U2 Cell Structure - Microscopy

OCR A A-Level Biology

magnification formula

magnification = image size/actual size

or I/AM

benefits of light microscope

• - easily available

• - cheap

• - can be used out in the field

• - can observe both living and dead specimens (although sometimes we use a stain which kills cells)

draw and label a microscope

benefits of having both objective and eyepiece lens

• allows much higher magnification

• reduces chromatic aberration

what adjustments would need to be made for an opaque specimen

illumination from above, in some microscopes

describe the dry mount preparation

• solid specimens viewed whole / in thin slices (sectioning)

• specimen on centre of slide with cover slip

• examples include hair, pollen, muscle tissue, plants

describe the slide preparation that could be used for a small water flea (2)

wet mount preparation

• suspend specimen in liquid such as water or an immersion oil

• place cover slip at an angle

• for example aquatic samples and living organisms

describe the slide preparation for soft samples/root tips

squash sample (?)

• wet mount is prepared

• press on cover slip with lens tissue

• depending on the material, can avoid damage to cover slip by squashing sample between two slides

• for example, root tips or soft samples.

describe smear slides prep

• edge of slide is used to smear the sample creating a thin even coating on another slide

• then place cover slip on

• for example is blood

describe the difference between brightfield microscopy and the other type of microscopy (2)

BRIGHTFIELD: objects are dark and the field is light; can be used to observe unstained microorganisms

WIDE FIELD: whole sample illuminated at once

identify if there images were made by a SEM, TEM or optical microscope

1- TEM

2- SEM

3- SEM

4- optical

5- TEM

describe how an image with a light microscope would look

• low contrast as most cells do not absorb a lot of light

• resolution is limiting factor by wavelength and diffraction of light passing through the sample

why do we use (differential) stains

• to see transparent structures (cytosol etc)

• stains increase contrast as different components take up stains to different degrees, enhancing visibility

• highlighting specific structures

• SPEC: to identify different organelles and cell types

how to prepare a slide for staining

(i.e. how do you properly stain a slide)

• Place on slide and allow to dry

• It is then heat fixed by passing through a flame

• the specimen will then adhere to the microscope slide and will then take up stains

identify the key structures on the pic

• pink circles: RBC (erythrocytes)

• purple: white blood cells (leucocytes)

• surrounding grey: plasma

exmaples of positively and negatvively charged dyes

+VE: crystal violet, methylene blue

-VE: nigrosin, congo red

what is the difference between positive and negatively charged dyes

positively charged dyes stain cell components.

negatively charged dyes stay outside cells and leave cells unstained

define differential staining

and give two examples

- can distinguish between two types of organism, or different organelles within a tissue sample

gram staining technique and acid-fast technique are both differential staining methods.

suggest and explain a method for staining a slide to differentiate thin-walled and thick-walled bacteria

• gram staining technique

• separates bacteria into gram+ and gram-

• apply crystal violet to specimen, then iodine

• - wash slide with alcohol

• gram+ will retain the stain and stay blue/purple

• gram- have thinner cell walls so lose the stain

• stain with a counterstain (safranin dye)

• the bacteria appear red

• gram+ are susceptible to penicillin which inhibits cell wall formation

• gram- have thinner cell walls so are not susceptible

why is iodine applied in the gram-stain technique?

why is alcohol added?

why is safranin used?

Iodine - it fixes the dye

alcohol - removes the dye from thin cell walls

safranin - stains bacteria pink but not in the presence of crystal violet

acid fast technique

• - differentiates species of mycobacterium from other bacteria

• - lipid solvent carries carbol fuchsin dye into cells

• - wash cells with dilute acid-alcohol solution

• - mycobacterium aren't affected by solution and retain stain, which is bright red

which technique?

acid-fast

which technique

gram stain (duh)

define fixing a slide

using chemicals like formaldehyde to preserve specimens in as near-neutral state as possible

(preparing for staining and killing the specimen and keeping structures)

what is sectioning

• specimens dehydrated with alcohols,

• then placed in a mould of wax/resin

• to form a hard block,

• then sliced with a microtome knife

fill this table

difference between transmission electron microscope and scanning electron microscope - how they work

• SEM - beam of electrons sent across the surface of a specimen and the reflected electrons are collected. creates image by collecting and deflecting reflected electrons

• TEM uses electrons passing through the sample and focused to create an image

compare the results from a SEM and TEM

SEM has lower resolution + magnification, but TEM has twoD images

SEM? OR TEM?

TEM

SEM? OR TEM?

SEM

define magnification

how many times larger an image is than the actual size of the object being viewed

define resolution

the minimum distance between 2 objects where they can still be seen as 2 different objects

THE ABILITY TO SEE MORE DETAIL / SEPARATE TWO OBJECTS accPMT

why is resolution limited in light microscopes

• light waves diffract when they pass near physical structures

• the structures present in the specimens are close and the light reflected from individual structures can overlap due to diffraction

how do electron microscopes solve this problem

electron beams are still diffracted, but the shorter wavelength means individual beams can be closer before they overlap

problems with electron microscope

• specimens can be damaged by electron beam

• artefacts (structures produced due to the preparation process)

why is there a vaccuym inside an electron microscope

to ensure electron beams travel in straight lines

specimen preparation for an electron microscope

• fixing using chemicals/freezing

• staining with heavy metals

• dehydration with solvents

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criteria for graphs

S ize of points - 50%

P oints plotted correctly

L ine of best fit

A xes with labels and units

T itle that’s descriptive

N o fluffy lines, extrapolation or tthick crosses

define artefact

give an example

visible structural detail caused by processing the specimen which is not a feature of the specimen

e.g. bubbles under the coverslip

give an artefact example for electron microscopes

loss of continuity of membranes, distortion of organelles, empty spaces in the cytoplasm of cells

how does a laser scanning confocal microscope work

• Specimen is stained with a dye,

• a laser is moved across the specimen, causing the dye to fluoresce /give off light.

• This light is passed through a narrow hole through a detector which generates an image

what is LSCM used for now

non invasive so used in diagnosis of eye disease, new dRuGs and endoscopic procedures

future uses for advanced optical microscopy

virtual biopsies particularly for skin cancers.

what is a eyepiece graticule?

what is a stage micrometer?

disc with a scale of 1-100 that represents different measurements with each magnification

microscope slide with a scale in micrometres, each division usually 10 micrometers. it calibrates an eyepiece graticule

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how to calibrate a lens

• put the stage micrometer in place and eyepiece graticule in… well, the eyepiece

• get the scale on the micrometre in focus

• align the scales and take a reading

• remove the stage micrometer

what are the units of the EG and the SM

EG- none

SM- micrometres… duhh

how many times do you calibrate the graticule scale

3x. for each objective lens separately.

what is the length of a usual SM division

1 division = 10 micrometers

20 EG = 10 SM

20 EG = 100 MICROMETRES

1 EG = 5 MICROMETERS

The magnification factor is 5. this is also the number of micrometers per division, or µm/division on the EG

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magnification factor for the x40 lens

x40=0.5

using this value, find the diameter (roughly) of this atom

1 div = 0.5µm

100 div = 50µm

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how to use a microscope - longhand

• stage in lowest position

• lowest objective lens

• put slide

• stage up but doesn’t touch slide

• stage down until you can see specimen

• fine focus up

• for more detail objective lens up and

OCR accepted figures for the magnification and resolution of

• light microscope

• SEM

• TEM

MAG

• LIGHT: 1000

• SEM: 100,000-500,000

• TEM: 500,000 - 2mil

RES

• LIGHT: 200nm

• SEM: 3-10nm

• TEM: 0.2-0.5nm

TEM - INSIDE THE CELL because SEM electrons bounce off it. sem only shows surface

light microscope (1)

graticule (1)

compare and contrast SEMs and TEMs (5)

similarities

• both use electrons

• both use vacuums / dead tissue only

• both have higher magnification and resolution than light microscopes

differences

• electrons are transmitted through the specimen in TEM (internal structure visible); in SEM they are reflected off the surface (surface)

• SEM 3D; TEM 2D

• TEM gives higher resolution and maximum magnification

• SEM can be used on thicker specimens than TEM

diff between prokaryotes and eukaryotes

prokaryotes:

• single celled with simple structure

• undivided internal area called the cytoplasm (composed of cytosol, which is made of water salts and organic molecules)

• DNA not contained in the nucleus.

• only 1 molecule of DNA, a supercoiled compact chromosome.

• the genes on the chromosome are grouped into operons so are switched on or off at the same time

• smaller ribosomes (70S)

• have a cell wall made from peptidoglycan (murein)

• thinner flagella, without 9+2 arramgement

• energy to rotate the filament is supplied from chemiosmosis not ATP

• flagellum attached to cell membrane by a basal body and rotated by a molecular motor

eukaryotes:

• multicellular organisms

• more complicated internal structure with a membrane-bound nucleus (nucleoplasm) and cytoplasm with more membrane-bound components

• DNA in nucleus as multiple supercoiled chromosomes

• chromosomes wrapped around histones (this complex called chromatin, which coils and condenses to form chromosomes)