1. Microscopy - the Art & Science of making fine detail visible

2. Magnification - producing an image which is larger than the original object

(What’s minification?)

M = Si = Di where S = size I = image

So Do D = distance O = object

Equation 1. Magnification

a. The object is the specimen.

b. The image is the representation of the specimen. It has its own physical reality and properties. Our job as microscopists is to reproduce the object as faithfully as possible in the image.

c. Magnification is a relationship of either:

1) the size of the image to the size of the object or

2) the distance from the image to the lens (Di) divided by the distance from the object to the lens (Do)

3. Resolution = a distance: the smallest separation between two features that still permits you to image them as two independent features.

Resolution is determined by the numerical aperture of the objective.

R = 1.22 l _ where 1.22 = shape factor

NAo+NAc l = wavelength (lambda)

NA = Numerical Aperture

o, c = Objective, condenser

Equation 2. Resolution

Caveat: This equation applies only to techniques derived from brightfield microscopy. Fluorescence is not a resolution-limited technique. Rather, it is detection limited.

4. Contrast - Visibility against the background

C = I_b – I_owhere I_b= Intensity of the background

I_b I_o = Intensity of the object

Equation 3. Contrast

5. Measurement - “IDKYCDT!” [1]

A Quick Tour Around the Microscope

(Diagram to be handed out by the instructor)

1. Three key optical components

a. Objective – the “mastermind” for the microscope

1) establishes the first step of magnification (see largest number on the engravings)

2) sets the resolution for the whole system (see the “/ --“ on the engravings)

3) may contribute to contrast (see the special nomenclature on the engravings)

b. Condenser – the “C” exercise

1) Controls the angle at which the light approaches the specimen and, therefore the

coherence (the degree to which light waves are in step with one another).

(Do the “Coherence Exercise”)

a) Coherence determines the quality of the edge information.

b) Physically, the angle is controlled by the aperture iris (demonstrate)

2) The second component of resolution

3) May contribute to contrast techniques

c. Eyepieces

1) Responsible for the second step of magnification (see the large number engraved on the objective)

2) Responsible for establishing the field of view (the diameter of the territory you are actually observing

a) feel the edge of the small baffle about ½” up the inside wall of the underside of the eyepiece

b) look for the field number engraved on the barrel (diameter across the opening, expressed in mm.

FOV = field number/Mag _objective

Equation 4. Field of View

3) May be the site of a reticle (a target used for photography or measurement)

Figure 1. Two typical eyepiece designs

2. Two key control mechanisms

a. Condenser aperture iris or just aperture iris (AI)

1) Controls the angle of light approaching the sample

2) Responsible for coherence control

b. Field iris (FI)

1) Controls the size of the illuminated disk on the specimen and therefore the territory you will observe

2) Also controls glare and haze

3. Some important distances:

Figure 2. Some important distances

Koehler illumination and you: 4 critical steps for alignment

Start by setting the focus ring on the eyepieces to “Zero” and setting the width of the binoculars for comfortable viewing (one, round circular view).

1. Start by setting the focus on the eyepieces to the “zero” point and the binoculars so that you are viewing comfortably.

2. The Objective

· Using the coarse and fine focus, focus the objective to the specimen.

· Always FOCUS AWAY

3. The “Condenser”

a. Close the field iris so that it is visible within the field of view

b. Using the condenser focus control, focus the image of the field iris.

c. Using the condenser centration controls, center the image of the field iris.

d. Open the field iris so that it is just outside the field of view (glare control).

e. Adjust the condenser/ aperture iris for the “Oomph” position (crispest image and optimum contrast)

Text Box:

Figure 3. Establishing Koehler

(L-R):

§ Out of focus/decentered image of field iris;

§ Centered focused image of field iris;

§ FI opened just outside of field of view and CAI set for “Oomph” position)

Text Box:

A handy tip: if you are fighting glare and haze, move the feature of interest to the center of the field and encircle it with the field iris.

4. The Eyepieces (oculars)

a. Adjust the interpupillary distance so that you see one, single, round field of view

§ Interpupillary distance is literally the distance between the centers of your pupils when you are looking straight ahead. You can measure this distance (in mm)with a ruler on your partner.

5. You

· Chair height?

· Back support?

· Room lighting?

Handy tips and other tidbits:

1. If you are fighting glare and haze, move the feature of interest to the center of the field and encircle it with the field iris. (Why does this technique work to reduce glare?)

2. Regarding the condenser/aperture iris: most books will tell you to remove the eyepiece, look down the “back focal plane of the objective” to see the aperture iris and adjust that iris so that the back focal plane is approximately 2/3 to ¾ open. Notice that the function of the aperture iris is to control coherence for optimal resolution and edge information in the sample yet, using this method, there is no reference to the sample.

3. Interpupillary distance is literally the distance between the centers of your pupils when you are looking straight ahead. You can measure this distance (in mm) with a ruler on your partner.

· Note that there is frequently a scale on the binocular body of the microscope that has numbers ranging from approximately 55 (mm) to 72 (mm). This is the interpupillary distance scale. Once you have measured your “ID” you can set this distance on any microscope for more comfortable working. You should set this distance every time you approach a microscope.

4. The diopter setting is the focus adjustment on the eyepieces. It takes into account the power differences between your two eyes.

5. Your dominant eye is the “lead horse” in your two-eye team. If you play a sport or shoot a gun, it is the targeting eye [2].

6. Adjusting the diopter setting when both eyepieces are adjustable:

· Rotate the focus control on the eyepiece for your dominant eye so that it reads “0” if there is a number or until the white or silver ring is just covered up. While looking with just your dominant eye, use the fine focus on the microscope to make sure that the microscope is really in focus for that eye.

· Close your dominant eye and, using just your non-dominant eye, adjust the focus on its eyepiece until the microscope image is sharp for that eye.

7. Adjusting the diopter setting when you have only one adjustable eyepiece:

· While looking through just the fixed eyepiece, use the fine focus on the microscope to make sure that the microscope is really in focus for that eye.

· Close that dominant eye and, using the adjustable eyepiece, focus it until the microscope image is sharp for that eye.

Lab 1. Anatomy and Physiology of the microscope

1. With your lab partner, locate each of the following on your microscope:

a. On-off switch	e. Diopter adjustment

b. Objective	f. Interpupillary adjustment

c. Condenser	g. Aperture iris

d. Eyepieces	h. Field iris (available?)

2. In your own words, describe the functions of each of those components

Objective

Condenser

Eyepieces

Field iris

Aperture iris

Diopter adjustment

Interpupillary distance

Lab 2. Setting up Koehler illumination

1. In your own words, summarize the steps for establishing Koehler illumination (refer to your earlier notes if necessary:

2. Using a 10x objective, set up Koehler illumination with the samples provided. Make sure that the instructor confirms that you have correctly set Koehler.

Checklist:

q Is your field uniformly illuminated?

q Is the field of view adequately bright?

q Are the edges of the features in your specimen crisp, sharp, and clean?

q Are YOU comfortable?

3. Without changing any of your settings, rotate the next highest magnification into position. Re-establish Koehler. What adjustments did you have to do to the condenser/ aperture iris and field iris and why?

4. Depending if you did this process on an upright or inverted light microscope, go to the other type of microscope and repeat steps 2 and 3. Describe any differences below.

1. Normal cleaning

· Puff - dry air to remove loose dirt

· Huff - moist air (deep in lungs) to deposit film of moisture to remove soluble dirt

· Swirl - pure cotton swab, from the center outward

Use once only. Remember, you are cleaning a delicate, dielectric film, not just glass

2. Oil and other immersion media

· Drag

· Swirl with moistened (not dripping) cotton swab

3. Stand, stage, and non-optical components

· Use a soft cloth dampened with a dilute solution of a mild detergent. Rinse with a dampened cloth and wipe dry.

1. The Nature of Light

Figure 4. The Nature of light

a. Light is Electromagnetic radiation: it has both electrical and magnetic characteristics.

b. A light wave can be described as the path defined by the movement of an electrical vector, building up first in the positive direction then decreasing; building up in the negative direction then decreasing.

c. Direction of travel – the direction in which the light wave is moving away from its source. You see me because light is traveling from me to you.

d. Direction of vibration – the direction in which the light wave is oscillating. The direction of vibration is always at right angles to the direction of travel but it could be at any or even all angles of rotation (North-South, East-West, or any angle in between).

e. Amplitude – the maximum displacement or “height” of the wave above or below the reference bar. The intensity of the light is proportional to a²

f. Wavelength – the distance it takes for a wave to go through one cycle (ex: peak to peak, trough to trough). The wavelength describes color and energy

Figure 5. The Spectrum

g. Memory clue: ROY G BIV

Text Box: (Colors of the rainbow are red-orange-yellow-green-blue-indigo-violet)

2. Light-Light Interactions – Interference

In the process of interference, the light waves add their energies to form a resultant wave

In order for interference to take place, the waves must be

· Traveling in the same direction, space, and time

· Vibrating in the same plane

· Having the same wavelength

· HELPFUL: Same approximate amplitude

a. Constructive Interference (nl)

Figure 6. Constructive Interference

· What happens if two green waves undergo constructive interference?

b. Destructive Interference (n/2 l)

Figure 7. Destructive Interference

· What happens if two green waves undergo destructive interference?

c. The Color Wheel

1) Primary colors for LIGHT:	Red	Green	Blue
2) Secondary colors for LIGHT:	Cyan	Magenta	Yellow
3) R+B+G = white

Figure 8. The Color Wheel

Light-Matter Light Interactions

1. Refractive index (ri, n)

a. A measure of matter’s electrical character

b. Mathematically, the ratio of the velocity of light in air versus the velocity of light in a material

ri = n = V air____ where velocity of light in air(vacuum) = 300,000 km/s

V material

Equation 5. Refractive Index

(1) What is the refractive index of air?

(2) What is the refractive index of glass? (velocity in glass = 200,000 km/sec)

(3) What is the velocity of light in water? (refractive index = 1.33)

c. Refraction versus reflection

Figure 9. Snell’s Law

Equation 6. Snell’s Law

Imaging Theory Part I: Geometric Optics

1. How lenses work

Figure 10. How Lenses Work

a. A focal point is a specific point at which light converges to form an image

b. A focal plane is a two-dimensional array of focal points

c. FFP = Front Focal Plane, the plane on the incoming side of the lens

d. BFP = Back Focal Plane, the plane on the emerging side of the lens

e. do = distance from lens to original object

f. di = distance from lens to image

g. (Reminder: OBJECT and IMAGE are two different entities!!!)

Magnification = di/do = si/so

1/f = 1/di + 1/do

Equation 7. Lens Makers’ Formula

2. Four cases of lenses[3]

· Place the object inside FFP/Virtual image

· Move the object to the FFP/Image at infinity

· Move the object beyond FFP/Image at finite location

· Object at Infinity/Image at BFP[4]

Figure 11. Four Cases of Lenses

3. Aberrations - Lens imperfections & their corrections

· Aberrations come in three basic “flavors”: Chromatic, Spherical, and Field Flatness

a. Chromatic Aberrations– As the name implies, chromatic aberrations involve the separation of white light into its colors (dispersion). Chromatic aberrations are a direct result of refraction and come in two forms:

(1) Axial chromatic aberrations occur along the axis and result from RRR. Since red resists refraction, it focuses farther down the axis of the microscope. This effect is very pronounced with simple Abbe condensers in transmitted light. To test: close the field iris so that only about ½ of the field is illuminated. When the iris is in focus, you may see either no ring at its edge or a yellow ring. Lowering the condenser moves that lens away from the specimen. At some point, you will encounter the longer focal distance for red light, producing a red ring at the edge of the condenser. Moving the condenser closer to the specimen will bring the blue light into focus.

(2) Lateral chromatic aberrations occur across the axis and result from a combination of light encountering different angles at different points along the curve of the lens (Snell’s Law) and RRR. Since the blue light will be focused in closer to the axis and the red light further away, there will be a smaller blue image ringed by a larger red image. This difference in size is sometimes called the chromatic aberration of magnification.

Figure 12. Chromatic and Spherical aberration

b. Spherical aberrations are a direct result of Snell’s Law and are due to the fact that lenses are ground as parts of spheres. As light approaches the lens, it encounters different angles and undergoes different amounts of refraction. Peripheral rays see the sharpest curve and refract the strongest, bring them to a focus closer to the lens. Rays closer to the optic axis of the microscope see a nearly flat surface and refract the least, causing them to focus farther away. Since the rays are focusing at a variety of locations, no clear image is formed. A strong symptom of spherical aberration is a milky or hazy image.

c. Field flatness is also due to the fact that lenses are ground as part spheres. The major symptom is fall-off or lack of focus at the edge of the field of view.

d. Other aberrations include “coma” and “astigmatism”. In coma, points in the object are imaged as smears or blobs in the image. In astigmatism, vertical and horizontal lines will focus at different planes.

4. Corrections

Lens classification:	Chromatic	Spherical
achromat	R+B	R+B
fluorite	R+B+G	R+B
apochromat	R+B+G+UV	R+B+G

Table 1. Lens classifications

Figure 13. Cutaway of a highly corrected, plan-apochromatic lens

(image courtesy of Carl Zeiss, Inc.)

Conjugate Focal Planes

1. Conjugate Focal Planes: Object located in lower plane is imaged in next plane in the series.

a. Very important for trouble shooting

b. Demonstrate location of these planes in the microscope, starting from the light source

Illuminating set (left hand)	Specimen set (right hand)
	Retina/receiver*
Ramsden disk (exit, eyepiece)
	PIP (eyepiece seat)
BFPo (rear of objective)
	Specimen
FFPc (condenser iris/aperture iris)
	field iris
Filament

Table 2. Conjugate Focal Planes - The Two-Handed Exercise

*includes film plane, detector in camera (digital/video), detector in spectrometer (UV-VIS, FTIR, etc.)

Lab 3. Demonstration of Conjugate Focal Planes in the Microscope

Note: Koehler must be established before starting this exercise. Use a well-defined specimen without much depth.

1. Finding the focal planes in the illumination system

a. Close the aperture iris and use it as a screen. If possible, remove the diffuser from the light path. Looking outside the microscope, use a dental mirror to see if the image the coiled filament of the lamp appears on the closed leaves of that iris.

If there is diffuser in the system, you may only see a disk of light.

b. Remove the eyepiece and peer down into the BFPo (deep down in the tube).

1) Do you see the lighted disk/filament or the sample?

2) Open and close the aperture iris. Describe what you see in the BFPo.

3) Open and close the field iris. Describe what you see in the BFPo.

c. Using a piece of lens tissue as a screen, find the Ramsden disk or exit pupil of the microscope.

1) How far is this plane located above the top of the eyepiece? How does this relate to the engravings on the eyepiece?

2) Do you see the lighted disk/filament or the sample?

3) Open and close the aperture iris. Describe what you see in the Ramsden disk.

4) Open and close the field iris. Describe what you see in the Ramsden disk.

5) When you sit down at the microscope, where does the Ramsden disk sit on your eye? (cornea, vitreous humor, retina?)

d. Starting with the filament, summarize the planes located in the illumination set.

e. Note especially the relationship between the FFPc and the BFPo.

2. Finding the planes in the specimen set.

a. Looking in the microscope, open and close the field iris. Describe the effect on the image of the specimen.

b. Repeat the experiment opening and closing the aperture iris. Describe the effect on the image of the specimen.

c. Remove one of the eyepieces and stretch a piece of lens tissue over the opening to act as a screen. Describe what you see at the PIP.

1) Open and close the field iris and describe what you see.

2) Open and close the aperture iris and describe what you see.

d. Where is the last plane in this series?

e. Starting with the lamp collector lens, summarize the planes that form the specimen set.

f. When we set up Koehler Illumination, why do we use the field iris as the target for focusing the condenser?

3. Why do we call these sets “conjugate focal planes”?

4. If you need to insert a measuring target (ruler, grid, etc.) into the microscope so that you would see its image superimposed on the image of the specimen, where could you put it?

Imaging Theory Part II: Diffraction Theory

1. Diffraction Theory - the other half of the story

a. The image is only a reproduction of the object. Our job is to make it the BEST reproduction.

b. The Diffraction Pattern is the result of interference from wavelets of light emerging from various points in the specimen. It can be seen in the BFPo

(a) (b)

Figure 14. The Diffraction pattern of a simple object (ex: a grating)

(a) Simple grating, as seen in the regular view of the microscope

(b) Diffraction pattern of that grating, as seen in the BFPo

c. The Diffraction Pattern carries the code (Fourier Transform) from the object to form the image, including the background, orientation, object spacing, and edge information

d. The Zero order is responsible for the background information (VERY IMPORTANT)

e. Any two adjacent diffraction spots are necessary for spacing

f. Any three adjacent spots are necessary for edge information (the more spots, the more faithful the edge)

2. The importance of Numerical Aperture (NA)

a. Numerical aperture is a measurement of the collecting angle of the objective.

Figure 15. Numerical Aperture

NA = n sin a	where	NA = Numerical Aperture
		n = refractive index of immersion fluid (air, oil, etc.)
		a = angle of last ray to be collected by objective
		wd = working distance between the front of the lens and the top of the specimen

Equation 8. Numerical Aperture

Text Box:

Figure 16. Two Numerical Apertures (Diagram courtesy of Carl Zeiss, Inc.)

Text Box:

Figure 17. Ability NA to collect diffraction pattern

b. Impact of NA on resolution:

R = 1.22 l/(NAo + NAc)

c. Impact of NA on edge information and quality of image

d. Impact of NA on fluorescence

e. What is the best choice for YOUR application?

Lab 4. Reading your microscope

1. Write down each of the engravings on your microscope and indicate what they refer to. Make sure that you also include engravings on the eyepieces.

2. For what application was this microscope purchased?______________________________

_________________________________________________________________________

3. Were these optics a good match for this application, especially in terms of magnification, resolution, correction, and contrast techniques? Defend your answer.

Putting the Basics to Work: Troubleshooting

1. The mystery of the moving shadow*

a. Things in focus are in the specimen set

b. Things out of focus are in the illumination set

2. Not enough resolution

a. The importance of NA, revisited

b. To immerse or not to immerse?

3. Not enough contrast

a. The power of the condenser aperture/aperture iris

b. Reducing glare with the field aperture

c. Spherical aberration

Becoming a Better Consumer:

Matching your Microscope to your Application & Budget

1. The stand

a. Current application(s)?

b. Future growth?

2. The Optics

a. Inverted or upright?

Figure 18. Light path of inverted microscope (L) and upright microscope (R )

b. Magnification? (Transmitted light: typically 5x/10x/20x/40x/60x/100x)

c. NA? (remember impact on resolution, edge information and ability to capture fluorescence)

d. Corrections? Color (chromatic), Spherical, and Field flatness

e. Budget?

f. Contrast techniques?

1) Components to generate (Darkfield, Fluorescence, Polarized Light, DIC)

2) Non-interfering optics (Fluorescence, Pol)

3) Correct illuminator (HMC, Pol, Fluorescence)

g. 3D or not? (Stereo microscope or compound; confocal? AFM?)

Matter

1. Refractive index revisited

Reminder: refractive index is a measure of Matter’s electrical character and is expressed by the ratio of the velocity of light in vacuum (air) divided by the velocity of light in the material under study.

ri = n = Vair

Vmat.

Isotropic

Anisotropic

Isotropic = same electrical field in

all directions

Anisotropic = NOT the same

= “Electrical grain”

Amplitude Objects (Absorbing objects)

Filters!

(a) Red filter

(b) Neutral density filter (nd)

(a)

(b)

Phase Objects

P = n x t

Optical Path Difference:

OPD = P₁ – P₂ = t(n₁- n₂)

(a) Films, glass

(b) Step (reflection

(a)

(b)

(d) Phase gradient, slope (Cells!!!)

j = a (n₁ - n₀)]

(c )

(d)

Table 3. Methods for classifying Matter

2. Further discussion on optical path:

a. The optical path is literally, the optical distance which light encounters moving through a system. It is based on both the physical thickness and the refractive index. As light passes through the material, it is slowed down compared to its neighbor, passing through air. As a result, the two waves emerge out of step with each other: they have undergone a phase shift. As a result, the object is called a phase object.

b. If you are measuring the optical path difference due to a step-type feature in reflected light or for the thickness of a thin film, divide the measurement by half to account for the bounce:

OPD = 2t

Equation 9. Step heights and thin film thickness calculated from OPD

c. The OPD can be visually estimated from Newton’s colors (see Michel-Levy Charts). For example, if a film of soap or oil (ri ~ 1.5) displays a rich magenta color, it indicates an OPD of approximately 560nm. Substituting the OPD and relative refractive indices of air and the oil into the standard equation:

OPD = t (n₁ – n₂)

560nm = t (1.5 – 1.0)

t = 280nm

Because the beam in the film went twice as far, we need to divide by 2: t=140nm or

0.140mm.

Light-matter Interactions

1. Absorption - selective reduction of one or more wavelengths

2. Reflection - bouncing of light from the surface

a. In specular reflection, the bounce is “well controlled” resulting in a clean, well formed image

(i. e., a mirror)

b. In diffuse reflectance, irregularities or texture on the surface break up the photons (packets of light), resulting in haze and glare.

3. Refraction - bending of light when it approaches a boundary between 2 different refractive indices (RRR = Red Resists Refraction)

4. Diffraction - bending of light at edges (BRD = Blue Resists Diffraction)

5. Polarization - selective transmission or reflection of only one direction of vibration

6. Fluorescence - Energy in/Energy out

7. Scatter is a combination of two or more of these phenomena

Bringing out the Best

A note before we begin:

The ultimate response to a contrast technique is determined by the sample itself. As the microscopist, you have to jobs:

· Set up the technique correctly

1) Make sure that you are using the correct components.

2) Optimize the alignment,

· Observe the response thoughtfully

1) Think about which light-matter interaction is in action.

2) Watch for artifacts or combined effects.

For each of the contrast techniques, we will be asking ourselves the following questions:

1. What is the technique detecting or enhancing: edges, angles (gradients), or a body effect?

2. What is the mechanism at work?

3. How do we do that technique?

4. What does the image look like?

5. What are the effects on (a) Resolution and (b) Contrast?

6. What are the likely artifacts?

7. Can we do it ourselves or do we need components built by the manufacturer?

Contrast I - Quick, Easy, and Free

1. Changing refractive index (35 mm slides: salt in air/ salt in oil)

a. Detects: Edges

b. Mechanism: Difference in refractive index

c. How do you do it? Immerse the sample in the fluid of choice

Air 1.00

Water 1.33

Glycerin 1.47

Immersion oil* 1.515

Cargille Liquids Full range

Corn oil?

Mineral oil?

Silicon oil?

d. What does the image look like?

1) The greater the difference in refractive index, the darker the boundary

2) Caution: Too great a difference creates excessive contrast and obliterates true edge

e. Resolution vs. Contrast? The resolution is the same as typical brightfield image

f. Can you do it yourself? Quite easily

2. Using colored filters

a. Detects: whole objects

b. Mechanism:

Same color suppresses (S-S); Opposite color enhances (The color wheel, revisited)

c. How do you do it?

Filters can be inserted anywhere in the light path.

Suggestions:

· Keep filters away from the lamp, which gets very hot and can actually either burn through the filter (if a gel filter) or bleach it (if glass).

· To reduce the potential for artifact (dust, scratches, etc), If possible, place the filter in an illuminating plane, rather than a specimen plane.

d. What does the image look like? The whole image takes on the color of the filter.

· Objects having the same color or having a secondary color containing the color of the filter will merge with the background.

· Objects have color opposite from the color of the filter will appear darker as their colors are absorbed by the filter.

e. Resolution vs. Contrast? The resolution is the same as typical brightfield image

f. Can you do it yourself? Quite easily

3. Axial illumination

a. Detects: Edges

b. Mechanism:

1) Based on diffraction in the image plane itself.

2) The more axial the illumination, the more coherent the beam.

3) The more coherent the beam, the more defined the diffraction and the more evident the

constructive (bright fringe) and destructive (dark fringe) interference.

c. How do you do it? Simply close down the aperture iris.

d. What does the image look like? Edges will get darker and thicker as you close the aperture iris (destructive interference). At some point, you will also be able to see the bright fringe, just outside the dark fringe (constructive interference).

e. Impact on resolution and contrast?

1) Enhanced resolution but, because you are closing down the NA of the condenser, decreased resolution

2) The more axial the illumination, the deeper the “waist” at the focal point.

Text Box: Result: Greater depth of field (important for thick samples, big particles or when one structure overlays another)

3) Caution: “Ringing” (bright-dark fringes at edges) will occur if you go too far.

f. Can you do it yourself? Absolutely.

4. Oblique illumination - Slightly offset the Zero order

a. Detects: Gradients

b. Mechanism:

1) Various gradients will refract light into or out of the collecting angle of the objective. If collected, that gradient will be bright. If lost, that gradient will be dark.

2) Very refractive index dependent (Snell’s Law, revisited)

c. How do you do it?

Anything which shifts the beam off-axis will work:

1) Off-set pinhole

2) Off-set phase ring

3) Business card

4) Even your thumb!

Figure 19. Oblique Illumination

d. What does the image look like?

The bright:dark shading provides cues to your eye-brain combination which create the impression of three-dimensionality.

e. Impact on resolution and contrast?

Interestingly, because you are selecting a population of offset Zero orders and rejecting the on-axis Zero orders that normally swamp this special population, you can improve resolution and contrast at the same time. The BFP on the back shows the first orders (“X”) lost in conventional, on-axis imaging. The BFP on the right reveals the effect of shifting the zero order to the right, capturing a first order (remember you only need two adjacent diffraction spots to generate spacing information).

Figure 20. Impact of oblique illumination on the capture of the diffraction pattern

f. Can you do it yourself? Absolutely.

g. Comments: Note that this technique is very directional. If you align the gradients so that they are parallel with the offset, there will be nothing to refract the light out of the collecting window. Result: no contrast. This directionality can be used selectively to enhance or suppress contrast.

Lab 5. Contrast Enhancement I: Managing Refractive Index

Equipment: None

Sample: Slide with crystals mounted in (a) air (b) immersion oils

Microscope Set-up: Koehler Illumination, 10x objective

Objective: Control refractive index to enhance or suppress contrast

Procedure: Observe the crystals in both mounting media and compare the contrast. Note especially the resolution and edge information.

Questions:

1. The refractive indices of air, salt, and immersion oil are 1.00, 1.47, and 1.51, respectively. Explain your observations in terms of the refractive index differences between salt and its two mounting media.

2. Is resolution affected by the change in refractive index?

3. Which of the two images would you prefer to use for automated particle counting. Defend your answer

.

Lab 6. Contrast enhancement II: Effects of staining and filtration

Equipment: A choice of colored filters (red, green, blue, magenta)

Samples: Softly stained sample

Microscope Set-up: Koehler Illumination, magnification to fit the sample.

Objective:

Control color contrast through staining and use of filters to enhance or suppress information

Procedures:

1. Observe the sample in normal brightfield. Insert the red filter over the light port and describe what happens to the contrast. Repeat the process using a green filter.

2. Using the softly stained sample, look at the color wheel and determine which filter will enhance a specific feature. Draw the color wheel and, in your comments, indicate (a) which feature and what color you want to enhance, (b) which color filter you chose, and (c) the effect.

Question:

1. How do you choose colors to

a. enhance contrast? b. suppress contrast

2. On which capability of human perception does this technique capitalize?

3. Does this technique bring out edges, angles, or body effects?

Darkfield illumination - Really offset the Zero order

1. Detects: Anything that will scatter light (Detection limited, not resolution limited)

2. Mechanism:

a. Light comes down the outer channel of the condenser. The angle of approach at the sample is so high that the background illumination escapes collection. Since that light is lost, the background is black.

b. Scratches, pits, edges, inclusions, and other scattering structures in the specimen will scatter light Only the light that enters the objective will be used to form the image.

Figure 21. Darkfield Illumination

3. How do you do it?

a. Use a patch stop placed in the FFPc .

b. What does the image look like?

1) Bright objects on a dark background.

2) Also, because anything that can scatter light may contribute to the image, this technique will often seem to have infinitely deep depth of field.

4. Impact on resolution and contrast? Darkfield is a detection technique; not resolution based. You will actually be able to discern particles far below the limit of resolution. Since you do not have enough of the diffraction pattern to collect spacing (let alone edge) information, you cannot make any comment about size, shape or other features. However, it only takes 2-3 photons to reach your eye for you to discern that something is there.

5. Can you do it yourself? Very easily

6. Comments: Because this technique is based on scatter, may have color as an artifact. Check in

BF first.

Phase Contrast

1. Detects phase objects

2. Works best with very small optical path differences

a. Ideally, OPD = l/8

b. Causes l/4 shift between sample lt. & background lt.

3. Has three components:

a. Annulus or ring in the condenser (FFPc)

b. Phase plate in the objective (BFPo)

c. Green filter to control l (usually 546 nm)*

4. Operates as follows:

a. In specimen:

1) Light going through background is undisturbed

2) Light going through sample lags l/4 (n x t)[5]

Figure 22. Phase Contrast

b. At phase plate:

1) Sample light passes through extra glass adding an additional l/4 lag

2) Background light passes through phase ring where it encounters a neutral density filter which reduces intensity to 15% of original. Result: amplitude of background wave ~= amplitude of sample wave

c. At PIP:

1) Sample light meets background light. They interfere to form the image

2) l/4 + l/4 = l/2 à destructive interference

5. Modestly tunable

a. Adjust ri of mounting medium

b. Use green filter (which l?)

Lab 7. Contrast enhancement III: A comparison of Axial, Oblique, Darkfield, and Phase

Samples: Microscope Set-ups:

a. Axial and Oblique: Start with Koehler Illumination, magnification appropriate to sample

b. Darkfield: Start with Koehler Illumination. Look for a device in the FFPc to generate Darkfield.

c. Phase: Start with Koehler illumination. Look for devices in both the FFc and the BFPo to generate Phase contrast.

Objectives:

1. Compare contrast, resolution, and detectability of axial, oblique, darkfield, and phase contrast to standard brightfield

2. Explain the steps necessary to achieve each contrast technique

3. Using a simple diagram, discuss how each technique operates, which component of the sample it brings out (edges, angles, or body effects), and which of our visual capabilities it uses (color or intensity).

Procedures:

1. Axial and Oblique illumination.

From previous labs, you know how to adjust the microscope for these two techniques and how the light beam changes. Review those concepts if necessary. Start with Koehler Illumination then adjust the microscope to achieve each of these two techniques. Carefully observe what happens to edges, gradients, and the body of the sample.

Please take the time to make a quick, labeled sketch of both the optical set ups and the image. In your observations, include comments about how each technique affects contrast, resolution, and detectability.

2. Darkfield illumination.

Use the same sample you used for Axial and Oblique so that you can compare the results of your darkfield experiment. Again, make a quick, labeled sketch of both the optical set up and the image. In your observations, include comments about how darkfield affects contrast, resolution, and detectability.

3. Phase contrast

Continue to use the same sample. Again, make a quick, labeled sketch of both the optical set up and the image. In your observations, include comments about how phase contrast affects contrast, resolution, and detectability.

Diagrams: (OA = optical axis of the microscope)

1. Axial

2. Oblique

3. Darkfield

4. Phase Contrast

Summary Questions:

1. Which of these four techniques is most effective for:

a. Bringing out general information

a. Detecting small pits and bits of debris

b. Bringing out edge information

c. Bringing out surface information

2. Explain the difference between “resolution” and “detection”. Can you really make a statement about resolution for a Darkfield image?

3. Explain why a green filter improves a Phase contrast image.

Advanced Contrast I: Fluorescence

1. Detects: selective chemical environment – availability of mobile p electrons (alternating or conjugated C-C and C=C bonds) (a “body” effect rather than edge, phase, or gradient)

Text Box:

2. Mechanism: Energy in/Energy out

a. Short energy beam (light, heat) hits

sample, activates mobile p electrons.

b. They absorb the energy and jump from their normal molecular energy levels to an “excited state”.

c. They will find a mechanism for releasing the absorbed energy and returning to their original energy states. We engineer the system so that they return the energy as light (fluorescence).

d. A small amount of energy is always converted to heat in this process, typically causing a shift from shorter wavelength to longer wavelength (the Stokes Shift).

Figure 23. Stokes’ Shift

3. How do you do it?

Text Box:

Figure 24. Key components for Fluorescence (courtesy, Carl Zeiss, Inc.)

a. Key components:

1) Exciter filter – isolates the particular wave length necessary to activate the fluorochrome

2) Dichroic beam splitter – the optical “gate”. The specification for this beam splitter is given in terms of a dividing wavelength. All shorter wavelengths will be reflected by the mirror (ex: the excitation light) and all longer wavelengths will pass through (i. e., the emitted fluorescence)

3) Barrier filter – a filter whose job it is to absorb any extraneous excitation, reflected, or unwanted fluorescence. This filter cleans up the signal so that the background is black and the fluorescence signal is highly visible.

4) High numerical aperture objectives - need to collect as much of the emitted fluorescence as is possible.

5) Correct lamp – see “light budget” below[6]. Typically, epi illumination (epi=”on top of”)

6) Responsive sample – only materials with mobile p electrons that are available for excitation will respond to fluorescence.

b. The “Light Budget” – spectral characteristics of each component in the system, from light source to filters to sample to detector, must match for optimum fluorescence.[7]

4. The Stokes Shift - energy dissipated before fluorescence

d = (1/l_ex + 1/l_em) x 10⁷

Equation 10. Stoke’s shift

a. Stokes fluorescence (“the usual”) - emission occurs at longer wavelengths than excitation

b. Anti-Stokes fluorescence - extra thermal energy absorbed; emission occurs at shorter wavelengths than excitation

c. Resonance fluorescence (FRET) – emission occurs at same wavelengths as excitation

5. The sample drives the process

a. Autofluorescence

b. Immunofluorescence

6. The importance of chemical structure[i]

a. Presence of easily excited p electrons (i. e., highly conjugated system - large number of alternating single and double bonds) with rigid, planar structures

b. Rule of Thumb I: The greater the degree of conjugation, the greater the quantum yield and the longer the wavelength of the emitted light.

Examples:	Structure:	Fluorescence:
Benzene		Ultraviolet
Naphthalene		Ultraviolet
Anthracene		Blue
Naphthacene		Green
Pentacene		Red

Table 4. Greater conjugation shifts the wavelength of the emitted light

c. Rule of Thumb II: The more rigid and planar the structure, the more intense the fluorescence

Examples:

Structure:

Emitted fluorescence:

Fluorescein

Intense Green

Phenolphthalein

Weak Red

Table 5 . Greater rigidity and planarity generates more intense fluorescence

7. Factors influencing fluorescence

a. pH - alters ionic association; affects both fluorescence spectrum (color) and quantum efficiency (intensity)

b. temperature - more movement, more likely interaction with neighboring molecules, less likely to fluoresce (related to rigidity)

c. viscosity or rigidity of the medium (i. e., mounting medium) - more movement, more likely interaction with neighboring molecules, less likely to fluoresce (related to rigidity)

d. binding to macromolecules (i. e., how the fluorochrome is bound to the substrate) – changes spectrum (both excitation and/or emission maxima), intensity, degree of fluorescence polarization, and temperature coefficient of fluorescence

e. metachromasia - same dye gives two different colors due to formation of dimers or polymers

ex: Acridine Orange (red and green)

8. Decay, Fading, photobleaching, and quenching

a. Decay - rapid , progressive drop in intensity after irradiation stops

b. Photobleaching - loss of intensity during irradiation

1) Frequently involves reaction of excited fluorophore and oxygen, resulting in a non-fluorescing product

2) Depends on

§ Nature of fluorophore:

porphyrins (fast) ; lignin and Acriflavine (slow)

metachroasis:

red version of Acridine orange = fast;

green version of Acridine orange = slow

§ Chemical environment:

creation of reactive intermediary + availability of oxygen

§ Intensity & Quantum Efficiency (E/time) of excitation:

a. no photobleaching during first few milliseconds*

b. rapid photobleaching during next few seconds

c. slower photobleaching

*(basis for success of confocal microscopy)

c. Fading - loss of intensity over long term (ex: storage)

d. Recovery - partial or complete restoration of intensity after photobleaching

e. Quenching - rapid reduction in fluorescence

1) Presence of other molecules in the system generates a competing, deactivating process

2) Affected by

§ Temperature:	Higher T, lower Fl (usually about 1% per degree but can vary up to 5%)
§ Impurities:	Highly variable; may have +/-/no effect
§ Oxygen:	conc. of ~ 1 mM reduces intensity ~ 20%
§ Concentration:	Lower Fl at both very low and very high concentrations
§ Prior treatment:	ex: hematoxylin, crystal violet

9. Which light source is best for your application?

a. Conventional halogen

b. HBO

c. XBO

10. A word about filters

Figure 25. Filters are the heart of the fluorescence system

(image Courtesy of Carl Zeiss, Inc.)

Figure 26. Choosing filters for a specific fluorochrome

(Images courtesy of Carl Zeiss, Inc.)

a. Exciter and barrier filters – driven by the fluorochrome

b. Dichroic filters – driven by the fluorochrome

c. Common filter terminology

1) KP = Kurz pass - passes only to the short/high energy side of the spectrum

2) LP = Long pass - passes only to the long/low energy side of the spectrum

3) BP = Band pass - passes only within a specific, narrowly defined band

4) WP = Wide pass - passes a broad band of the spectrum

5) DF = Discriminating filter with very steep sides and especially deep

attenuation

6) CWL = Center Wavelength

7) FWHM = Full Band Width at Half Maximum Transmission

ex: 488 DF 22 CWL

488 CWL = Central wavelength: 488 nm (blue light)

DF = discriminating filter

FWHM = 22 nm (passes only 11 nm on either side of 488nm)

11. Fine tuning your optics for fluorescence

a. Glassware in the fluorescent train must not respond to fluorescence (Use fluorites or specified plan apos)

b. Use the highest numerical aperture to gather the greatest amount of light

Intensity is directly proportional to NA⁴ !

c. Use the lowest magnification possible, both at the objective and at the eyepiece

Intensity is inversely proportional to M²

Magnification¹	NA¹	OBI²	Rel. Brightness 1³	Rel. Brightness 2⁴	Rank Order
10	0.25	3.9	1.0	3.7	15
10	0.50	62.5	16.0	59	5=
16	0.40	10.0	2.6	9.4	14
16	0.50	24.4	6.3	23	9=
20	0.50	15.6	4.0	15	13
20	0.75	79.1	20.3	74	3
25	0.60	20.7	5.3	19	11
25	0.80	65.5	16.8	61	4
40	0.75	19.8	5.1	19	12
40	1.00	62.5	16.0	59	5=
60	1.40	106.7	27..3	100	1
63	1.25	61.5	15.7	58	7
63	1.40	96.8	24.8	91	2
100	1.25	24.4	6.3	23	9
100	1.40	38.4	9.8	36	8

¹ Nominal Values. Actual objective magnification and numerical apertures may vary

considerably from the values engraved upon the objective barrel.

² OBI = Objective Brightness Index = (NA⁴/Mag²)x10⁵

³ Relative Brightness compared to the 10x/0.25NA objective

⁴ Relative Brightness compared to the 60x/1.4NA objective

Table 6. Effect of Objective Magnification and Numerical Aperture on Relative Intensity[ii]

Data for 15 commonly used objectives from the Zeiss and Nikon ranges

(Table modified slightly for purposes of this discussion)

d. For UV excitation, use quartz

Remember: your optics have spectral response, too. Most standard microscope optics only pass between approximately 380nm and 2200nm.

12. Detectors: There’s more here than meets the eye

a. Eye: 400-700nm

b. Camera: depends; frequently has strong response in the infrared (use a heat filter to suppress)

c. Photomultiplier tube (PMT): visual range to near infrared; can be expanded with PbS detectors

d. Always ask for technical specifications then TEST with your sample.

13. Set-up:

a. Before beginning, make sure that all fluorescence filter cubes are out of the optical path. If you are using an arc source for the epi illumination, turn it on before turning on any other electrical equipment, especially computers. Check with the manufacturer as to warm-up time.

b. You may or may not be able to see your cells or particles under brightfield, but if can, establish Koehler as usual, using transmitted light.

c. Block off the transmitted light beam and open the shutter for the epi illumination.

d. Insert the appropriate filter cube and observe.

14. What does the image look like?

a. Because the filter cube blocks all the incoming illumination, the background is black.

b. Because the sample selectively responds, it will be bright and have a characteristic color.

15. Resolution v. contrast?

a. As with darkfield, fluorescence is a detection technique. You can often image cellular components or small particles well below the limit of resolution (ex: microtubules in the cytoskeleton at 0.10 mm or 100 nm).

b. Because of the bright object on dark background, contrast can be greatly enhanced. However, some fluorescence is very faint. Suggestions: darken the room and dark-adapt your eyes or use a cooled camera that can integrate images.

16. Can you do it yourself? Yes, but it would be a stretch. For optimum results, use manufacture-suggested components.

17. Comments:

The chemical environment may affect the Stoke’s shift. Check for pH, presence of interfering ions such as Mg++, NA+, etc.

Also, some materials exhibit very large Stokes’ Shifts. Ceramics, for example, may excite in the blue region (~450nm) but emit in the red region (~600nm).

Filter technology is highly developed. Companies such as Chroma and Omega Filters (both in Brattleboro, VT) offer superb product lines, technical support, and engineering services to optimize filters for your specific application.

Some good references on fluorescence:

Rost, F. W. D., Fluorescence Microscopy, Cambridge U Press, New York, 1992

Rost, F. W. D., Quantitative Fluorescence Microscopy, Cambridge U Press, New York, 1991

Shotton, D, ed. Electronic Light Microscopy, Wiley-Liss, New York. 1993

Slavik, J., M., Fluorescence Microscopy and Fluorescent Probes, Plenum Press, New York, 1996

Taylor, D. L, and Wang, Y., eds., Fluorescence Microscopy of Living Cells in Culture:

Part A: Fluorescent Analogs, Labeling Cells and Basic Microscopy, Methods in Cell Biology, Vol. 29. Academic Press, San Diego, CA, 1989

Part B: Quantitative Fluorescence Microscopy --- Imaging and Spectroscopy, Methods in Cell Biology, Vol. 30, Academic Press, San Diego, CA, 1989

Wang, X. F., and Herman, B., Fluorescence Imaging: Spectroscopy and Microscopy, John Wiley & Sons, New York, 1996

Lab 8. Contrast Enhancement: Fluorescence

(Our thanks to Reinhard Enders for the initial exercise on which this one is based)

Specimen: Any sample which will respond to fluorescence (ex: H&E or trichrome stained

tissue preparation)

Microscope Set-up:

Koehler Illumination, 40x objective;

Microscope fitted with epi illuminator, fluorescence filters; arc lamp illumination source.

Note: “epi” refers to light coming from above

Objectives:

1. Describe what sorts of materials respond to fluorescence microscopy.

2. Using a simple diagram, discuss the location and how each of the following fluorescent microscopy component operates: arc lamp, excitation filter, dichroic beam splitter, filter, and fluorochrome in the sample.

3. Adjust the “light budget” (filters, optics, stain, and illumination) to optimize the fluorescence image.

4. Discuss the influence of numerical aperture and total magnification on image intensity.

5. Evaluate light sources, especially in terms of their spectral characteristics, and their application to fluorescence.

6. Explain the differences and limitations of incident (epi) illumination vs. transmitted light fluorescence microscopy.

7. Explain the safety precautions necessary for fluorescence microscopy

8. Align a fluorescence system for optimum performance.

Procedure:

1. Locate each of the following components in the fluorescent microscope then sketch a diagram of this system:

a. Epi illuminator, its lamp and controls (field iris and aperture iris)

b. Filter cube (label excitation, dichroic, and barrier filters)

2. Set up the microscope for transmitted Koehler Illumination (suggested: 10x objective). Observe the sample in standard brightfield. In a simple sketch, note any identifying features and colors.

3. Block the transmitted light path and switch to fluorescence mode.

Question: What step did you have to take to achieve this switch?

4. Switch to a 40x objective. Record and explain any differences in intensity.

5. (Optional). If there is an automatic camera attached to this system, measure the brightness differences between the 10x image and the 40x image:

Objective	Exposure Time
10x
40x

6. If available, compare the intensity observed in objectives of equal magnification but different NA’s. Several examples are given below. The instructors will indicate which sets are available.

Magnification/NA	Relative Intensity	Cost
40x/0.65
40x/0.85

Question:

If the manufacturer’s literature is available, look up the difference in price between the

objectives you used and make a comment about the practicality of one objective over another in terms of cost and effectiveness.

7. If they are available, compare fluorescent images at the same magnification but with different objective-eyepiece combinations. (Note: A mag changer can be used to mimic different eyepiece magnifications)

Obj Mag/NA

Eyepiece Mag:

M_total*

Observations

(Relative intensity of image)

*M_total= M_obj X M_eyepiece X M_intermediate

Question: From these and your earlier observations, what is the best choice in optics for a fluorescence system?

8. (Optional) If they are available, compare the efficiencies of (a) a wide band, (b) semi-narrow, and (c) narrow band excitation filter. Use one diagram to sketch the curves for these three filters.

9. CAUTION: THIS EXERCISE IS TO BE DONE ONLY WITH AN INSTRUCTOR PRESENT!

b. Examine the internal construction of the lamp for the Fluorescence microscope. What safety precautions are designed into the lamp housing and why?

a. Go through lamp alignment with the instructor. List the steps you followed:

Summary Questions:

1. Case 1: your specimen is tagged with FITC. There is no autofluorescence in the sample but the emission from the FITC is extremely poor and the fluorescence fades rapidly. Which components could you use to optimize the system? Justify your choices

a. Light source:

a. Filter set:

b. Objective:

c. Eyepieces:

d. Transmitted light or incident fluorescence?

e. Other accessories?

2. Case 2: You need the very best intensity from your fluorescence image but need narrow band excitation between 480nm and 495 nm. Which light source should you use and why?

3. Case 3: You are interested in imaging your sample using its autofluorescence. What information about this fluorescence will you need in order to equip your microscope for this task?

Advanced Contrast II: Polarized Light

A lecture demonstration – kits will be provided by the instructor. You will also need a Sharpie marker. Also, strip the coatings off both sides of all the BIG filters in your kit. For the smaller ones, strip the coatings just half way down on both sides. Note: these kits are yours to keep, with our compliments.

Since this particular topic requires a bit more science before we apply it to microscopy, we’ll do a series of exercises at your desk to demonstrate the basic principles then show you how it works in your microscope.

4. Ordinary v. Polarized Light

Figure 27. Ordinary v. Polarized Lights

a. Ordinary light contains waves that are vibrating in all directions. Note that all these vectors are perpendicular to the direction of travel.

b. Polarized light contains only one permitted direction of vibrations

c. Ordinary light can be converted to polarized light by a number of light-matter interactions.

5. Creating polarized light

a. Polarization by reflection

You’ll need a clean glass slide from your kit, one of the gray polarizing filters, and a Sharpie. Place the slide flat on the desk in front of you, in a horizontal position. If you view the slide at about 45 degrees, you should be able to catch an image of the overhead lights.

1) Light is polarized on reflection. Can you determine the direction of vibration carried by the reflection? If not, why not?

Hold one of the gray polarizers in front of your eye, as though it were part of a pair of sunglasses. While looking through it at the reflection, rotate the polarizer. You will notice that in some positions, the reflection is somewhat brighter and in others, it is somewhat dimmer.

2) Explain your observations in terms of permitted directions of vibration.

3) Based on your explanation, you should be able to use a double-headed arrow to mark the permitted direction of vibration emerging from the reflection on your glass slide. ( )

b. Polarization by absorption

1) Also, based on your observations, you should be able to use a double-headed arrow to mark the permitted direction of vibration on your polarizing filter.

2) Double-check your results against the Instructor’s polarizer.

c. Parallel and crossed polars

1) Stack your two gray polarizing filters and look through them at a source of illumination.

2) Hold one stationary and rotate the other. Notice that the resulting intensity changes. Using a simple diagram, explain your observation in terms of permitted direction of vibration.

3) When you align the two directions of vibration, you are setting parallel polars. When you rotate one polar so that the directions of vibration are at right angles, you have crossed polars.

4) Mark your second polar with its permitted direction of vibration. Check it against the instructor’s polarizers.

6. Different Optical Paths (Overhead demo: microwell plate)

a. Review of isotropic v. anisotropic materials

1) Isotropic materials exhibit the same electrical field in all directions, therefore exhibits the same refractive index in all directions.

2) Anisotropic materials exhibit different electrical fields with direction, therefore exhibit different refractive indices at specific directions, much as wood exhibits a “grain”.


a. Isotropic	b. Anisotropic

Figure 28. Difference in electrical fields between isotropic and anisotropic materials

3) Because anisotropic materials exhibit at least 2 different, defining refractive indices (n1 and n2), we say they are bi – refr – in – gent

That is:

gent = having the property of

bi = 2

refr in = refractive indices

b. When you put polarizing filters into your microscope, you automatically define an electric vector, E, as the light passes through the first polarizer.

c. When that vector encounters a material which is anisotropic, it is split into two components, e and o, which vibrate at right angles to each other.

1) Each vector encounters a different electrical field as it travels through the material. The result: each will have its own optical path, OP = t x n.

2) The difference in refractive indices, |n₁ – n₂|, is called birefringence, and is a optical property of the material.

3) The ray encountering the higher refractive index will slow down compared to the other and emerge later. Logically, we call that component the slow ray and its partner, the fast ray.

4) This difference in velocity will cause one ray to lag behind the other. The lag is called retardation, G, and depends on three factors:

c) t, the thickness of the material

d) D, the birefringence

e) the orientation or position of the material between crossed polarizers.

Figure 29. Retardation

5) Retardation, G (gamma), is the mathematical difference in the optical paths:

G = OP1 – OP2

Since both rays travel through the same thickness but are experiencing refractive indices oriented at right angles to each other, this equation can be written as:

G = t D = t |n₁ – n₂|

Equation 11. Retardation

7. Retardation and Pol Colors - An interference effect

B G R

450 550 650 900 1100 1350 1650 1800

*Legend* :		450 nm blue
		550 nm green
		650nm red

Figure 30. Evolution of Pol colors

(Original diagram, courtesy of Jan Hinsch)

a. Conditions: (Fresnel-Arago’s Third Law)

1) Incoming light must be plane polarized ----> a coherent beam

2) Split into e and o within the birefringent material. These two beams are vibrating at right angles to each other, both when they are in the material and when they emerge.

3) When the light passes through the second polarizer (Analyzer), the beams are brought back into the same plane and now meet criteria for interference. However, the permitted direction of vibration is 90 degrees to that of the polarizer so destructive interference occurs at full l intervals rather than at l/2.

b. What does the analyzer (the second polarizer) “see”

Destructive interference will remove one sector of the Color Wheel, leaving the two other primary colors à their secondary resultant.

Minus Green = MaGenta

8. The Michel-Levy Chart

a. The retardation axis

b. The birefringence axis

c. The thickness axis

9. Testing retardation

a. Cross your polarizers

b. Insert one of the two clear plastic strips between the two polarizers, on a diagonal (Remember, orientation is a key component of retardation. This position is called the “position of brightness” and produces maximum polarized light effect).

c. Compare the color you see to the colors on the Michel-Levy chart and estimate the retardation: _________________nm

d. Repeat the experiment with the other plastic strip. Again, estimate the retardation: ___________________nm

10. Larger and smaller Optical Path Differences (addition and subtraction)

a. Cross your polarizers

b. Stack your two clear plastic strips, one on top of the other, and insert on the diagonal.

1) Estimate the retardation: _________________nm

2) Using the larger retardation you measured above, determine if this new value moved up the Michel-Levy chart (additional retardation) or down the chart (a position of subtraction)

c. Addition occurs when the faster ray from one strip is aligned with the faster ray from the second. The result is the addition of the retardation from Strip 1 + Strip 2

d. Subtraction occurs when the slower ray from one strip is aligned with the faster ray from the second, canceling part of the original retardation.

11. Repeat the experiment, but this time, cross the two clear plastic strips.

a. Estimated retardation: ______

b. Addition or subtraction: _____

12. Conclusion?

Tools:

Old fashioned cellophane tapes

Overhead transparencies

Set of polarizers.

Protocol:

Have the kids lay down layers and layers of tape on the overhead. Typically, the tape has about 80-100nm retardation, so as they layer it in different orientations and thicknesses, it will produce a huge variety of colors from the Michel-Levy chart. After they are all done, have them hold their masterpiece up to the light, between crossed polars.

As they get older and more experienced, they can determine how many layers it will take to make white for clouds, blue for sky, red for houses, turquoise for water and can build “magic” pictures by layering then carefully cutting and placing stacks of tape.

13. Polarized light microscopes typically come with a set of plates of known retardation:

a. 550 nm = First Order Red Plate, Full wave plate, or “sensitive tint” plate

b. 146nm = Quarter order plate

14. Retardation plates can also be made of variable retardations. When inserted into a special slot in the microscope, in a position of subtraction, they can be used subtract out ALL of the retardation from the sample. This phenomenon is called compensation and the test plates used for this purpose are called compensators.

Advanced Contrast III: DIC (aka Nomarski)

1. Detects: Gradients

2. Mechanism: Shear (differentially) & Shift

a. Beam splitters shear the incoming wave front by an amount smaller than R_objective

b. Viewing the sample between crossed polars brings out optical path differences (retardations)

c. For better discrimination: Use a compensator to shift one wave front with respect to the other (adds optical path)

Figure 31. Mechanism for DIC

1) Typically, the compensator is set so that Gradient 1 is 146nm, soft dove gray.

2) Since Gradient 2 has a smaller OPD, it exhibits a Pol color closer to Zero Order Black.

3) Since Gradient 3 has a larger OPD, it exhibits a higher Pol color, typically on the order of 1^st Order White.

3. What does the image look like?

If the compensator is tuned as described in the Figure above, the bright/dark cues prompt our eye-brain combination to interpret the scene as having a high degree of three-dimensionality.

4. How do you do it?

Key components - The “Foster DIC Sandwich”

1) Crossed polars (P₁ and P₂)(”bread”)

2) 2 Prisms[8] (“butter” & “mayo”):

BS₁ = beam splitter;

BS₂= beam recombiner

3) Sample (“meat”)

4) Compensator (“lettuce”)

Figure 32. DIC components

5. Set-up: (This approach is unique to Ms. Foster and assures that you will not fall into the trap of interpreting polarized light responses as DIC).

a. Before you begin, make absolutely sure that both polarizers and all beam splitters are out of the optical path.

b. Establish Koehler using the Brightfield setting on the condenser.

c. Insert the polarizer and analyzer. Cross and observe the sample for any polarized light response. If a rotating stage is available, confirm your observations on rotation.

d. Insert the two beam splitters.

e. Insert the compensator. Tune the background to ~146nm soft dove gray. Adjust for optimum contrast.

6. Resolution v. Contrast? DIC is the most elegant of all techniques, providing superb resolution with magnificent contrast

7. Can you do it yourself? No.

8. Comments

a. Because there is a specific direction of shear, this technique is highly directional. To take advantage of DIC, the gradients should sit at right angles to the direction of shear. As with HMC and Oblique illumination, this facility can be used to increase or suppress contrast.

b. While DIC produces beautifully 3-dimensional images, it does not tell you “which way is up”. There are two solutions:

1) Use the shading cues from an internal reference (either a structure you know or one you have planted in the specimen) or

2) Find a structure that can act as a reference. Focus first on the flat “plain” of the sample and focus away. If it is a mountain, the peak will come into focus after the plain. If it is a valley, the “peak” will go further out of focus.

c. DIC is well known for its ability to optically section. That it, it produces images with very shallow depth of field, allowing you to investigate even closely spaced layers in your sample. To maximize, open the condenser aperture fully.

d. Because this technique depends on polarized light, it will not work properly with birefringent materials. Despite your best efforts to insert the proper bits and pieces in the microscope, the sample will produce polarization effects in lieu of the expected DIC response. As with all things in the microscope, the Sample is the Boss.

e. Depending on the type of compensator available in your microscope, you may be able to tune the background to higher Pol colors. Experimentation is usually rewarding: the images are artistically beautiful and make good candidates for the cover of the annual report. Closing the condenser aperture slightly may also improve the color saturation. Note: scientifically, dove gray is the optimum setting.

Lab 9. Investigating DIC

Specimen: Use the same cheek cell prep you used for the other contrast techniques

Microscope Set-up:

Koehler Illumination, 40x objective;

Microscope fitted with DIC

Objectives:

1. Set up DIC, starting from Koehler Illumination

2. Tune DIC for optimum results

3. Interpret the DIC image relative to peaks and valleys

4. Trouble shoot the DIC image, especially in terms of potential interference from anisotropic materials.

Procedure:

1. Setting up and fine-tuning DIC

a. Before you begin, make absolutely sure that both polarizers and all beam splitters are out of the optical path.

b. Establish Koehler using the Brightfield setting on the condenser.

c. Insert the polarizer and analyzer. Cross and observe the sample for any polarized light response. If a rotating stage is available, confirm your observations on rotation.

d. Insert the two beam splitters.

e. Insert the compensator. Tune the background to ~146nm soft dove gray. Adjust for optimum contrast.

2. Investigating the range of the compensator

a. Adjust the compensator to its lowest then highest settings. Record your observations below.

b. Using the compensator, set the background to the color shown in the left column. Record the resulting image changes in the right two columns.

Background tuned to:	Color, left edge	Color, right edge	3d impression?
Zero order black
1^st order white
1^st order dove gray
1^st order red

Can your compensator go beyond Zero order black? If so, tune the background to the “other” First order gray and comment on the affect on 3-D, up and down.

3. Identifying Up and Down

a. Locate a known feature. Observe the shadowing.

Is this feature a peak or a valley? ___________________________

Which side is shadowed? _________________________________

b. Locate an unknown feature. Using both the shadowing method and the fine focus method, determine if it is a peak or a valley. Record your observations.

4. Looking for “false positives” (optional)

a. Use a sample which has a mixture of isotropic and anisotropic materials.

b. Stop when you reach the crossed-polar step.

1) Observe which samples are bright and which are dark. Make a quick diagram of part of the field of view to show this phenomenon.

2) Using the Michel Levy chart, estimate the retardation of 2 or three of the bright objects.

c. Insert the beam splitters and tune the compensator to the optimum setting.

1) What has happened to the polarization color of the bright objects you described above? (Reminder: you can determine the retardation introduced by the compensator by checking the background color against the Michel-Levy chart)

2) What is the difference in 3D appearance between the objects that appeared dark and those that were bright between crossed polars? Briefly, explain this observation.

[2] Two quick exercises to find your dominant eye:

a. Make a triangular window by putting your two index fingers together and your thumbs together. Hold the window at arm’s length and center a distant object in the window, making sure that you look with both eyes.

Close your right eye. If the object moves significantly, you are right eye dominant. Repeat

the test by closing your left eye. If you were right eye dominant, the object should stay fairly

centered.

b. Make an “O” with your fingers and your thumb. Locate a distant object by looking through the resulting tube with both eyes. Slowly move the tube toward your face, always looking at the distant object with both eyes. You will find that the tube will automatically migrate toward your dominant eye. (This technique was provided by a past student who was a State of Pennsylvania champion sharpshooter).

[3] In a ray diagram you can use two construction lines to locate the image:

Draw Line 1from the point of interest (tip of the arrow), directly at the lens then through the BFP.

Draw Line 2 from the point of interest through the center of the lens.

An image will form wherever the two lines intersect.

[4] This situation is difficult to draw. As the object moves further and further away, the image becomes smaller and moves toward the BFP. When the object is truly at infinity, the image is reduced to a point. If the object is on the optic axis, that point will fall on the optic axis. If the object is off the optic axis, the point will be imaged across the optic axis, still at the BFP.

[5] Note: only very specific materials respond to phase because their optical path (product of their “nxt” ) produces the required l/4 retardation

[6] A word about lamp alignment. We may have a chance to see an arc lamp housing in lab and learn how to align it. Typically these lamps have both a real image and a mirror image which need to be positioned. CAUTION!!! (1) Always wear protective goggles when installing a new arc (2) NEVER look directly at the arc! and (3) never touch an arc source with your bare fingers; always wear gloves.

[7] For comparison of spectra, see Optimizing Light Microscopy for Biological and Clinical Laboratories, P. 76.

[8] Most companies use Wollaston prisms, referring to the cut used to create them… an invention of Dr. Georges Nomarski. Leica uses a different engineering, typically the Smith approach.

Today’s Schedule ...	Welcome! To begin the afternoon, we’ll take a few moments to get to acquainted, then begin the program. There will be 15-minute breaks mid-session for each block longer than 2 hours.

Ask us questions ...	Microscopy is full of new ideas and vocabulary. This is the place to ask questions and practice those new ideas. Our speaker will be available during breaks to answer your questions and help you solve those sticky application problems.

Put it in your own words...	This workbook is yours. We actively encourage you to take notes in your own words, draw sketches, and do whatever is necessary to convert our words into yours.

A pat on the back...	Our thanks to Minnesota State for inviting us here and supporting this teaching effort with staff and equipment. When you get back to your own lab, show your appreciation: share your knowledge. Most importantly, congratulate yourself for taking time to sharpen your microscopy skills.

Optimizing Microscopy and Fluorescence & Ratio Imaging

Copyright 2004 Microscopy/Microscopy Education, Inc. Springfield, Massachusetts

a. The object is the specimen.

b. The image is the representation of the specimen. It has its own physical reality and properties. Our job as microscopists is to reproduce the object as faithfully as possible in the image.

c. Magnification is a relationship of either:

1) the size of the image to the size of the object or

2) the distance from the image to the lens (Di) divided by the distance from the object to the lens (Do)

a. Objective – the “mastermind” for the microscope

1) establishes the first step of magnification (see largest number on the engravings)

2) sets the resolution for the whole system (see the “/ --“ on the engravings)

3) may contribute to contrast (see the special nomenclature on the engravings)

b. Condenser – the “C” exercise

1) Controls the angle at which the light approaches the specimen and, therefore the

a) Coherence determines the quality of the edge information.

b) Physically, the angle is controlled by the aperture iris (demonstrate)

2) The second component of resolution

3) May contribute to contrast techniques

c. Eyepieces

1) Responsible for the second step of magnification (see the large number engraved on the objective)

2) Responsible for establishing the field of view (the diameter of the territory you are actually observing

a) feel the edge of the small baffle about ½” up the inside wall of the underside of the eyepiece

b) look for the field number engraved on the barrel (diameter across the opening, expressed in mm.

3) May be the site of a reticle (a target used for photography or measurement)

a. Condenser aperture iris or just aperture iris (AI)

1) Controls the angle of light approaching the sample

2) Responsible for coherence control

b. Field iris (FI)

1) Controls the size of the illuminated disk on the specimen and therefore the territory you will observe

2) Also controls glare and haze

1. Start by setting the focus on the eyepieces to the “zero” point and the binoculars so that you are viewing comfortably.

2. The Objective

3. The “Condenser”

a. Close the field iris so that it is visible within the field of view

b. Using the condenser focus control, focus the image of the field iris.

c. Using the condenser centration controls, center the image of the field iris.

d. Open the field iris so that it is just outside the field of view (glare control).

e. Adjust the condenser/ aperture iris for the “Oomph” position (crispest image and optimum contrast)

4. The Eyepieces (oculars)

a. Adjust the interpupillary distance so that you see one, single, round field of view

5. Your dominant eye is the “lead horse” in your two-eye team. If you play a sport or shoot a gun, it is the targeting eye[2].

· Close your dominant eye and, using just your non-dominant eye, adjust the focus on its eyepiece until the microscope image is sharp for that eye.

· While looking through just the fixed eyepiece, use the fine focus on the microscope to make sure that the microscope is really in focus for that eye.

· Close that dominant eye and, using the adjustable eyepiece, focus it until the microscope image is sharp for that eye.

a. Light is Electromagnetic radiation: it has both electrical and magnetic characteristics.

b. A light wave can be described as the path defined by the movement of an electrical vector, building up first in the positive direction then decreasing; building up in the negative direction then decreasing.

c. Direction of travel – the direction in which the light wave is moving away from its source. You see me because light is traveling from me to you.

d. Direction of vibration – the direction in which the light wave is oscillating. The direction of vibration is always at right angles to the direction of travel but it could be at any or even all angles of rotation (North-South, East-West, or any angle in between).

e. Amplitude – the maximum displacement or “height” of the wave above or below the reference bar. The intensity of the light is proportional to a2

f. Wavelength – the distance it takes for a wave to go through one cycle (ex: peak to peak, trough to trough). The wavelength describes color and energy

g. Memory clue: ROY G BIV

2. Light-Light Interactions – Interference

a. Constructive Interference (nl)

b. Destructive Interference (n/2 l)

c. The Color Wheel

1) Primary colors for LIGHT:

2) Secondary colors for LIGHT:

3) R+B+G = white

a. A measure of matter’s electrical character

b. Mathematically, the ratio of the velocity of light in air versus the velocity of light in a material

a. A focal point is a specific point at which light converges to form an image

b. A focal plane is a two-dimensional array of focal points

c. FFP = Front Focal Plane, the plane on the incoming side of the lens

d. BFP = Back Focal Plane, the plane on the emerging side of the lens

e. do = distance from lens to original object

f. di = distance from lens to image

g. (Reminder: OBJECT and IMAGE are two different entities!!!)

2. Four cases of lenses[3]

a. Very important for trouble shooting

b. Demonstrate location of these planes in the microscope, starting from the light source

1. Finding the focal planes in the illumination system

a. Close the aperture iris and use it as a screen. If possible, remove the diffuser from the light path. Looking outside the microscope, use a dental mirror to see if the image the coiled filament of the lamp appears on the closed leaves of that iris.

b. Remove the eyepiece and peer down into the BFPo (deep down in the tube).

1) Do you see the lighted disk/filament or the sample?

2) Open and close the aperture iris. Describe what you see in the BFPo.

3) Open and close the field iris. Describe what you see in the BFPo.

c. Using a piece of lens tissue as a screen, find the Ramsden disk or exit pupil of the microscope.

1) How far is this plane located above the top of the eyepiece? How does this relate to the engravings on the eyepiece?

2) Do you see the lighted disk/filament or the sample?

5. Your dominant eye is the “lead horse” in your two-eye team. If you play a sport or shoot a gun, it is the targeting eye [2].

e. Amplitude – the maximum displacement or “height” of the wave above or below the reference bar. The intensity of the light is proportional to a²