Microscopy

Question 1

Resolution definition?

Answer 1

the ability to distinguish between to separate points/objects

Question 2

Magnification definition?

Answer 2

Is the number of times larger an image is compared to the original object

Question 3

Magnification calculation?

2 calculations

Answer 3

Mag=size of image/actual object

total mag= mag of ob lens x mag of eyepiece lens

Question 4

How does a light microscope work?

Answer 4

It uses a focused light and lenses to magnify a specimen, usually a cell, by sending up light through the objective lens

Question 5

What is the magnification of a light microscope?

Answer 5

around 1500x = a low mag

Question 6

What is the resolution of a light microscope?

Answer 6

around 200nm = low resolution

Question 7

What can you see with a light microscope?

Answer 7

Most bacteria and organelles like mitochondria, plus the human egg

Question 8

What are the advantages of a light microscope?

6 points

Answer 8

1) cheap to buy and operate
2) relatively small
3) both living and dead specimens can be viewed
4) Little expertise required to set up and use
5) Original colour of the specimen can be viewed
6) Used to study whole living specimens

Question 9

What are the disadvantages of a light microscope?

4 points

Answer 9

1) poor mag
2) poor resolution= structures closer then 200nm together will be seen as one object
3) specimen can be disfigured during prep
4) 2D images

Question 10

How does a laser scanning microscope work?

Answer 10

Uses laser scanning beams to scan a specimen usually tagged with fluorescent dye. The laser causes the dye to give off light which is then focused through a pinhole onto a detector. The detector is hooked up onto the computer to produce a 3D image

Question 11

What is the magnification of a laser scanning microscope?

Answer 11

1000x

Question 12

What is the resolution of a Laser scanning microscope?

Answer 12

512 x 512 pixels

Question 13

What can be seen under a Laser scanning microscope?

2 points

Answer 13

1) used in medical professions

2) used to observe fungal filaments within the cornea of a patient with a cornea fungal infection

Question 14

What are the advantages of a laser scanning microscope?

4 points

Answer 14

1) Images are high in resolution and contrast
2) Have depth selectivity= can focus on structures at different depths
3) adjust magnification electronically
4) 3D images can be viewed in color

Question 15

What are the disadvantages of a laser scanning microscope?

Answer 15

1) poor magnification
2) limited number of excitation wavelengths, which occur over very narrow bands and are expensive to produce
3) harmful nature of high intensity laser irradiation to cells and tissue
4) high cost to operate and use

Question 16

How does TEM work?

2 definitions

Answer 16

1) An electron source at the top of the microscope emits electrons that travel through a vacuum in the column of the microscope. Electromagnetic lenses are used to focus the electrons into a very thin beam and this is directed through the specimen of interest
2) The beam of electrons passes through the specimen, which is stained with metal salts, some electrons pass through and are focused on the screen/ photographic plate

Question 17

What is the Magnification of TEM?

Answer 17

2 million x

Question 18

What is the resolution of TEM?

Answer 18

less than 1nm

Question 19

What can be seen with TEM?

Answer 19

the features of very small specimens

Question 20

What are the advantages of electron microscopes?

4 points

Answer 20

1) extremely good mag
2) wide range of applications that can be utilized in many different scientific fields
3) provide information on element and compound structure
4) Images are high quality and detailed

Question 21

What are the disadvantages of an electron microscope?

7 points

Answer 21

1) Unable to analyse live specimens= electrons are easily scattered by other molecules
3) artefacts can be left over from sample preparation
4) expensive
5) Large
6) special operators are required to use
7) Limited to samples that are thin enough to let the electrons pass through- TEM

Question 22

How does SEM work?

Answer 22

A beam of electrons is scanned across the specimen. This knocks off electrons from the specimen, which is gathered in the cathode ray tube to form an image

Question 23

What is the magnification of SEM?

Answer 23

1 million x

Question 24

What is the resolution of SEM?

Answer 24

1-20 nm

Question 25

What can be seen with SEM?

Answer 25

cell structures i.e the cell wall; nucleus; vacuoles, mitochondria e.t.c

Question 26

What are the differences between SEM and TEM?

4 points

Answer 26

1) SEM provides detailed images of the surfaces of cells. It focuses on the sample surface and composition
2) SEM cannot see images to the same level as TEM but is faster, less restrictive and sometimes can be performed with little or no sample prep
3) TEM gives info on internal structures ( can see the structures in more detail), however it is limited to samples that are thin enough to let the electrons pass through
4) SEM provides images in 3D and colour

Question 27

What is a haemocyter?

Answer 27

Glass microscope slide used to count cells

Question 28

How is cell concentration determined?

Answer 28

average cell count x 10(4) x dilution factor

Question 29

What is flow cytometry?

Answer 29

analysis technique used to determine cell phenotype and health