CLS 1113 Introduction to Clinical Laboratory Practice

1. CLS 1113Introduction to Clinical Laboratory Practice Microscopy Brunzel, Chapter 1

2. Always... use lens paper to clean lens surfaces. use a lens cleaning solution. protect the microscope from dust and debris when not in use. Document cleaning and have your scope serviced annually. Never... use gauze or tissue on lenses. touch optical surfaces. wear excessive makeup disassemble the microscope. Do’s and Don’ts of the Microscope

4. Fundamental Knowledge • Resolving Power- • Ability to see two adjacent objects as separate and distinct. • Numerical Aperture • Numerical designation of the resolving power of each lens and the condenser.(The higher, the better) • 10x (.25) • 40x (.40) • 100x (1.25)

5. Resolving Power • The ability to distinguish two very small and closely spaced objects as separate entities. • It depends on: • Wavelength of light • Numerical Aperture

6. Resolution • R = 0.612 x λ / NA • R = resolving power • λ = wave length of light • NA= numerical aperture of objective lens • As NA increases resolution distance gets smaller – you can distinguish a smaller distance between two objects: Greater Resolution and magnification!

7. Numerical Aperture • Numerical aperture (NA) is inscribed on the lens and condenser specific for each. • NA = N x sinµ • N = refractive index of optical medium • µ = angle of light made by the lens (aperture angle) • The higher the NA the better the lens light gathering properties.

8. Numerical Aperture • NA can be increased by: • Changing the refractive index of the optical medium • Increasing the aperture angle • Immersion oil - more image information is captured by the lens, and more image detail can be observed.

9. Brightfield Microscopy • Specimen appears dark against a bright background. • Three types • monocular, binocular, and trinocular • Utilizes Kohler Illumination

11. Darkfield Microscopy • A bright specimen appears on a dark background. The illuminator is directed through the specimen, not behind it. • Due to the dark background, the specimen can be viewed with greater contrast. • An oil immersion condenser and very clean slides are required.

13. Fluorescence Microscopy • Light of a specific wavelength is exposed to the specimen. If a particular fluorescent tag is present, the light is absorbed and emitted at a longer wavelength. The technologist will then be able to see the light emitted from the substance. • Uses: • herpes testing • chlamydia testing

16. Phase Contrast Microscopy • There are substances in urinalysis that have a very low RI, similar to that of air. Because of this fact, they are very difficult to view with out adding stain to the sample. • Phase contrast microscopy overcomes this problem.

18. When wavelengths of light are “in phase”, the intensity of light is the greatest. • If the wavelengths are slightly asynchronous the light intensity will be dimmed to a certain degree. • If the wavelengths are exactly opposite of each other, they will cancel each other out and no light will be evident. • The variations in the specimens will demonstrate different intensities of light. • REVIEW DIAGRAMS ON PAGE 14 IN THE TEXT.

22. Polarizing Microscopy • Regular light waves travel in all directions. When light is polarized, it is limited to one direction. • If we limit the light hitting our specimen to waves that are traveling in one direction, what will happen if they hit something that is birefringent. • In addition, what if we place another filter to cancel out the light we initially let through?

24. We would get something that looked like this.