Biol 240 Unit 1

3 Major Functions of a Microscope

1. Magnification

2. Resolution

3. Contrast

Magnification

• How much larger the image appears compared with the actual object

• Example: 400x magnification = image appears 400 times larger

• Increasing magnification doesn’t enhance detail

Resolution

• What is it

• Formula/Determined by

• More detail and sharper structures/Ability do discern fine details

• Resolution - wavelength/2

• Ability to distinguish two very close points as separate objects

• Determined by the wavelength of light (or energy) used for illumination. For light microscopy, the limit is ~200nm.

Contrast

• What is it?

• How is produced?

• Telling structures apart. Difference in intensity between the image and the background.

• Produced in the specimen by staining with colored dyes that absorb light by using special optical techniques, or by using fluorescent probes.

• Difference in appearance (brightness/color) between the object and its background or between different structures

• Cells are often naturally transparent, so microscopy techniques/stains are used to increase contrast

• Doesn’t necessarily increase resolution

A researcher observes two proteins located extremely close together on a cell membrane. Under the microscope, the region is clearly visible against the surrounding membrane, but the two proteins appear as one single spot rather than two separate spots.
Which property of the microscope most directly limits the researcher’s ability to distinguish the proteins?

Resolution

A student examines an unstained living cell. The nucleus and cytoplasm are within the microscope’s ability to distinguish spatially, but they have nearly identical brightness and therefor are difficult to tell apart.
Which would most directly improve the image?

a. increasing magnification
b. improving contrast
c. improving resolution
d. using a larger image display

Improving contrast

A microscope image is enlarged from 400x to 100x. The image becomes much larger, but previously blurry structures remain blurry.
Which statements could correctly describe what happened?

1. Magnficiation increased

2. Resolution necessarily increased

3. The microscope may heave reached its resolution limit

4. Making an image larger does not necessarily reveal additional detail

5. Contrast must have decreased

1. Magnification increased

2. Making an image larger does not necessarily reveal additional detail

Two microscopes examine the same pair of organelles, which are 180 nm apart.

• Microscope X produces a bright, easily visible image, but the organelles appear as one object

• Microscope Y produces a dimmer image, but the two organelles can clearly be distinguished as separate objects

Which microscope has better resolution, and what observation proves it?

Y, because it distinguishes two closely spaced objects

A student says:
“I can’t distinguish the two organelles in this image, so the microscope has poor resolution.”
The professor says this conclusion is not necessarily justified. Which situation would best support the professor?

1. The organelles are 50nm apart and appear as one continuous object.

2. The organelles are sufficiently far apart to be resolved, but their signals are almost identical to the surrounding cytoplasm.

3. Increasing magnification makes the merged organelles appear as a larger merged structure.

4. The microscope’s resolution limit is 200nm, while the organelles are 100n apart.

2. The organelles are sufficiently far apart to be resolved, but their signals are almost identical to the surrounding cytoplasm.

A researcher is examining two proteins in a membrane. In the original imgae, she sees one faint patch. After changing the microscopy conditions, she sees one very bright, sharply visible patch. She concludes “The new method improved out ability to resolve the two proteins.”

Is her conclusion justified?

1. Yes, because the object became easier to see

2. Yes, because greater contrast necessarily produces greater resolution

3. No, the increased visibility demonstrated improved contrast, but there is no evidence that the two proteins can now be spatially distinguished.

4. No, the result demonstrated increased magnification instead.

1. No, the increased visibility demonstrated improved contrast, but there is no evidence that the two proteins can now be spatially distinguished.
   
   You would need evidence of two separate, distinct structures.

How Scientists Prepare Tissues to be Viewed Under Microscopes

3 Steps:

• Process

  • Function

• Fixation - Preserving specimen

  • Cells/tissues normally start breaking down once they’re removed from an organism

  • Fixation preserves the cellular structures and essentially locks them in place

  • Freezing the cell’s structures in their current arrangement / dead tissue / preserving structure, not keeping cells alive

• Embedding - Giving tissue support

  • Tissues get surrounded by something that becomes slid, like paraffin or plastic/resin, to avoid it deforming or falling apart

  • Soft tissue embedded inside a solid block that allows you to cut extremely thin slices without destroying the organization of the tissue

• Sectioning - Cutting it into thin slices

  • Machine, microtome, takes embedded tissue block and uses a knife to produce extremely thin sections that can be placed onto a microscope slice

  • A thin section allows us to observe all individual cells-structures inside of it, unlike a thick chunk.

  • Many microscopy methods need a sufficiently thin slice for illumination or an electron beam to pass through and produce a useful image.

• Fix → Embed → Section → View

• Preserve → Support → Slice → Microscope

Histological Stains

Why?

Most cell structures don’t naturally look dramatically different form one another, so scientist use stains that selectively bind to particular cellular molecules

Tissue gets treated with different staining to label other cellular/tissue components

Why/How do different things stain differently?

Different cellular structures contain different molecules with different chemical properties

A stain has particular chemical properties that cause it to interact preferentially with certain molecules

Different molecular composition → Different affinity for stains → Different colors → Increased contrast

Adds boundaries, improved contrast

Problem with Staining

• Traditional staining usually requires killing the cells

• What if you want to watch a cell while its alive?

Differences in Refractive Index

• Allows you to generate contrast without killing a cell

• Different parts of a cell have different refractive indices

  • How much a material slows/bends as light passes through it

  • ie. Cytoplasm, nucleus, organelles, surrounding fluid have slightly different compositions and thus affect light differently

Specialized Microscopes:

1. Phase-contrast microscopy

2. Differential Interference Contrast (DIC)

Phase-Contrast microscopy

• What

• How

• Pros

• Cons

Phase contrast uses differences in how light travels through cellular structures to create differences in brightness

Invisible difference in light → visible light/dark difference

Pros: Food for transparent cells, lets you watch cells change/move over time

Cons: Can produce halos around structures; less useful for thick specimens, doesn’t tell you what a structure is

Differential interference Contrast (DIC)

• What

• How

• Pros

• Cons

DIC Also exploits differences in how light travels through different parts of the cell, but uses interference between light paths to generate contrast

Creates 3D/Shadowed appearance

Pros: Very sharp boundaries and excellent contrast, useful for fine Cuellar details, 3d appearance

Cons: More complicated/expensive optics, 3D appearance is artificial and may be misleading, doesn’t specifically identify molecules

Distinction of choosing between PC and DIC

What kind of image you want

• General observation of living cells

• Very clear cell boundaries/edges

• Track cells moving/dividing over time

• Fine structural/morphological differences

• Cheapest/simplest setup

• 3D like view of cell shape

What kind of image you want

• General observation of living cells
  Phase contrast

• Very clear cell boundaries/edges
  DIC

• Track cells moving/dividing over time
  Phase contrast

• Fine structural/morphological differences
  DIC

• Cheapest/simplest setup
  Phase contrast

• 3D like view of cell shape
  DIC

What is the purpose of Florescence Microscopy?

It lets you locate specific molecules of structures inside cells by attaching florescent labels to them and then detecting the light those labels emit

What is the core mechanism in Flurescence?

Flurophores

What are Fluorophores?

• How do they work

• Explain Excitation/Emission

• Relevance to wavelengths

A molecule that can absorb light energy and release some of the energy as light

ie: Specific wavelength of light comes in → Fluorophore absorbs it → Fluorophore becomes excited → Some energy is lost —> Fluorophore emits lower energy light

Excitation = Light IN
Emission = Light OUT

Higher energy = shorter wavelength
Lower energy = longer wavelength

Why does the microscope need different wavelengths?

• Brief on how it works

• Contextualize it with its background

• Takeaway point

To separate what dye is absorbed/reflected

Graph:

• Blue curve represents wavelengths that excite the particular dye, green curve represents the light that the dye admits

• Shining blue light at specimen → fluorophore absorbs blue → fluorophore emits green → Microscope detects green

• Microscope ignores the light used to excite the sample, instead showing the light coming back from the fluorescent molecules

• This is also why florescent structures are able to appear brilliantly colored against a dark background

  • Microscope doesn’t illuminate with light

  • We shine the appropriate excitation wavelength onto specimen

  • Only fluorophores capable of absorbing that wavelength become excited and emit their own

• TAKEAWAY: Fluorophore absorbs energy and emits a new photon at a different wavelength

Fluorophores/Colors

How are different colors of fluorescence produced?

• The chemical properties of a dye determines its excitation and emission

Different fluorophores have different molecular structures, so they absorb and emit different wavelengths

• Label different things with different fluorophores

• Color = which florescent label is associated with what cellular target

Molecular Specificity: Colors

• Why are colors helpful?

• Besides the obvious: Being able to visualize structures

• Them being labeled differently in the same cell lets us see their spatial relationships

• Does not tell us about whether they specifically interact, only can tell us things like co-localization

Florescence microscopy with Living vs. Fixed Cells

• Living cells: Relevance?

• Fixed cells: Advantage

Living cells

• Fluorescence microscopy can be used on living cells with the appropriate florescent labels

• Allows us to watch biological processes change over time

• IE. Where proteins move, how mitochondria moves, how ER shapes, what happens during cell division

• Dynamic information

Fixed Cells

• You’re able to use labeling techniques that may not work as easily in living cells and lets you examine structures very clearly

• Cannot examine change, only preserved structural and molecular localization

How does the dye know where to go?

• 2 ways

• Florescent antibodies

  • Antibody recognizes protein X

  • Fluorophore attaches to antibody

  • Antibody binds to protein X

  • Shine excitation light

  • Fluorophore emits

  • You can visualize protein X location

• Genetically coded fluorescent proteins (Like Green fluorescent protein)

  • GFP can be fused to a protein they’re interested in

  • Protein + GFP, cell produces fluorescent fusion protein and allows researchers to track where protein X goes in living cells

A dye absorbs most strongly around 480 nm and emits around 650 nm.
Which has more energy?

480 nm

Shorter wavelength = Higher energy

A scientists uses two fluorophores

Protein A = Green
Protein B = Red

Some regions contain only green and others only red. What might this mean?

The proteins have different cellular distributions. Some regions may overlap which suggests co-localization, but doesn’t prove interaction.

A researcher wants to observe mitochondria moving around a cell for 30 minutes. Would a fixed-cell technique work?

No.

Light Path of a Florescence Microscope: Entire Path

• Why is it set up in a certain way

• How its set up: Orderly

  • Every mechanic

  • Every process from mechanic

  • Complete path of light

• Microscope needs to control which wavelengths travel where

  • Light Source → Excitation filter

    • Chooses light going INNNNNN

    • Selects/filters the wavelength/range of wavelengths capable of exciting our fluorophore

  • Dichroic Mirror

    • Treats different wavelengths differently

    • Reflects certain wavelengths while transmitting others to the objective lens

  • Objective

    • Magnifies and Focuses light onto the specimen

    • (Recall we filter what light does in)

    • Collects the emitted fluorescent light coming back from the specimen

    • Light travels through objective in both directions

      • Going down = Excitation light → Objective → Specimen

      • Coming back = Specimen fluorescence → Objective → Specimen

  • Fluorophore

    • Shorter wavelength excitation light absorbs

    • Fluorophore excited

    • Energy lost

    • Longer wavelength emission light released

  • Emission Filter

    • After light comes back through the Dichroic Mirror, the filter selects wavelengths that we want our detector to receive

    • Helps mitigate stray excitation light, unwanted wavelengths, background light

    • Extra specificity

    • CLEANS UP FROM THE FLUOROPHORE

  • Detector

    • Appropriate fluorescent light emissions reach the detector which measure the fluorescence and allows an image to be produced

    • Fluorophore present → Emission → Detector receive signal → Bright

    • Little/no emission = little/no signal → dark

1. Immunofluorescence consists of

2. Fluorescent protein tagging consists of

1. Immunofluorescence: Antibodies and Fluorescent marks

2. Fluorescent Protein Tagging: Genetically encoded fluorescent proteins like GFP

1. What is an antibody

• Structure

• Mechanism

• Production

• Structure

  • 2 Heavy chains + 2 Light chains

• Mechanism

  • Have high affinity to particular antigens

• Production

  • Can be produced in animals like rabbits, mice, sheep, goats, and humans

Antibody Structures: Variable Regions

• Location

• Importance

• The tips of the Y

• Determine what the antibody recognizes and binds to

Antibody Structures: Constant

• Location

• Importance

• Everything but the Tips

• Not really relevant

Antigen

Molecule/Structure recognized by an antibody

• Ex: Tubulin, Golgi protein

How Variable Regions Work

• Antibody recognizes protein

• Antibody binds protein

• Fluorescent marker becomes associated with that antibody

• Fluorescence appears when antibody is accumulated

• We infer the location of the protein

  • Antibody = Specificity ; Fluorophore = Visibility

Antibodies and Antigens: Detailed Example

• Part 1: Antibodies

• Inject protein A into a rabbit (Antigen A)

  • Rabbits immune system recognizes and generates antibodies against protein/Antigen A

• Researchers collect blood from the rabbit and separate out the serum containing antibodies

  • Serum contains tons of different antibodies, not just the one we want

  • We want to isolate the protein A antibodies

• Attach Protein A to beads

  • Pour the rabbit serum through

  • Antibodies that don’t recognize protein A don’t bind

  • Antibodies that do recognize protein A bind strongly and stay stuck in the column

• Disrupt the interaction and release the antibodies by using high salt

  • Collect a purified serum enriched for antibodies that recognize antigen

Antibodies and Antigens: Detailed Example

• Part 1: Primary/Secondary Antibodies and Immunocytochemistry/Immunofluorescence

• Primary antibody recognizes biological target

  • In our previous explained example: Rabbit antibody directed against Antigen/Protein A = Primary Antibody

  • Primary antibody binds directly to Antigen A

• Secondary Antibody recognizes the Primary antibody

  • Marker-coupled antibody that recognizes rabbit antibodies

  • Secondary fluorescence reveals antigen