MICROSCOPY 🔬

MicRoscopy

PRESENTATION

GUIDED BY: DR. DEEPAK WADHAWAN

Presented by: TANISHAROLL NO. : 217208 Class : : msc. Zoology (2nd sem)

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Contents

1. INTRODUCTION

2. CHARACTERISTICS

3. HISTORY

4.TYPES

5. APPLICATIONS

6. DISADVANTAGES

7. CARING OF MICROSCOPE

Introduction

MICROSCOPY - Technique of seeing small objects. MICROSCOPE - The instruments used to detect small sized objects. NEED - Required because human eye cannot resolve two points and most of the cells that are smaller in size.

CHARACTERISTICS

• Microscope possesses two peculiar characteristics.

RESOLVING POWER

MAGNIFICATION

Resolving power

• Resolving power -

Ability of microscope to distinguish two objects lying very close to each other.

(LR)

Minimum distance between two points at which they can be seen as separate.

• Limit of Resolution

For EM

• Water-1
• Oil-1.5

By Abbe's equation

Capable of objective to render outline of image of an object clearer.

• Numerical Aperture (NA) -

MAGNIFICATION

• Magnification -

Ratio of visible size of an object to its actual size.

• Optical Tube length
• Focal length of object
• Magnifying power of eye piece

• Depends upon

Calculated by multiplying magnifying power of objective lenses by that of eye piece

• Total Magnification -

Magnification of LIGHT MICROSCOPE is 3000 times.

m= height of image height of object

Magnification of ELECTRON MICROSCOPE ranges from 100000. -600000.

HISTORY

1590

1609

1625

1673

1931

1932

1981

TY P E S

1. Bright field microscopy
2. Dark field microscopy
3. Phase Contrast microscopy
4. Fluorescence microscopy
5. Confocal microscopy

Optical/Light Microscopy

1. Transmission electron microscopy(TEM)
2. Scanning electron microscopy (SEM)

Electron Microscopy

Scanning Probe Microscopy

1. Scanning tunneling microscope (STM)
2. Atomic force microscopy (AFM)

MICROSCOPY

BRIGHT FIELD MICROSCOPY

• Produces dark image against a Brighter background.
• Has several Objective lenses.
• Two Types 1.) SIMPLE MICROSCOPE 2.)COMPOUND MICROSCOPE

1). SIMPLE microscopE

• Contains a single magnifying lens.

m= 1+D Where, D- Least distinct vision f f- Focal length.

• PRINCIPLE - When a sample is placed within focus of microscope virtual, direct and magnified image is obtained at the least distinct vision from Eye.

• APPLICATIONS -

• Used by Watchmakers
• Used by Jewellers
• Used by Dermatologists

BRIGHT FIELD MICROSCOPY

2.) COMPOUND microscopE

• Series of lenses for magnification.

• PRINCIPLE - When a tiny specimen is placed just beyond the focus of its objective lens, virtual, inverted and highly magnified image is obtained at the least distinct vision from Eye.

• METALLURGY .

• Useful in FORENSIC LABORATORIES.

• APPLICATIONS -

DARK FIELD MICROSCOPY

• DISCOVERY - J.J Lister (1830)

• Results of brightly illuminated specimen surrounded by a dark field.
• Optical system to enhance the contrast of unstained body.
• A bright field microscope is adapted to dark field microscope by adding a special disk known as STOP to condenser.

STOP - blocks all light entering objective lens except light that is reflected of the sides of specimen itself.

DARK FIELD MICROSCOPY

REQUIREMENTS/REQUISITES FOR DARK FIELD MICROSCOPE

• Dark ground condenser
• High intensity lamp
• Funnel STOP

• APPLICATIONS -

• Syphilitic infections
• Treponema pallium
• Leptospirosis
• Endospore

Used to study

Treponema pallium

• Specimen needs to be strongly illuminated which can damage the delicate sample.

• DISADVANTAGES -

Leptospirosis

PHASE CONTRAST MICROSCOPY

• DISCOVERY - F. Zernike (1930)

• It is special kind of modified light microscope employed to study the instant living cells or tissues.
• Also known as ZERNIKE MICROSCOPE.

• PRINCIPLE - The small phase changes in the light rays, induced by differences in the thickness and refractive index of the different parts of an object, can be transformed into differences in brightness or light intensity.
• Uses a special adapter that slows down the wavelength of light by 1/2(phase shift) results in the cell having different refractive index then it's surroundings.

PHASE CONTRAST MICROSCOPY

REQUISITES FOR PHASE CONTRAST MICROSCOPE

• Annular diaphragm or condenser annulus
• Phase plate

• Diagnosis of tumour cells
• Enables visualisation of internal cellular components (cheek cells)
• Observation of cells cultured in Vitro

• APPLICATIONS -

Used in

• Not ideal for thick specimens.
• Decreases resolution.

• DISADVANTAGES -

Human cheek cells under PCM

FLUORESCENT MICROSCOPY

• DISCOVERY - Haitinger and Coons

• Also known as EPIFLUORESCENT MICROSCOPE.
• Exposes specimen to UV Violet or blue light.
• PRINCIPLE - Employed to study the properties of certain organic compounds that show phenomena of fluorescence naturally if not it is stained with fluorescent dyes called FLUOROCHROMES.

FLUORESCENT MICROSCOPY

REQUISITES FOR FLUORESCENT MICROSCOPE

• Utilises Mercury vapour arc lamp for lighter source.
• Employs three set of filters to alter the light that passes up through instruments to Eye.

• APPLICATIONS

• Localising cellular structure like chloroplast fluoresce a deep red colour .
• Identify strains of bacteria.
• Immunofluorescence.

• DISADVANTAGE

• Low resolving power
• Undergoes photo bleaching.

IMMUNOFLUORESCENCE

CONFOCAL MICROSCOPY

• DISCOVERY - Marvin Minsky (1957)

• Updated version of fluorescence microscopy.
• It is an optical imaging technique for increasing optical resolution and contrast of the micrograph
• Uses pinhole screen to produce high-resolution images.
• Eliminates out of focus.
• So images have better contrast and are less hazy.
• A series of thin slices of the specimen are assembled to generate a 3-D image.

• Two pinholes are typically used
• A pinhole is placed in front of the illumination source to allow the transmission only through a small area through which illumination is imaged onto focal plane of the specimen that excited in this manner at the focal plane which is image onto confocal pinhole placed right in front of detector.

• PRINCIPLE -

RAY DIAGRAM FOR CONFOCAL MICROSCOPY

CONFOCAL MICROSCOPY

REQUISITES FOR CONFOCAL MICROSCOPE

• Utilise Detector, confocal pinhole,illumination pinhole,dichroic mirror and objective lens.

• APPLICATIONS

• Confocal microscopy allows analysis of fluorescent labelled thick specimen without physical sectioning
• Three-dimensional reconstruction of specimen.
• Improved resolution.

3-D Restruction of specimen

• DISADVANTAGE

• It has inherent resolution limitation due to diffraction. The maximum Best resolution of confocal microscopy is about 200 nm.
• Undergoes photo bleaching.

Kidney cells (fluorescence v/s confocal microscope.)

ELECTRON MICROSCOPY

• DISCOVERY - Knoll & Ruska (1931)

• EM used a beam of highly energetic electrons to examine objects on a very fine scales.

• Denser the material , greater is the scattering of electron irrespective of chemical composition.

• PRINCIPLE -

• MAGNIFICATION-

• 600,000 times .

• Resolving power of EM is 250 times than that of light microscope

• RESOLVING POWER-

• TYPES OF EM -

1. TEM
2. SEM

RAY DIAGRAM FOR ELECTRON MICROSCOPY

TRANSMISSION ELECTRON MICROSCOPY

• DISCOVERY - Knoll (1935)

• Stream of electrons is formed.
• Accelerated using positive electric potential.
• Focused by metallic aperture and electro magnets. Interaction occurs inside the irradiated sample which are detected and transformed into image.
• Projector lens forms image on fluorescent viewing screen.
• TEM forms a 2-D image

• PRINCIPLE -

• MAGNIFICATION-

• 10,000 to 100,000 times .

• 2.5 nm

• RESOLVING POWER-

SCANNING ELECTRON MICROSCOPY

• DISCOVERY - Knoll (1935)

• PRINCIPLE -

• A beam of electron is generated by a suitable source, typically a tungsten filament or a field emission gun.
• Electron beam is accelerated through a high voltage and pass through a system of aperture is an electromagnetic lens to produce a thin beam of electrons.
• The beam scans the surface of the specimen and electrons and acted from the specimen by the action of scanning beam and collected by a suitable position detected.

• MAGNIFICATION-

• 1,000to 10,000 times .

• Thousand times better than light microscope.

• RESOLVING POWER-

SEM

TEM

VS

• Electron beam passes through the object.
• Resolving power is more that is 10 Armstrong
• Depth of focus is less.
• I 2-D image is formed.
• Used to study the ultrastructure of cell and cell organelle lease.
• Primary electron beam undergoes differential scattering by the components of object.

• Electron beams scans the surface of the object.
• Resolving power is low that is 10 nm.
• Depth of focus is more.
• 3D images formed.
• Used to study the surface features of the objects.
• Secondary electron scattered from the surface of specimen form the final image.

SCANNING TUNNELING MICROSCOPY

Gerd Binning & Heinrich Rohrer(1981)

• DISCOVERY -

• The principle of scanning tunneling microscope is in quantum mechanics that explains the wave and particle like behaviour of tiny particles like photons and electrons.
• The quantum mechanics phenomenon which explains tunnelling effect is the working principle of STM.

• PRINCIPLE -

It is the phenomenon where a particle tunnels through a barrier that is classically could not save Mount.

• TUNNELING

EFFECT -

SCANNING TUNNELING MICROSCOPY

COMPONENTS Of STM

• Scanning tip
• Piezoelectric scanner
• Distance control and scanning unit
• Vibration isolation system
• Data processing unit

• APPLICATIONS

• Widely applied in research and manufacturing in the shield spanning from biology to material science of micro electronics.
• Use to image topography.
• Used to measure Service properties.
• Imported tool in nanotechnology

Schematic view of STM

• DISADVANTAGE

• It is very expensive.
• It needs specific training to operate effectively. Need very clean surface,excellent vibrations control while operation,single atom tip.

STM image of a pure gold surface shows individual atom of gold arranged in columns.

ATOMIC FORCE MICROSCOPY

Binning (1986)

• DISCOVERY -

• A metal and diamond probe is gently forced down onto a specimen. As the probe moves along the surface of the specimen, its movements are recorded and a three-dimensional image is produced.
• AFM does not need or requires special specimen preparation.

• PRINCIPLE -

• AFM uses forces when two objects are brought within nano range of each other : probe in contact with surface-repulsive force, a few nanometre away-attractive force.
• AFM generates images by scanning a small cantilever over the surface of sample. The sharp tip on the end of the cantilever contacts the surface, bending the cantilever and changing the amount of laser light reflected into the photodiode. The height of cantilever is then adjusted to restore the response signal, resulting in the mayor cantilever height tracing their surface.

ATOMIC FORCE MICROSCOPY

COMPONENTS Of AFM

• Moving AFM tip.
• Force sensor.
• Vibration controller
• Computer

• APPLICATIONS

• Widely applied in research and manufacturing in the shield spanning from biology to material science of micro electronics.
• Use to image topography.
• Used to measure Service properties.
• Important tool in nanotechnology.

• DISADVANTAGE

• Limiting magnification rate.
• Tip or sample can be damaged.

AFM image shows long, strand-like molecules of nanocellulose

CARING OF MICROSCOPE

• Proper storage
• Handling
• Care of lenses
• Care of oil emersion objective
• Care of lamp

ThankYou!