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Z Old Biophysics I > Labs: Refractometry, Microscope, Measuring Devices > Flashcards

Labs: Refractometry, Microscope, Measuring Devices Flashcards

1
Q

What was determined in the refractometry experiment?

And how?

A
  • refractive indices of several glycerol solutions of known concentration were measured
  • the data was used to make a concentration vs. refractive index graph (linear relationship)
  • unknown concentrations of 2 glycerol solutions were determined by measuring their RIs and using this graph
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2
Q

What is absolute index of refraction?

Relative index of refraction?

A

Absolute - speed of light in vacuum divided by speed of light through given medium (n1 = C/C1)

Relative - ratio of indices of refraction of 2 media (n1/n2)

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3
Q

What is refraction?

A

a change in the direction of propagation of light that occurs when the light strikes a boundary surface between two media at any angle other than perpendicular

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4
Q

What is Snell’s Law?

A
  • the ratio of the sines of the angle of incidence and the angle of refraction equals the ratio of the speed of light (V) in the corresponding media
  • also equals the index of refraction of the 2nd medium (n2/n1)
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5
Q

What is the critical angle?

A

less dense to more dense medium - critical angle is the largest angle of refraction, corresponding to the grazing incident ray

more dense to less dense medium - critical angle is the incident angle which will produce a grazing refracted ray

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6
Q

What is a grazing ray?

A

a ray that is parallel to the interface between 2 media

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7
Q

What is total internal reflection?

A

When an incident ray moving from a more dense toward a less dense medium is of an angle greater than the critical angle, all the light is reflected back into more dense medium.

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8
Q

What is dispersion?

What causes it?

A
  • decomposure of white light into its component wavelengths
  • caused by the dependence of refractive index on wavelength
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9
Q

How is dispersion dealt with in the refractometer?

A

The compensator is a compound prism of two Amici prisms which restores white light from its components

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10
Q

What is Snell’s Window?

When does it occur?

A
  • a cone of light formed by refracted rays with an aperature angle 2x the critical angle
  • occurs as a result of light moving from less to more dense medium from all possible angles of incidence (as in from air into water)
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11
Q

What is the equation which relates concentration of solution to index of refraction?

A

n1 = n0 + K * c

n1 is RI of solution

n0 is RI of solvent

K is constant (dependent on solute)

c is concentration

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12
Q

How does the Abbe refractometer measure the refractive index of a solution?

A

it determines the position of the shadowline at the edge of the Snell’s window created when light passes through the solution and into a measuring prism of very high refractive index

(this allows determination of the critical angle)

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13
Q

What is the general layout of the Abbe refractometer?

Draw it.

A
  • two tubes with eyepieces: one aimed at the prisms and one aimed at a scale
  • a mirror reflects light into the two prisms (illuminating and measuring) between which a drop of solution is placed
  • scale is connected to prisms via an axis so that when prisms are turned to show the shadowline, refractive index can be read from scale
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14
Q

How are the results from the refractometer lab graphed?

A

Concentration as independent variable on the X axis

Refractive index as dependent variable on Y axis

(shows a linear relationship with direct proportionality)

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15
Q

What was the goal of the the microscope measurement?

A

A sample of frog RBCs diameters were measured using the micrometer

mean, standard deviation, standard error, reference + confidence intervals were determined

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16
Q

What is fermat’s principle?

A

Light will take the path between two points that keeps travel time to a minimum

(ie longer distance in the medium of lower refractive index)

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17
Q

What is the optical axis?

A

a line connecting the centers of curvature of the spherical surfaces of a lens

(in other words, an imaginary line through the center of the lens, perpendicular to its surface)

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18
Q

What are converging and diverging lenses?

Their shapes?

A

Converging - biconvex, focus light onto one point

Diverging - biconcave, scatters light

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19
Q

What is focal length?

A

the distance along the optical axis between the focal point and the principal plane (middle of lens)

20
Q

What is a real vs. a virtual image?

A

real - an inverted image created on the opposite side of a lens from its source object which can be projected onto a screen

virtual - an upright image created on the same side of a lens as its source object which can not be projected onto a screen

21
Q

What is the focal point?

A

the point at which a converging (biconvex) lens focuses light

22
Q

In a simple image formation diagram, what are the names and paths of the 3 main rays coming from an object point?

A
  1. parallel ray - parallel to optical axis, deflected through focal point on opposite side
  2. central ray - straight through center of lens
  3. focal ray - through proximal focal point, deflected parallel to optical axis
23
Q

What is the lens makers equation?

A

f is focal length

n is relative RI of lens

R1 & 2 are the radii of curvature of the lens (negative if concave)

24
Q

What are the five scenarios of an object’s distance from the lens in relation to once (F) and twice (2F) the focal point ?

What kind of image does each produce?

A
  1. Closer than F - virtual, upright, magnified
  2. At F - virtual, upright, at infinity (refracted rays are parallel)
  3. Between F and 2F - real, inverted, magnified
  4. At 2F - real, inverted, same size as object
  5. Beyond 2F - real, inverted, diminished
25
Q

How is the magnification of a lens determined?

A

it is the ratio if the image size to object size

(or image distance to object distance)

M = I/O = i/o

26
Q

What is the lens equation?

A
27
Q

Describe the path of light from a specimen to the observer in a simple light microscope, considering just the two main lenses.

A
  • light reflects from the object (between F and 2F) through the objective lens to form a real, inverted, magnified image
  • the eyepiece lens then uses this “intermediate” image as an object (at < F) to form a virtual, inverted, magnified image
28
Q

Draw the diagram of how a specimen is magnified by a simple light microscope.

A
29
Q

How can the eyepiece scale of the microscope be calibrated?

A

Using a stage micrometer with known distance between marks

  • line up stage micrometer and eyepiece scale
  • pick length of eyepiece scale where marks align with stage micrometer
  • divide known length between stage micrometer marks by number of eyepiece units to get the calibration factor
30
Q

What is standard error vs. standard deviation?

A

**standard error ** - the standard deviation of the sample mean from the population mean

standard deviation - the amount of variation of a sample of data from the average value

31
Q

What is reference interval vs. confidence interval?

A

**reference interval ** - mean plus or minus 2 times standard deviation

confidence interval - mean plus or minus 2 times standard error

32
Q

What is Huygen’s Principle?

A

waves propagate such that each point on a primary wavefront is the source of spherical secondary wavelets with the same speed and frequency

33
Q

What is interference?

What are the two kinds of it?

A

interference is the process that occurs when two waves meet each other

constructive - two waves in phase (peak aligned with peak) will combine and strengthen each other

destructive - two waves out of phase (peak aligned with trough) will cancel out (if of equal amplitude)

34
Q

What is Abbe’s Principle?

A

an optical system can resolve only the details of a specimen which diffract light such that at least the 1st order diffraction maxima contribute to image formation

35
Q

What is an optical grating?

What are two different kinds of them?

A

an object with periodic optical properties

amplitude grating - transmission properties vary periodically (ex: transparent slits between opaque grating elements)

phase grating - transmission % of grating remains same, but refractive index (and thus phase of transmitted light) changes periodically

36
Q

What is the double slit experiment?

A

two slits in an optical grating between a light source and screen act as two different sources of secondary wavelets which interfere with one another to create a pattern of light and dark bands on the screen

37
Q

What is the name of the light bands of an interference pattern created by an optical grating?

And how are they numbered?

A

Diffraction Maxima

  • middle is 0th order maximum, next out are 1st order maxima, then 2nd order, etc.
38
Q

What is Abbe’s Formula?

A

delta is minimum resolvable distance

n is RI of immersion medium

omega is half-aperature angle of light entering objective lens

39
Q

What is the numerical aperature of an objective lens (in relation the the special microscopes experiment)?

A

the divisor of the abbe formula (which is the product of the index of refraction of the immersion medium and the sine of the half-aperature angle)

n sin ω

40
Q

What is the resolving power of a microscope?

How is it calculated?

A
  • the microscope’s ability to create a clear image in which very small details can be distinguished
  • it is the reciprocal of the limit of resolution
    equation: f = 1/δ

f is resolving power

δ is minimum resolvable distance

41
Q

What is an immersion objective microscope?

A

an immersion medium of high RI is placed between the specimen and objective lens in order to increase the # of diffraction maxima which contribute to image formation

42
Q

What is a stereomicroscope?

A

a microscope which has two separate objective and eyepiece lenses equipped with prism systems which create upright, unmirrored images that allow for more a more 3D view of the specimen

  • used in microsurgery
43
Q

What is a **dark field illumination **microscope?

A
  • a special condenser is used which blocks the central beam
  • the specimen is illuminated from sides by oblique rays
  • image is formed by diffraction rays
  • used to observe colloid particles
44
Q

What is a fluorescence microscope?

A
  • light source provides UV and visible excitation & filters are used to select proper wavelength
  • native/stained specimens absorb light and emit visible fluorescence
  • staining involves use of fluorescent dyes called fluorochromes
  • can reveal presence of single fluorescing molecule (visible as center of shiny disk)
45
Q

What was the goal of the special microscopes lab?

How was it achieved?

A

to prove Abbe’s formula and the relationship between wavelength and resolving power

by using a light microscope modified with a monochromatic light source to observe an optical grating

46
Q

How is standard error calculated?

A

standard deviation divided by sq rt of n

Z Old Biophysics I (6 decks)

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