Labs: Refractometry, Microscope, Measuring Devices Flashcards
What was determined in the refractometry experiment?
And how?
- 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
What is absolute index of refraction?
Relative index of refraction?
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)
What is refraction?
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
What is Snell’s Law?
- 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)
What is the critical angle?
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
What is a grazing ray?
a ray that is parallel to the interface between 2 media
What is total internal reflection?
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.
What is dispersion?
What causes it?
- decomposure of white light into its component wavelengths
- caused by the dependence of refractive index on wavelength
How is dispersion dealt with in the refractometer?
The compensator is a compound prism of two Amici prisms which restores white light from its components
What is Snell’s Window?
When does it occur?
- 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)
What is the equation which relates concentration of solution to index of refraction?
n1 = n0 + K * c
n1 is RI of solution
n0 is RI of solvent
K is constant (dependent on solute)
c is concentration
How does the Abbe refractometer measure the refractive index of a solution?
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)
What is the general layout of the Abbe refractometer?
Draw it.
- 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
How are the results from the refractometer lab graphed?
Concentration as independent variable on the X axis
Refractive index as dependent variable on Y axis
(shows a linear relationship with direct proportionality)
What was the goal of the the microscope measurement?
A sample of frog RBCs diameters were measured using the micrometer
mean, standard deviation, standard error, reference + confidence intervals were determined
What is fermat’s principle?
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)
What is the optical axis?
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)
What are converging and diverging lenses?
Their shapes?
Converging - biconvex, focus light onto one point
Diverging - biconcave, scatters light
What is focal length?
the distance along the optical axis between the focal point and the principal plane (middle of lens)
What is a real vs. a virtual image?
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
What is the focal point?
the point at which a converging (biconvex) lens focuses light
In a simple image formation diagram, what are the names and paths of the 3 main rays coming from an object point?
- parallel ray - parallel to optical axis, deflected through focal point on opposite side
- central ray - straight through center of lens
- focal ray - through proximal focal point, deflected parallel to optical axis
What is the lens makers equation?
f is focal length
n is relative RI of lens
R1 & 2 are the radii of curvature of the lens (negative if concave)
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?
- Closer than F - virtual, upright, magnified
- At F - virtual, upright, at infinity (refracted rays are parallel)
- Between F and 2F - real, inverted, magnified
- At 2F - real, inverted, same size as object
- Beyond 2F - real, inverted, diminished
How is the magnification of a lens determined?
it is the ratio if the image size to object size
(or image distance to object distance)
M = I/O = i/o
What is the lens equation?
Describe the path of light from a specimen to the observer in a simple light microscope, considering just the two main lenses.
- 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
Draw the diagram of how a specimen is magnified by a simple light microscope.
How can the eyepiece scale of the microscope be calibrated?
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
What is standard error vs. standard deviation?
**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
What is reference interval vs. confidence interval?
**reference interval ** - mean plus or minus 2 times standard deviation
confidence interval - mean plus or minus 2 times standard error
What is Huygen’s Principle?
waves propagate such that each point on a primary wavefront is the source of spherical secondary wavelets with the same speed and frequency
What is interference?
What are the two kinds of it?
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)
What is Abbe’s Principle?
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
What is an optical grating?
What are two different kinds of them?
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
What is the double slit experiment?
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
What is the name of the light bands of an interference pattern created by an optical grating?
And how are they numbered?
Diffraction Maxima
- middle is 0th order maximum, next out are 1st order maxima, then 2nd order, etc.
What is Abbe’s Formula?
delta is minimum resolvable distance
n is RI of immersion medium
omega is half-aperature angle of light entering objective lens
What is the numerical aperature of an objective lens (in relation the the special microscopes experiment)?
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
What is the resolving power of a microscope?
How is it calculated?
- 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
What is an immersion objective microscope?
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
What is a stereomicroscope?
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
What is a **dark field illumination **microscope?
- 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
What is a fluorescence microscope?
- 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)
What was the goal of the special microscopes lab?
How was it achieved?
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
How is standard error calculated?
standard deviation divided by sq rt of n
