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    Lbybi13 N01a Cristobal HW#1 (Guide Questions)

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    Earl Dominic Cristobal
    1. Contrast in microscopy makes minute specimen details visible by creating differences in light intensity between images and backgrounds. It is achieved by factors like absorption, brightness, scattering. 2. Contrast can be improved by optimizing apertures, filters, illumination techniques like dark ground or using phase contrast microscopy. 3. Both shorter wavelengths and higher numerical apertures provide better resolving power or ability to distinguish close objects. Oil immersion further increases numerical aperture above 1, improving resolution.

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    Lbybi13 N01a Cristobal HW#1 (Guide Questions)

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    Earl Dominic Cristobal
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    1. Contrast in microscopy makes minute specimen details visible by creating differences in light intensity between images and backgrounds. It is achieved by factors like absorption, brightness, scattering. 2. Contrast can be improved by optimizing apertures, filters, illumination techniques like dark ground or using phase contrast microscopy. 3. Both shorter wavelengths and higher numerical apertures provide better resolving power or ability to distinguish close objects. Oil immersion further increases numerical aperture above 1, improving resolution.

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    LBYBI13 N01A CRISTOBAL HW#1 (GUIDE QUESTIONS)

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    1. Contrast in microscopy makes minute specimen details visible by creating differences in light intensity between images and backgrounds. It is achieved by factors like absorption, brightness, scattering. 2. Contrast can be improved by optimizing apertures, filters, illumination techniques like dark ground or using phase contrast microscopy. 3. Both shorter wavelengths and higher numerical apertures provide better resolving power or ability to distinguish close objects. Oil immersion further increases numerical aperture above 1, improving resolution.

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    21 views2 pages

    Lbybi13 N01a Cristobal HW#1 (Guide Questions)

    Uploaded by

    Earl Dominic Cristobal
    1. Contrast in microscopy makes minute specimen details visible by creating differences in light intensity between images and backgrounds. It is achieved by factors like absorption, brightness, scattering. 2. Contrast can be improved by optimizing apertures, filters, illumination techniques like dark ground or using phase contrast microscopy. 3. Both shorter wavelengths and higher numerical apertures provide better resolving power or ability to distinguish close objects. Oil immersion further increases numerical aperture above 1, improving resolution.

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    CRISTOBAL, Earl Dominic Palad

    LBYBI13 (N01A)

    MICROSCOPY: GUIDE QUESTIONS


    1. Why is contrast also important in microscopy?
     Contrast in microscopy is very important as it makes the minute details of the specimen
    become visible enough when viewed under a microscope. It is the difference in light
    intensity between the image and its adjacent background in relation to its overall
    background intensity. With the naked eye only able to see 2% of minimum contrast value
    (in general) to distinguish relative features of the image against its background, contrast
    produced in the specimen by the absorption of light, brightness, reflectance,
    birefringence, light scattering, diffraction, fluorescence, or color variations is the basis of
    imaging specimens in brightfield microscopy. The ability of a detail to stand out against
    its background or its other adjacent details is a measure of a specimen contrast. (Hoffman
    & Davidson, 2018)

    2. How can contrast be achieved in using the microscope?


     Contrast can be achieved depending on the type of microscope, but in theory, it can be
    achieved by improving the placement of suitable apertures or filters within the optical
    path, either in the illuminating system alone (dark ground or Rheinberg illumination), or
    in conjugate planes in the imaging system (e.g., for phase contrast, differential
    interference contrast or polarized light microscopy). (Sanderson, 2000) Other factors
    might also come into play such as the amount of light, nature of the specimen being
    viewed, position, etc.

    3. What is the relationship of wavelength and numerical aperture in getting a good resolving
    power?
     In theory, since shorter wavelengths yield to a higher resolution (University of
    Cambridge, n.d.), the basis of resolving power, numerical aperture determines the
    resolving power of an objective, but the total resolution of a microscope system is also
    dependent upon the numerical aperture of the substage condenser. Although other factors
    might come into play like the correct alignment, right objective lenses, or the specimen
    being stained, considering wavelength and numerical aperture, the higher the numerical
    aperture of the total system, the better the resolution. (Abramowitz & Davidson, n.d.)

    4. How does oil increases resolution? Explain your answer.


     In microscopy, oil immersion is the usage of oil to increase the resolving power of the
    picture when viewed under a microscope, that is achieved by immersing both
    the objective lens and the specimen in a transparent oil of high  refractive index, thereby
    increasing the numerical aperture of the objective lens. Oil eliminates or reduces the
    reflective light bouncing outside the specimen and focuses the light on the glass slide
    making the specimen high in resolution, which further makes the image clearer when
    viewed under a microscope. (Martin, 1966) In theory, lenses reconstruct the light
    scattered by an object wherein, ideally, all the diffraction orders have to be collected.
    (Fankhauser, 2004) The resolution of a microscope is the minimum separation needed
    between two objects under examination in order for the microscope to discern them as
    separate objects. If two objects are separated by a distance shorter than the minimum
    distance, they will appear as a single object in the microscope. Now, objectives can have
    a numerical aperture of up to 0.95. Because sin α 0 is always less than or equal to unity
    (the number "1"), the numerical aperture can never be greater than unity for an objective
    lens in air. If the space between the objective lens and the specimen is filled with oil
    however, the numerical aperture can obtain values greater than unity. This is because oil
    has a refractive index greater than 1 (Cargille, 1985), which is why microbiologists use
    the Oil Immersion Objective (OIO) lens, enhanced with oil, in viewing or observing
    microorganisms such as bacteria.
    SOURCES
    Hoffman, R. & Davidson, M. (2018). Contrast in Optical Microscopy. Fsu.Edu.
    https://micro.magnet.fsu.edu/primer/techniques/contrast.html

    Sanderson, J. (2000). The Theory of Contrast Control in the Microscope. Microscopist.Co.Uk.


    http://www.microscopist.co.uk/wp-content/uploads/2017/04/Theory-of-Contrast.pdf

    Martin, L. C. (1966). The Theory of the Microscope. Blackie; First Edition.

    Abramowitz, M. & Davidson, M. (n.d.). Numerical Aperture and Resolution. Olympus-Lifescience.Com.


    Retrieved October 18, 2021, from https://www.olympus-lifescience.com/en/microscope-
    resource/primer/anatomy/numaperture/

    Cargille, J. (1985). Immersion Oil and the Microscope. Archive.Org.


    https://web.archive.org/web/20110911190610/http://www.cargille.com/immersionoilmicroscope.
    shtml

    Fankhauser, D. (2004). USE OF OIL IMMERSION OBJECTIVE. Archive.Org.


    https://web.archive.org/web/20020612041253/http://biology.clc.uc.edu/Fankhauser/Labs/Microsc
    ope/Oil_Immersion.htm

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