a system of lenses to magnify and resolve image details usually invisible to the naked eye. Reflected light microscopy relies on the observed specimen to have a highly reflective surface, while other light microscopy techniques employ a different fundamental system. [1][10][11] In this lab, we make use of transmitted light microscopy – a practical and common technique made possible by sample illumination using a light source on the opposite side of the specimen from the objective. [2] Relatively, this technique was discovered only very recently and has by and large become just as heavily used as reflected light microscopy. The chronology of the transmitted light microscope is rooted in the evolution of the optical microscope, but branches off in 1893 when August Kӧhler began to work with sample illumination and made use of light that interfered with the sample, rather than the light being absorbed and reflected.[3] Transmitted light microscopy is heavily favored to observe biological sample and in Figure 1, we can see how light is transmitted through the sample and into an eyepiece, instead of being reflected off the surface.[4]

Figure 1 Unstained (left) and stained (right) biological sample.
In 1953, this technique saw a crucial jump in development and began exploring phase contrast techniques.[5] Bright-field and dark-field techniques also allowed for contrast between specimen and background for better identification of sample characteristics, but they did could not show all aspects of a specimen.[6] By 1955, polarized light and differential interference microscopy also became additions to the transmitted light family. These techniques use similar yet more advanced fundamentals – they will be discussed further in the proceeding section.[2]
Theory
Similar in structure to the reflected light microscope, the transmitted light microscope puts a different theory into practice by using an illumination source opposite to the objective lens on the other side of the specimen. Major components of this type of compound microscope are almost identical to that of the reflected light microscope except for a few exceptions. It consists of an illumination source, a condenser, a polarizer, a field diaphragm ring, a stage, the objective lens, the ocular lens, and the fine and coarse focus knobs. As we can see from Figure 2, the major differences between a reflected light microscope and a transmitted light microscope are the location of the illumination source and the presence of a polarizer. [7][8]

Figure 2 Reflected Light Microscope (Left) and Transmitted Light Microscope (Right)
To understand the basics of a transmitted light microscope we can simply follow the optical pathway of the light. Initially, an illuminator produces light to be shone onto the specimen. The intensity and area of focus of this light is controlled by the condenser to attain maximum illumination. Light rays are conducted in such a way as to allow for polarization or redirection. The light, then, interferes with the specimen that is placed on the stage and transmits through it; the specimen may be moved in the X and Y directions on the stage. Then, the light is taken in by the objective lens and is refracted and magnified through a compound lens setup to finally reach the ocular lens. The fine and coarse focus knobs may be adjusted with different objectives. [2][9]
To fully fathom the inner workings of a transmitted light microscope we must delve deeper into its underlying principles. For example, the amount that an image is being magnified is the product of both the magnification of the objective lens and the magnification of the ocular lens, and not any one of those individually. [10]
A major principle of this technique is the resolution. The resolution is the shortest distance between two points that can clearly be observed as separate. It is highly dependent on the wavelength of light that hits the sample and the quality of the lens. Usually a smaller wavelength yields a better resolution. All microscopes are limited by the diffraction of light. A circular aperture with a perfect lens can only focus so much until light itself hinders the focus; and this limitation creates a blurry pattern, shown in Figure 3, called the Airy Disk. The circular pattern is extremely dependent on the numerical aperture, or N.A, of an objective lens and the wavelength of light. The limiting distance is given by the formula:
.[11] [12] [13]

Figure 3 Airy Disk Region
The numerical aperture of an objective lens is usually located on the body of the objective lens. Using a formula to find the numerical aperture gives us the magnitude of how much light can be collected and how well the image can be resolved by the objective lens. Looking at Figure 4, it is measured by taking the sin of the half angle of the maximum cone of light allowed by the objective and multiplying it by the index of refraction of the material.[10] [13] In order to fully utilize oil immersion techniques in transmitted light microscopy, it is imperative we know how to read an objective lens, show in Figure 4. Its numerical aperture and magnification are located right in the center since they are the most important factors. We are also given the immersion medium to use to increase resolution of an image; this will be discussed further when analyzing techniques. Another important variable to note is the coverslip thickness. This tells exactly what thickness coverslip we can use to cover our sample to attain a better resolution using immersion oil. [11]

Figure 4 Numerical Aperture and Body of Objective Lens

Also the depth of field plays an important role to determine the focal plane. Figure 5 shows us the DOF is the distance from above the focal plane to below it which can give a practical image. A larger depth of field allows for more focus of light on a larger area, whereas a narrower one allows for focus on specific areas but it leaves the rest of the image blurry. [10]

Figure 5 Depth of Field Ranges
Reference: Hamed Hosseinibay, Lab Module #1
Many times, to increase the resolution of an image, the use of a high refractive index immersion oil is necessary, especially with high objective lenses. The greater magnification is more sensitive to the differences in the indices of refraction between the specimen and the air. To counter this, we place a coverslip with the same refractive index as the immersion oil atop the specimen. Then, a few drops of the oil are placed on the coverslip and the objective is emerged into the immersion oil. By doing this, the refractive indices of all mediums remain equal and constant so the image is able to better resolve itself by allowing more transmitted light in. A good representation of a specimen and objective in immersion oil is shown here in Figure 6.[11]

Figure 6 Refractive Indices with Immersion Oil

Figure 7 Constrast-Enhancing Techniques in Optical Microscopy

Techniques:
Transmitted light microscopy is riddled with many techniques that play a huge role in making famous discoveries and characterizing organic and inorganic materials. One such common method is bright-field microscopy – also called Kohler Illumination. This technique is characterized by observing specimens against a bright background. This contrast is caused by optical disparities between the interaction of wavefronts and different refractive indices. Only part of the transmitted light is absorbed by denser areas, giving off brighter intensities. It is much easier to observe outlines of biological matter, but it fails to identify key areas of living matter without the use of dyes, like in Figure 7a. This in turn, caused a huge necessity for fluorescent microscopy, which uses dyes and monochromatic wavelengths to properly magnify and identify a specimen. Contrastively - no pun intended - dark-field imaging uses oblique illumination to create a high contrast image against a black, or dark, background. In this technique, the condenser sends a cone of light through the specimen; the rays are then diffracted, refracted, and reflected by optical disparities. These faint light rays are then absorbed by the objective lens for viewing, but too much light scattering, however, can obscure the image and decrease contrast. Dark-field imaging is a useful tool to observe micro-organisms at low and high magnifications as is evident in Figure 7e. [2][11][14][15]

Phase contrast is yet another important transmission technique. A special condenser is required in order to retard the light waves passing through the specimen. This technique uses very clever optical tricks to translate phase shifts into visible changes in a specimen. As light enters and travels through a living specimen, various entities cause the light waves to lag, or retard, more than the others. Figure 8 shows how this technique takes those minute differences and translates them to amplitude changes in the light waves allowing us to view the topography of the specimen. The main idea for employing this method is that many thin, unstained cultures do no absorb visible light and thus cannot be vied under conventional bright-field and dark-field imaging, which is why phase contrast is needed to highlight invisible features shown in Figure 7b. [2][14][15]

Figure 8 Phase Contrast Imaging of Transparent Thin Specimens
Birefringent specimens – specimens with two indices of refraction – are usually analyzed using polarized light microscopy. This technique requires a polarizer, which is placed below the condenser, and an analyzer, which is placed above the objective, orthogonal to the polarizer. Directly transmitted light can be optionally blocked to allow for higher contrast with this setup. The polarizer and analyzer first orient light in different directions and then recombine them respectively to ensure the light waves have the same amplitudes. This is an excellent technique for samples such as plant cell walls or muscle tissue, which are birefringent as in Figure 7f. [2][14][15]

Figure 9 Polarized Light Microscope Optical Pathways and Components

DIC, or differential interference contrast, uses the same methodology as polarized light but it also requires the use of Nomarski Prisms, or birefringent crystal wedges, given in Figure 9. DIC is characterized by plane-polarizing the light path then shearing it using Nomarski prism. Once the light lasses through the specimen it is recombined by the analyzer and the shear is taken off the light to analyze minute thickness gradients and slight changes in indices of refraction. This technique is helpful in observing very thick tissue samples such as in Figure 7c, since it creates a more 3D image of the specimen. [2][14][15]

Figure 10 DIC Nomarski Prism

Hoffman Modulation is not that different from differential interference contrast. In fact, it is an inferior, but cheaper, substitute to DIC. It uses a modulator, inserted into the rear focal plane of the objective lens, which modulates based on the intensity of light coming in. The modulation produces some contrast but the overall image quality in Figure 7d is filled with optical artifacts, making this technique an undesired substitute to DIC. [14][15]

The theory behind transmitted light opens expands the visible world for us by enabling us to observe micro-organisms. Transmitted light microscopy, without a doubt, can employ a handful of useful techniques which are only limited by the resolution we can achieve due to light diffraction. Electron microscopes remove that limit and give us a glimpse into the nano-scale world.

[1] Hale, Alan. "Types of Microscopes." Microscopes. 2007. Print. [2] "What Is Light Microscopy?" What Is Light Microscopy? Web. 26 Jan. 2015. <https://www.jic.ac.uk/microscopy/intro_LM.html>. [3] Davidson, Micahel, and Thomas Fellers. "UNDERSTANDING CONJUGATE PLANES AND KÖHLER ILLUMINATION." 4. The Florida State University. Web. <http://www.microscopyu.com/pdfs/KohlerIllumination.pdf>. [4] "Brightfield Illumination." Olympus Microscopy Resource Center. Web. 25 Jan. 2015. <http://www.olympusmicro.com/micd/anatomy/micdbrightfield.html>. [5] "The Nobel Prize in Physics 1953." The Nobel Prize in Physics 1953. Web. 27 Jan. 2015. <http://www.nobelprize.org/nobel_prizes/physics/laureates/1953/>. [6] Nature.com. Nature Publishing Group. Web. 26 Jan. 2015. <http://www.nature.com/milestones/milelight/full/milelight08.html>. [7] "Nikon MicroscopyU | Fundamentals of Digital Imaging." Nikon MicroscopyU | Fundamentals of Digital Imaging. Web. 25 Jan. 2015. <http://www.microscopyu.com/articles/digitalimaging/digitalintro.html>. [8] "CX31-P Polarized Light Microscope Configuration." Olympus Microscopy Resource Center. Web. 27 Jan. 2015. <http://www.olympusmicro.com/primer/techniques/polarized/cx31polconfiguration.html>. [9] Spector, David L. "Light Microscopy." Basic Methods in Microscopy: Protocols and Concepts from Cells : A Laboratory Manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory, 2006. Print. [10] Hosseinibay, Hamed. "Introduction to Optical Microscope." Lab Module #1. UC Riverside, Riverside. 14 Jan. 2015. Lecture. [11] Hosseinibay, Hamed. "Introduction to Optical Microscope." Lab Module #2. UC Riverside, Riverside. 21 Jan. 2015. Lecture. [12] Education in Microscopy and Digital Imaging." ZEISS Microscopy Online Campus. Web. 17 Jan. 2015. <http://zeiss-campus.magnet.fsu.edu/articles/basics/resolution.html>. [13] "Education in Microscopy and Digital Imaging." ZEISS Microscopy Online Campus. Web. 18 Jan. 2015. <http://zeiss-campus.magnet.fsu.edu/tutorials/basics/numericalaperturelightcones/indexflash.html>. [14] "Education in Microscopy and Digital Imaging." ZEISS Microscopy Online Campus. Web. 27 Jan. 2015. <http://zeiss-campus.magnet.fsu.edu/articles/basics/contrast.html>. [15] "Review of Transmitted Light Microscope Techniques." Review of Transmitted Light Microscope Techniques. Web. 27 Jan. 2015. <http://microscopy.berkeley.edu/courses/tlm/bf_review/LM_review.html>.