Scribd
Upload
36 views33 pages

Microscope Teqnique 2014

The document discusses various microscopy techniques used to study cells. It begins by explaining that animal cells are too small to see with the naked eye, requiring microscopes. It then covers: - The components and workings of light/compound microscopes and electron microscopes. - How staining and sample preparation allows visualization of internal cell structures. - Specialized microscopes like fluorescent, phase-contrast, and dark-field that don't require staining. - Key microscopy terms like resolving power, magnification, and how techniques see different sized structures.

Uploaded by

Irwan Anggara
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
36 views33 pages

Microscope Teqnique 2014

The document discusses various microscopy techniques used to study cells. It begins by explaining that animal cells are too small to see with the naked eye, requiring microscopes. It then covers: - The components and workings of light/compound microscopes and electron microscopes. - How staining and sample preparation allows visualization of internal cell structures. - Specialized microscopes like fluorescent, phase-contrast, and dark-field that don't require staining. - Key microscopy terms like resolving power, magnification, and how techniques see different sized structures.

Uploaded by

Irwan Anggara
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
You are on page 1/ 33

Microscope Technique :

Studying cells by visual means

Dwi Ari Pujianto


Department of Biology
The reasons why using the microscope

• A typical animal cell is 10-20 μm in diameter (one-fifth the


size of the smallest particle visible to the naked eye)

• Understanding the structural organization of cells 


How cells function

• Animal cells are colourless and translucent

• Discovery of internal features depends on the


development of microscope and staining methods
Scale between living cells and atoms
Sizes of various cellular and subcellular structures
that different types of microscopes can visualize

µm (micrometer) = 10-6 m

Nm (nanometer) = 10-9 m

A (Angstrom) = 10-10 m
Resolving Power (RP)

RP of human eyes ≅ 100 µm → able to differentiate 2 points with


the distance of 100 µm .

Animal cells & bacteria have the diameter of 10 – 20 µm → can’t be


differentiated by eyes → need supporting apparatus (magnification
lense/microscope) to strengthen RP.

RP of compound microscope is 0.2 – 0.8 µm → suitable for the


investigation of bacteria and mitochondria with the diameter of
0.5 µm.

RP of electron microscope is 2 – 4 nm → utilized for the


investigation of particles, organels & viruses with very small sizes.
Resolution

The differentiation result of microscopic settings that


ultimately give clear (sharp) image → resolution.

The microscope resolution is dependent on :


- λ = (wave length) of light used for investigation
- n = (refraction index) of light passing the media & direct it to
objective lens, it is usually increased by adding water or
emersion oil,
- sin θ (aperture) = Half the angular width of the cone of rays
collected by objective lens

Resolution (D) = C. λ c = constant (0,61)


n. sin θ
Resolution improved by adding immersion oil

Index refractive
Resolution (D) = 0,61. λ Air = 1
n. sin θ Glass = 1,51
Oil = 1,51
What are Microscopes?

Instruments which enable the human beings to see substances and organisms,
which cannot be seen with the naked eye

Three basic types


- light (optical) Differ in method of illuminating
- electron the objects

Light microscopes
- Simple  single lens
- Compound  It uses a combination of lenses.

Electron Microscopes
- It uses a beam of highly energetic electrons to illuminate the object
- Produce highly magnified images.
Microscope components

Eyepiece Lens: the lens at the top that you look through (10X or 15X)

Tube: Connects the eyepiece to the objective lenses

Arm: Supports the tube and connects it to the base

Base: The bottom of the microscope, used for support

Illuminator: A steady light source used in place of a mirror. If the


microscope has a mirror, it is used to reflect light from an external light
source up through the bottom of the stage.

Stage: The flat platform where you place your slides.

Revolving Nosepiece or Turret: Part of the microscope that holds two


or more objective lenses and can be rotated to change power.
Microscope components

Objective Lenses: Usually 3 or 4 objective lenses on a microscope


(4X, 10X, 40X and 100X), retractable

Condenser Lens  condenser lens is to focus the light onto the specimen

Diaphragm or Iris  adjust the amount of light projected upward into the slide
Objective lens terms
• Plan: Planakromat objective lens, with flat
surface
• Planapo: Planakromat with better correction
• Ph1: Phase-contrast object for diaphragma 1
• Ph2: Phase-contrast object for diaphragma 2
• Ph3: Phase-contrast object for diaphragma 3
Objective lens

Objective magnification = 100 x


Numerical aperture= 1,25
Tube length = 160 mm
Cover slip thickness = 0,17 mm
Immersion oil line

immersion oil

Total magnification = Objective X Ocular (eyepiece) magnification


Compound microscope :
# Consist of 2 functional lenses – objective & ocular
(eyepiece) lenses

# Utilized for investigation of 0.2 – 0.8 µm objects

# Resolving power (RP) – the capacity to differentiate


2 closely separate points → depend on the λ
(wave length) of light collected by the objective
lense.
Preparation/processing of cells/tissues for investigation with
compound microscope.

Cells/tissues are investigated for their structure & topography


situation in organs; cells/tissues are treated into static state →
fixative solutions (acid and/or organic solution) :
- alcohols
- formaldehides
- glutaraldehides

After fixation cells/tissues are cut/dissected in definite thickness


allowing light fluently pass through (4 – 8 µm).

Cells/tissues are embedded in supporting medium: waxes, paraffines


→ embedding tissues are cut by microtomes → exposed (laid) on
glass slide.
Cell/tissue staining

Cells/tissues are stained with specific dye/dyes in order them to be


differentiated (distinguishable) one to another.

Specific dye show affinity (tendency) to particular cell/component so


that one having more affinity to a dye would appear more
prominent (differently) than the other.

Ex : Hemotoxylin (acid dye) show good affinity to base molecules


/proteins which are abundant in the nucleus.
Eosin (base dye) show good affinity to acid molecules in the
cytoplasm.
Upon staining with HE → nuclei became blue
cytoplasms gain red colour

Specific staining for cell components investigation :

feulgen - DNA (nucleus)


methylen blue - proteins
Comassie blue - Protein
Peryodate schiff - carbohydrate
janus green - mitochondria
Modified compound microscopes

1. Flourescent microscope

Useful for the investigation of specific cell structures/molecules.


Cells (specimen) is stained by fluorescent dye → cell components attracted
fluorescent dye become fluorescent.

Fluorescent molecules attract high energy light (UV), eventually reflect


light with lower energy (visible).

Ex : Y chromosome react specifically to quinacrine → create F body &


fluoresce upon exposure to UV light.

Fluorescent conjugated Ab reacted to specific Ag in cells → cell


component (structure) containing Ag fluoresce under
investigation using fluorescent microscope.
The optical system of a fluorescence microscope
Fluorescent Dye
2. Phase-contrast microscope

Usually utilized for investigating cells in natural (life) condition

Light of the same phase are mutually promote (strengthen) to each other
once they recombine (interferenced) → become brighter
Light of different phases are undergoing subtraction to each other once
they recombine (interferenced) → become gloomy (less bright)

When light pass through cell components → the light shift their phases
synergistically to their refraction index .

Ex. When a cell is exposed by a group of light → light passing


through the thick region (nucleus) reduce the amplitude of wave
or even shift the phase relative to the original (neighbouring)
light; as this light allow to recombine with the original light, the
region appear gloomy surrounded by bright background.
Interference (recombination) of lights at the same phases →
strengthen to each other → increase light intensity (bright)

Interference lights at different phases → reduce (subtract) to each


other → decrease light intensity (gloomy)

Life cells investigated under phase-contrast microscope :


brighter outside (surrounding the cell)
gloomy in the cytoplasm & darker in the nucleus.
→ cell appear gloomy (darker) with bright background.
Bright-field microscopy Phase-contrast microscopy

Differential-interference-contrast Dark-field microscopy


microscopy

You might also like

pFad - Phonifier reborn

Pfad - The Proxy pFad of © 2024 Garber Painting. All rights reserved.

Note: This service is not intended for secure transactions such as banking, social media, email, or purchasing. Use at your own risk. We assume no liability whatsoever for broken pages.


Alternative Proxies:

Alternative Proxy

pFad Proxy

pFad v3 Proxy

pFad v4 Proxy