Microscopy of blood cells

Question 1

why is microscopy important

Answer 1

understanding the structure and organisation of cells and tissues important in the understanding of normal and pathological processes

Question 2

at the very start of setting up a microscope you…

Answer 2

start with low power 4x
put slide in clips 
turn on lamp 
adjust diustance between eye pieces to suit your own eyes 
make both eyepieces same focus

Question 3

focussing the image

Answer 3

main focussing knob
may be outer and inner for course and fine focussing (Nikon)
or single = fine first 1/4 then course (Leitz)

Question 4

focus the condenser

Answer 4

close field iris
adjust condenser focus knob until sharp image of edge of the disc of the light from the iris diaphragm
make in centre of field of view by the centring screws

Question 5

adjust the field iris

Answer 5

open iris until whole field of view only just illuminated

Question 6

adjust the condenser iris

Answer 6

stop with the condenser iris fully open

close until just begins to darken the image

Question 7

condenser iris control

Answer 7

adjusts the opening and closing of the condenser and its diaphragm

Question 8

turret objective lense

Answer 8

rotates to bring objectives of diff mag into the light path

Question 9

field iris

Answer 9

adjust opening and closing of field iris diaphragm

Question 10

centring screw

Answer 10

adjust the position of the condenser iris

Question 11

lamp v control

Answer 11

adjust the brightness of the image

Question 12

condenser focus knob

Answer 12

bring condenser into focus

Question 13

resolution

Answer 13

closest limit at which you can distinguish 2 adjacent small objects

Question 14

background behind setting up a microscope

Answer 14

basis from physics and the parameters that affect resolution
wl
optical quality of lenses and other components
refractive index of medium
geometry of light illuminating cone provided by the condenser lens - depend on way condenser is focussed and iris diaphragm adjusted
shown by numerical aperture- written next to mag

Question 15

how can you improve the refractive index

Answer 15

get highest resolution by using oil instead of air between objective lens and slide

Question 16

in practice what can you change to optimise resolution

Answer 16

set up condenser properly

Question 17

leaving the microscope

Answer 17

lowest power
V down
switch off lamp

Question 18

blue and pink dyes

Answer 18

haematoxylin and eosin

Question 19

investigating a slide

Answer 19

start at low mag to see what you want to look at

go up to 40x and look at individual cells

Question 20

nuclei images

Answer 20

not uniform
darker around edge - more densely packed DNA
dark spots in centre - nucleoli - assembly of ribosomes

Question 21

cytoplasm images

Answer 21

evidence of structures made of protein 
ER
Golgi 
mt 
lysosomes 
cytoskeleton 
cant distinguish without special stains

Question 22

what reduces resolution

Answer 22

changing the focus of the condenser

closing the condenser iris too far

Question 23

using stains

Answer 23

few cellular components absorb light at visible wl

H and E main stain

Question 24

haematoxylin

Answer 24

purple blue component
binds to acidic components of cells
bind DNA - show nucleus

Question 25

Leishman’s stain

Answer 25

similar to haematoxylin
show nucleus
red cells = red
white cells = blue because of nucleus

Question 26

eosin

Answer 26

pink stain

binds protein components

Question 27

H and E

Answer 27

general purpose stain

others used for particular chem constituents eg carbs

Question 28

alternative to stains

Answer 28

exploit interference properties to detect refractive index of cells compared to surrounding fluid
needs microscope fitted with special optics
important when examining living cells

Question 29

safety for taking a blood smear

Answer 29

blood is hazardous 
clean skin with alcohol swab 
don't share lancet 
use sharps
protect wound - plaster 
wear labcoat
wash hands if contact blood 
don't use mouth pipette
spills of blood decontaminated
dispose of anything that came into contact with blood

Question 30

why do you have to make smear quickly

Answer 30

blood clots quickly

Question 31

taking a blood smear

Answer 31

get blood using lancet - puncture fleshy pad by base of finger nail
place drop on clean slide
spread in film - use 2nd slide as spreader - blood drop behind the spreader otherwise cells damaged
dry smear by waving it in air
check it is thin and even

Question 32

stain the blood smear

Answer 32

place slide over draining dish
8 drops of Leishman’s stain so it covers smear
wait 1 min
8 drops buffer pH 6.8 -rock slide so it mixes in
wait 7mins
dry slide - tap on edge of filter paper and wave in air

Question 33

cell count

Answer 33

systematic

cover 20-50 cells

Question 34

numerical aperture

Answer 34

categorise the range of angles over which a system accept or emit light