Types of Microscopes + Cells or Debris?

CH2 Section2. Answer w/definition

Is this a cell or debris

cell

For (D) aka Limit of Resolution, you want a ____ numerator (lambda) and ____ denominator (obj lens + condesor)

small, large

Ways to Improve Limit of Resolution

1. Increase magnification

2. Shorten working distance (focal length)

3. Adding oil

If D is less than distance…..

2 cells seen as seperate

If D is more than distance…

2 cells blur into one

Is this a cell or debris

debris

Is this a cell or debris

cell

Is this a cell or debris

cell

Is this a cell or debris

debris

Is this a cell or debris

debris

Is this a cell or debris

debris

Is this a cell or debris

cell

Coccus/cocci

circular

bacillus/bacilli

rod

ovoid coccus

oval

vibrio/vibrios

curved rod

sprillium/spirilla

spiral

spirochete/spirochetes

flexible spiral

Strepto

chains

Staphylo

clusters

Diplo

2 cells connected together

Tetrad

4 cells connected together

Sarcina

8 cells in a cube

Single

no obv arrangement. Dependent on shape

If all the possible cells seem to be very small then its likely….

bacteria

If all the possible cells seem to be take up a large amount of the field of view then its likely….

debris

Bright-field Microscope

produces image w/bright background & dark or pigmented cells. Gives 2D image & stains the cell or background

Dark-field Microscope

Produces dark background with illuminated specimen. Gives 2D image

Phase-contrast Microscope

produces image in shades of grey, has the cells have a ring of light surrounding them. Gives 2D image

Differential Interference Contrast (DIC) Microscope

Produces specimen looking as shade of grey & its natural color. Gives 3D image

• uses 2 beams of light→travel through speciment @diff times, then rejoin in obj lens

• Provides interior view of eukaryotic cells

Fluroescent Microscope

Produces image appearing brightly colored w/black background. Has ambiguous imaging

• relies on adding fluorescent dye to chemically bond to certain molecules.

• Illuminated via UV + requires computer

• GFP

Scanning Electron Microscope (SEM)

Produces detailed image that gets darker the position of image the longer it takes electron to return. Gives 3D image & interior of cell isn’t seen

• Electron beam hits specimen →computer interprets & displays

Transmission electron Microscope (TEM)

involves cutting specimen into very thin slices, then directing electron at slice. Gives 2D image & interior of cell is seen

What are the 2 types of electron microscopes (uses electrons to create very detailed image thats interpreted by computer)?

Scanning electron microscope and Transmission electron microscope

Limit of Resolution

For a hypothetical microscope, you have some options listed below:

1. Objective lens of 40X with a NA of 0.85

2. Objective lens of 100X with a NA of 1.25

3. Condenser lens with an NA of 1.25

4. Wavelength of light around 350nm

5. Wavelength of light around 550nm

Q1: Using this information, which components would you include in your microscope to have the best resolution? Include calculations to support your choice.     

350nm/1.25 + 1.25 = 140nm

Limit of Resolution Formula

D = lambda / condensor + objective lens (want numerator smaller than denominator)

Limit of Resolution

For a hypothetical microscope, you have some options listed below:

1. Objective lens of 40X with a NA of 0.85

2. Objective lens of 100X with a NA of 1.25

3. Condenser lens with an NA of 1.25

4. Wavelength of light around 350nm

5. Wavelength of light around 550nm

Q2: If there were two cells that are 245nm apart, using your microscope, would these cells be seen as separate or will they blur into one? Explain your answer choice.

These cells would be seen as 2 seperate since distance is smaller than D, giving seperation. Any larger would blur into one

• D = 140nm

To improve numerical aperature you’d have to

shorten focal length

When naming cell morphology + arrangement, what goes first?

(a) arrangement + shape

(b) shape + arrangement

a

Staphylo cocci; brightfield

hint: thinking of layering (3D or 2D)

Single cocci; SEM

Diplo bacilli; Phase Contrast

Diplo bacilli; TEM

single spirillium; darkfield

Hint: they’re smaller cells

single vibrio; DIC

Limit of Resolution

You have two microscopes available to use:

1. Has a wavelength of 450nm, a condenser lens of 1.25 and two objective lenses: 40x/0.25 or 60x/0.65

2. Has two wavelength options either 655 or 555nm, a condenser of 1.25 and a 100x/1.25 objective lens 

Q1: Using this information, which microscope and set up would have the best resolution? Include calculations to support your choice.

555nm/1.25 + 1.25 = 222nm. Microscope B

Limit of Resolution

You have two microscopes available to use:

1. Has a wavelength of 450nm, a condenser lens of 1.25 and two objective lenses: 40x/0.25 or 60x/0.65

2. Has two wavelength options either 655 or 555nm, a condenser of 1.25 and a 100x/1.25 objective lens 

Q2: If there were two cells that are 375nm apart, using the best set up microscope, would these cells be seen as separate or will they blur into one? Explain your answer choice.

Based off of the Resolution (222nm), the two cells will seen as separate because the distance from the 2 cells (375nm), meets the at least 222nm (aka the limit/minimum)