Cell Biology Lecture 36: Microscopy

(Microscope Basics) What is enlarging an image called?

Magnification

(Microscope Basics) What is the ability to identify two objects as separate and distinct?

Resolution

(Microscope Basics) Are light or electron microscopes larger?

Electron Microscope

(Microscope Basics) Which type of microscope is used to see hair, blood cells, and bacteria?

Light Microscope

(Microscope Basics) Which type of microscope is used to see viruses, DNA, glucose, and atoms?

Electron Microscopes

(Microscope Basics) What is the source of illumination in light microscopes?

Ambient light, bulb, photons

(Microscope Basics) What is the source of illumination in electron microscopes?

Electrons

(Microscope Basics) What is the focus of the lens in microscopes?

To focus light/electrons

(Microscope Basics) What is the lens type in light microscopes?

Glass

(Microscope Basics) What is the lens type in electron microscopes?

Electromagnetic

(Microscope Basics) What is the magnification method in light microscopes?

Moving lenses

(Microscope Basics) What is the magnification method in electron microscopes?

Focal length changed by current through lens coil

(Microscope Basics) What is used for viewing in light microscopes?

Eyepiece and digital

(Microscope Basics) What is used for viewing in electron microscopes?

Fluorescent screen and digital

(Microscope Basics) Do light microscopes have a vacuum?

No

(Microscope Basics) Do electron microscopes have a vacuum?

Yes (entire electron path)

(Microscope Basics) Do light microscopes or electron microscopes use more lenses?

Electron Microscopes

(Microscope Basics) Does light in light microscopes travel as waves or particles?

Waves

(Microscope Basics) Does electrons in electron microscopes travel as waves or particles?

Both

(Microscope Basics) What allows the electrons to be deflected by electromagnetic fields?

Negative charge

(Microscope Basics) Do electrons or photons have higher energy?

Electrons

(Microscope Basics) What affects the limit of resolution?

Wavelength

(Microscope Basics) What is the wavelength range of light microscopes?

400 to 800 nm

(Microscope Basics) What is the wavelength of electron microscopes?

0.0037 nm

(Microscope Basics) What allows electron microscopes to image smaller things?

Shorter wavelength

(Electron Microscopes) What is used to magnify and resolve specimens in electron microscopes?

Electron beams focused by electromagnets

(Electron Microscopes) What are the two types of electron microscopes?

1. Transmission Electron Microscope (TEM)

2. Scanning Electron Microscope (SEM)

(Electron Microscopes) Which microscope generates cross sections of objects through the object?

TEM

(Electron Microscopes) Which microscope displays a 3D surface map?

SEM

(Electron Microscopes) What advantages do electron microscopes have over light microscopes?

1. Higher range of magnification

2. Higher resolution

(Electron Microscopes) Do SEMs show color without pseudocoloration?

No

(Electron Microscopes) Which electron microscope forms an image by the detector collecting scattered electrons from surface and coils scanning beams in a raster pattern?

SEM

(Electron Microscopes) Which electron microscope forms an image by transmitting electrons through a thin slice of sample?

TEM

(Electron Microscopes) Which electron microscope has a resolution of 0.5 nm?

SEM

(Electron Microscopes) Which electron microscope has a resolution of 50 pm?

TEM

(Electron Microscopes) Which electron microscope shows a 3D image?

SEM

(Electron Microscopes) Which electron microscope shows a 3D projection?

TEM

(Electron Microscopes) Which electron microscope has an accelerating voltage up to 30 kV?

SEM

(Electron Microscopes) Which electron microscope has an accelerating voltage up to 60 - 300 kV?

TEM

(Electron Microscopes) Which electron microscope has the sample positioned at the bottom?

SEM

(Electron Microscopes) Which electron microscope has the sample positioned in the middle?

TEM

(Electron Microscopes) Which electron microscope uses electromagnetic coils?

TEM

(Sample Preparation) Which microscope can image live cells?

Light Microscope (Less resolution)

(Sample Preparation) What is imaged in cells to learn the structure of cells?

Native State

(Sample Preparation) What defect is caused during preparation of samples?

Artefacts

(Sample Preparation) Are artefacts present when when the specimen is alive?

No

(Sample Preparation) What are the sample preparation techniques that kill the cell?

1. Chemical fixation with aldehyde

2. Fast freezing

(Sample Preparation) What is needed for chemical fixation with aldehyde?

1. Time for diffusion/reactions

2. Room temperature

3. Vacuum environment in microscope

(Sample Preparation) What is the advantages of fast freezing?

1. Preserves cell close to native state

2. Fast

(Sample Preparation) What is needed for fast freezing?

1. Keep sample frozen

2. Transform to room temp while preserving ultrastructure

3. Vacuum environment in microscope

(Sample Preparation) What is needed for working with frozen samples?

Special protocols and equipment

(Sample Preparation) Why must specimens be stained with electron dense stains in TEM?

Contrast of membranes

(Sample Preparation) Why must specimens be coated with heavy metal?

Prevent charging from build up of electrons on surface

(Sample Preparation) What must specimens be in vacuums for electron microscopes?

Stable

(Sample Preparation) What does fixation with aldehydes allow specimens to do?

Remain in same location as when it was alive (Crosslink proteins)

(Sample Preparation) What removes water in specimens for electron microscopes?

Dehydration with ethanol

(Sample Preparation) What must TEM samples be thin?

For electrons to pass through

(Sample Preparation) How are specimens prepared in TEM?

Embedded and cured into hard resin + ultrathin sectioning

(Sample Preparation) What are the disadvantages of chemical fixation of specimens?

1. Slow process

2. Changes structure of specimen

3. Removes proteins

4. Masks antigenic sites

(Sample Preparation) Does fast freezing change the structure of the specimen?

No

(Sample Preparation) Does fast freezing retain proteins?

Yes

(Sample Preparation) Does fast freezing mask antigenic sites?

No

(Correlative Microscopy) What does Correlating light microscopy with electron microscopy (CLEM) correlate?

1. Dynamic functional information from LM

2. High resolution structural information from EM

(Correlative Microscopy) Which markers for CLEM are particle-based?

FluoroNanoGold and Quantum Dots

(Correlative Microscopy) Which part of FluoroNanoGold easily enters cells and tissues?

Small gold label

(Correlative Microscopy) What does increasing size by gold or silver enhancement after labeling do for FluoroNanoGold?

Better detection

(Correlative Microscopy) Which CLEM marker is an inorganic semiconductor nanocrystal?

Quantum Dots

(Correlative Microscopy) What does quantum dots produce?

Bright stable fluorescence

(Correlative Microscopy) Can quantum dots traverse the membrane?

No

(Correlative Microscopy) What allows quantum dots to glow?

Stimilation by UV light

(Correlative Microscopy) What can be evaporated into coverslips?

Gold or carbon finder patterns

(Correlative Microscopy) Which finder pattern can be used as cell support and followed by plunge or high pressure freezing?

Gold finder grids

(Correlative Microscopy) Which low molecular weight fluorescent dyes allow the gross feature to be visible under LM through the blockface?

1. Acridine Orange

2. Saffranin O

(Correlative Microscopy) What are low molecular weight fluorescent dyes useful for?

Orienting complex tissue

(Correlative Microscopy) What is the most laborious part of CLEM?

Correlation

(Correlative Microscopy) What may shrink during resin embedding?

Tissue Blocks

(Correlative Microscopy) What may stretch or wrinkle during sectioning?

Ultrathin sections

(3D Imaging) Which microscope uses serial section on grids?

TEM

(3D Imaging) Which microscope uses serial blockface?

SEM

(3D Imaging) Which microscope uses focused ion beam?

SEM

(3D Imaging) Which microscope uses array tomography?

SEM