Chapter 1 Cells and Microscopy

Question 1

Transmission electron microscope

Answer 1

Source of radiation is a high voltage tungsten filament to produce beam of electrons
beam focused using electromagnets
x500000 max magnification
0.1 nm is the max resolution
no live specimen
sectional view of specimen
expensive
image can’t be viewed directly
staining required
2D image
high risk of distortion of material
time consuming and specific equipment needed

Question 2

Light microscope

Answer 2

Light is the source of radiation
beam is focused by condenser
x1500 max effective magnification
200nm max resolution
live specimen can be used
sectional and external view of specimen
cheap
image can be viewed directly
staining is not always required
2d image
low risk of distortion of material
quick preparation and low skill level

Question 3

Scanning electron microscope

Answer 3

source of radiation is high voltage tungsten filament to produce beam of electrons
beam focused by electromagnets
x100,000 max resolution
10nm is max resolution
no live specimen
external view only
expensive
image can’t be viewed directly
staining required
3d image produced
varies on risk of distortion of material
preparation can be quick

Question 4

Confocal laser scanning microscopy

Answer 4

source of radiation is high intensity light
beam focused by objective lens
x2000 max magnification
max resolution is 200nm
live specimen can be used
sectional and external view of specimen observed
expensive
image can’t be viewed directly
staining required
2d image
risk of distortion of material
complex preparation

Question 5

Fluorescence

Answer 5

emitting light by atoms so energy is released

Question 6

How does a CLSM work

Answer 6

A CLSM moves a single spot of focussed light across a specimen (point illumination)
laser passes through an illumination pinhole onto a dichroic mirror which reflects the single wavelength of light from the laser
dichroic mirror allows other wavelengths of light from the specimen to pass through
single wavelength of light that is reflected from the mirror causes the fluorochrome marker to fluorescence
light is then emitted from the specimen passes through a confocal pinhole aperture and passes to a detector.
only light radiated from very close to the focal plane is detected.

Question 7

Describe images obtained from a CLSM

Answer 7

high resolution as very thin sections of the specimen are examined and light from elsewhere is removed
two dimensional as the illuminating spot is moved across the surface of the specimen
three dimensional when images are created at different focal planes

Question 8

Why is the resolution of the CLSM same as optical LSM?

Answer 8

Both use light as a source of radiation

Question 9

Advantages of confocal micrscope

Answer 9

ability to eliminate or reduce background information to reduce image degradation
capable pf collecting a series of optical sections from thick specimens at various depths using optical sectioning
high resolution
3d reconstructions
ability to image living cells and tissues

Question 10

Describe how fluorescent markers or tags can be used in CLSM

Answer 10

Fluorochromes attached to an antibody that is specific for one antigen or tagged to a chemical that binds to a specific part of the cell surface membrane