DEPARTMENT OF BIOCHEMISTRY (PG) Bioanalytical Techniques ...PG)-10.p… · using a confocal...

2D Gel Electrophoresis

KONGUNADU ARTS AND SCIENCE COLLEGE

(AUTONOMOUS)

Coimbatore – 641 029

Bioanalytical Techniques

Dr. S. Viswanathan

U/TS/649

DEPARTMENT OF BIOCHEMISTRY (PG)

• INTRODUCTION

• DEFINITION

• HISTORICAL BACKGROUND

• VARIBLES USED IN MICROSCOPY

• COMPOUND MICROSCOPE - Structure and

Function

• USE OF MICROSCOPE

• VARIOUS TYPES OF MICROSCOPES

• CARE OF MICROSCOPE

2

INTRODUCTIONTO

MICROSCOPY3

• Understanding the

optical principles

and construction of

microscopes

• Role of microscopy

• Microscopic

techniques and

application

DEFINITION 4

• A microscope (Greek: micron = small and

scopos = aim)

• MICROSCOPE - An instrument for viewing

objects that are too small to be seen by the

naked or unaided eye

• MICROSCOPY - The science of

investigating small objects using such an

instrument is called microscopy

HISTORICAL BACKGROUND5

• 1590 - Hans Janssen and his son Zacharias

Janssen, developed first microscope.

• 1609 - Galileo Galilei - occhiolino or compound

microscope.

• 1620 - Christian Huygens, another Dutchman,

developed a simple 2-lens ocular system that

was chromatically corrected.

Anton van Leeuwenhoek

(1632-1723)6

• Anton van Leeuwenhoek is

generally credited with

bringing the microscope to

the attention of biologists.

• A tradesman of Delft, Holland.

1661 - He discovered bacteria, free-living

and parasitic microscopic protists, sperm

cells, blood cells, microscopic nematodes

etc.

Microscope used by

Anton von Leeuwenhoek7

An old pocket Microscope

9

VARIABLES

USEDIN

MICROSCOP

Y

MAGNIFICATION 9

• Degree of enlargement

• No of times the length, breadth or

diameter, of an object is multiplied.

MAGNIFICATION VS SHARPNESS

• USEFUL MAGNIFICATION AND EMPTY

MAGNIFICATION

• RESOLUTION – Ability to reveal closely

adjacent structural details as separate and

distinct

• LIMIT OF RESOLUTION (LR) – The min

distance between two visible bodies at

which they can be seen as separate and

not in contact with each other

10

Types of microscope Resolving power

Compound Microscope 200 nanometers

Scanning Electron

Microscope

10 nanometers

Transmission Electron

Microscope

0.2 nanometers

W = Wavelength

NA = Num aperture11

• LR = 0.61 x WNA

13

NUMERICAL APERTURE(NA)

• Ratio of diameter of lens to its focal length

• NA = n Sin θ/2n = refractive index,

θ/2

A

B

D

θ = angle of aperture (CAD)

C

• n of air = 1

• n of oil = 1.5

14

DEFINITION

• Capacity of an objective to render outline

of the image of an object clear and distinct

• Depends of elimination of Spherical and

Chromatic aberration

15

ABERRATION

• Chromatic aberration

• Correction of aberration – Achromatic

objective and Apochromatic objectives.

Blue focusRed focus

Incident

light

• Spherical aberration

Focus of marginal rays

Focus of axial rays

15

TYPES OF MICROSCOPE16

• Simple microscope

• Compound microscope

• Phase Contrast

Microscope

• Dark Ground Microscope

• Fluorescent Microscope

• Electron Microscope

• Others

COMPOUND

MICROSCOPE

17

Compound microscope made

by John Cuff 1750

19

PARTS OF COMPOUND MICROSCOPE

• Ocular (Eye piece)

• Body or Tube

• Coarse focusing knob

• Fine focusing knob

• Objective Lens

• Movable stage

• Condenser Lenses

• Field (Iris) Diaphragm

• Mirror and light source

20

OBJECTIVE LENS

• Mounted on Nose piece

• It forms magnified real image.

• Magnification of objective

= Optical Tube length

Focal Length

•

•

•

•

Scan - 4X

Low Power - 10X

High Power - 40X

Oil immersion - 100

TYPES

21

OIL IMMERSIONOBJECTIVE

Highest magnification

Oil prevents refraction of light outwards and

allows it to pass straight in to objective G

GLASS

OIL

AB

C

E

F

FBEG - OILD

ABCD - AIR

EYE PIECE21

•

•

Forms magnified virtual & erect image

TYPES

(a)

(b)

(c)

(a)

(b)

(c)

Monocular

Binocular

Trinocular

or

Huygenian

Ramsden

Compensating

22

24

PARTS OF MICROSCOPE (Cont)

Iris Diaphragm

Condenser

Field Diaphragm

Diaphragm control ring

Focusing knob

illumination source

ILLUMINATION - Lamp,

sunlight, battery operated

lamp, 60 W bulb, Quartz

halogen light.

FILTERS - Blue, Green,

Heat absorbing filters,

Barrier filters.

24

Multiple step operation employed to attain optimal

illumination:

1. Remove any diffusing filter.

2. Put a slide on the stage and focus.

3. Completely close the field diaphragm .

4.Move the condenser until the border or the iris

hexagon is neat and clear.5. Center if necessary.

6.Open the field diaphragm until the tip of the

hexagon touches the field limit

KOHLER’S ILLUMINATION 25

HOW A MICROSCOPE WORKS

?

26

28

OPTICAL PATH IN COMPOUND

MICROSCOPE

METHODOF USING

COMPOUND

MICROSCOPE

28

1. Grasp the microscopes arm with

one hand and place your other

hand under the base.

29

2. Place the microscope on a bench.

Adjust seat

3. Clean Lenses.

4. Turn the coarse adjustment knob

to raise the body tube

arm

arm

5. Revolve the nose piece to set low-power objective lens.

6. Adjust the Condenser lenses and diaphragm .

7. Place a slide on the stage and secure with stage clips.

8. Switch on the light at low intensity and then increase intensity.

30

9. Turn the coarse adjustment knob to lower the body tube until the low power objective reaches its lowest point.

10.Looking through the eyepiece, very slowly move

the coarse adjustment knob until the specimen

comes into focus.

11. Adjust distance between eye piece.32

12. Switch to the high power objective lens only

after adjusting condenser and iris diaphragm.

13. Place a drop of oil over specimen before using

oil immersion objective.

14. Lower the objective until oil makes contact with

objective.

15. Looking through the eyepiece, very slowly

focus the objective away from the slide i.e by

raising the objective lens.

32

HOW TO OBSERVE A SLIDE ?33

CAUSES OF ERROR IN

FOCUSING34

• Revolving Nose Piece is off centre

• Preparation is upside down

• Thick cover slip

• Dirt or Dried oil over Lens

• Air bubble in immersion oil

• Poor illumination – Condenser not fully

racked up

PHASE CONTRAST MICROSCOPE35

PHASE CONTRAST

MICROSCOPE36

• First described in 1934 by Dutch physicist

Frits Zernike

• Produces high-contrast images of

transparent specimens

• Advantage - Living cells can be examined

in their natural state

PRINCIPLE OF PHASE CONTRAST

MICROSCOPY37

• Unstained bacteria have constituents of

different refractive index .

• Diffraction of light

• Phase contrast microscope employs an

optical mechanism to translate minute

variations in phase into corresponding

changes in intensity of image.

39

REQUISITE FOR PHASE

CONTRAST MICROSCOPY

39

• ANNULAR DIAPHRAGM

• PHASE PLATE

CONDENSER ANNULUS 40

• The condenser annulus or annular

diaphragm is opaque flat-black (light

absorbing) plate with a transparent

annular ring.

• Produces hollow cone of light.

Condenser

annulus

41

PHASE PLATE 42

• Placed in back focal plane of objective.

• FUNCTION :-

1. Enhances phase difference by

retarding diffracted wave front by one

quarter of wavelength .

2. Reduces intensity of direct rays and

equalizes it with diffracted rays intensity.

44

Phase plate

IMAGES OF PHASE CONTRAST

MICROSCOPY

Clostridium botulinum45Spirilium volutans

46

COMPARISION OF IMAGES OF BRIGHT

FIELD AND PHASE CONTRAST

MICROSCOPY

USES OF PHASE CONTRAST

MICROSCOPY46

• Phase contrast enables visualization of

internal cellular components.

• Diagnosis of tumor cells .

• Examination of growth, dynamics, and

behavior of a wide variety of living cells in

cell culture

DARK GROUND MICROSCOPE 47

• Optical system to enhance the contrast of

unstained bodies .

• Specimen appears gleaming bright

against dark background

• PRINCIPLE OF DGI

OPTICAL PATH IN DARK GROUND

MICROSCOPY 48

REQUISITES FOR DARK GROUND

MICROSCOPY

• Dark ground

condenser

• High intensity

lamp

• Funnel stop

49

USES OF DARK GROUND

MICROSCOPY50

• Useful in demonstrating

-Treponema pallidum

- Leptospira

- Campylobacter jejuni

- Endospore

Treponema pallidum

FLUORESCENCE MICROSCOPY51

• PRINCIPLE

UV light

Fluorochrome

Visible

radiation

FITC EX - 495 nm EM - 520nm

TRITC EX – 540 nm EM – 590 nm

Texas Red Ex – 600 nm EM – 615 nm

• UV rays passes through exciter filter

• Dark ground condenser

• Micro organisms stained with fluorescent

dye, when examined under microscope

with ultraviolet light are seen as bright

object against dark background

52

USE OF FLUORESCENCE

MICROSCOPY53

• Auramine Rhodamine –Yellow Fl - Tubercle bacilli

• Acridine Orange R - gives

orange red Fl with RNA and

yellow green Fl with DNA

• QBC

• IMMUNOFLUORESCENCE

55

ELECTRON

MICROSCOPE

ELECTRON

MICROSCOP

E

55

• Electron Microscopes uses a beam of highly

energetic electrons to examine objects on a

very fine scale. This examination can yield

the info about

– Topography

– Morphology

– Composition

– Crystallographic structure

TYPES OF EM 56

• Transmission Electron Microscope (TEM)

• Scanning Electron Microscope (SEM)

58

TRANSMISSION

ELECTRON

MICROSCOPE (TEM)• Stream of electrons is formed

• Accelerated using a positive electrical potential

• Focused by metallic aperture and Electro magnets

• Interactions occur inside the irradiated sample which are detected and transformed into an image .

TEM

(Cont)

58

• Projector Lens forms

image on Fluorescent

viewing screen

• 2D Image

• Magnification

10,000 X to 100,000 X

• Scan a gold-plated specimen to give a 3-D

view of the surface of an object which is

black and white.

• Used to study surface features of cells and

viruses.

• Scanning Electron microscope has

resolution 1000 times better than Light

microscope .

SCANNING ELECTRON

MICROSCOPE

59

WORKING OF SEM 60

62

SEM IMAGES

Vibrio cholerae with

polar flagellaTreponema pallidum

63

INVERTED MICROSCOPE

• Used in metallurgy

• Examination of cultures in flat bottom

dishes

• Micro dissection

• Examination of parasites

• Observation of agglutination in serology

OTHER MICROSCOPES

STEREO MICROSCOPE

• Double Microscope

• Produces 3D images

POLARIZING MICROSCOPE

• Uses two Polariser

• Gives info about Birefringence of a body

• Used in Crystallography, Urine examination

• Apple Green Birefringerence in AMYLODOS64IS

• Uses a laser beam to illuminate

a specimen whose image is

then digitally enhanced for

viewing on a computer monitor.

• Laser beam scans single plane

of 1µm thickness.

CONFOCAL SCANNING

LASER MICROSCOPE64

Comparison of Depth of

Light Collection and

Image clarity

65

Light Microscope Confocal Scanning

Laser Microscope

PRINCIPLE OF CONFOCAL MICROSCOPY66

USES OF CONFOCAL

MICROSCOPE67

• Observing cellular morphology in

multilayered specimen

• Eg. used in diagnosing Ca cervix

• Evaluation and diagnosis of basal cell

carcinoma of skin

What is the advantage of

using a confocal

microscope?

68

• By having a confocal pinhole, the

microscope is really efficient at rejecting

out of focus fluorescent light so that very

thin section of a sample can be analyzed.

• By scanning many thin sections through a

sample, one can build up a very clean

three-dimensional image .

NEWER MICROSCOPE 69

• SCANNING PROBE MICROSCOPE -Class of

Microscope that measures surface features

by moving a sharp probe over object surface.

Used to visualize atoms and molecules

– Scanning Tunneling Microscope (STM)

– Atomic Force Microscope (AFM)

CARE OF THE

MICROSCOPE

70

• Handling

• Proper storage

• Care of Lenses

• Care of oil emersion objective

• Care of lamp