Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye (objects that are not within the resolution range of the normal eye). There are three well-known branches of microscopy: optical, electron, and scanning probe microscopy, along with the emerging field of X-ray microscopy.
BRIGHT FIELD MICROSCOPY by SIVASANGARI SHANMUGAM
bRIGHT FIELD MICROSCOPY is also called a compound microscope. The name bright - field is derived from the fact that the specimen is dark and contrasted by the surrounding bright viewing field.
Bright field microscopy, Principle and applicationsKAUSHAL SAHU
Introduction
History
Basic Component of Microscope
Light Microscopy
Types of Light Microscopy
What Are Bright Microscopy
Principle of Bright Microscope
Advantage
Disadvantage
Application
Conclusion
Reference
BRIGHT FIELD MICROSCOPY by SIVASANGARI SHANMUGAM
bRIGHT FIELD MICROSCOPY is also called a compound microscope. The name bright - field is derived from the fact that the specimen is dark and contrasted by the surrounding bright viewing field.
Bright field microscopy, Principle and applicationsKAUSHAL SAHU
Introduction
History
Basic Component of Microscope
Light Microscopy
Types of Light Microscopy
What Are Bright Microscopy
Principle of Bright Microscope
Advantage
Disadvantage
Application
Conclusion
Reference
LIGHT MICROSCOPY by SIVASANGARI SHANMUGAM
The optical microscope, The functions of a light microscope is based on its ability to focus a beam of light through, which is very small and transparent, to produce an image.
This presentation include information about electron microscope & types of electron microscope i.e. SEM (Scanning electron microscope) & TEM (Transmission electron microscope).
An electron microscope is a microscope that uses a beam of scattered electrons as a source of illumination. It is used to get information about structure, topology, morphology & composition of materials. It has many advantages. Basically there are 4 types of electron microscope but here we will discuss only 2 types.
Transmission electron microscopy is a microscopy technique in which a beam of electrons is transmitted through an ultra-thin specimen, interacting with the specimen as it passes through it. Its resolution & magnification is about 10,000,000x. There are 5 types of transmission electron microscope i.e. BFTEM (Bright field transmision electron microscope), DFTEM (Dark field transmission electron microscope), HRTEM (High resolution transmission electron microscope), EFTEM (Energy filtered transmission electron microscope), ED (Electron diffraction). there are 4 techniques of TEM i.e. negative staining, shadow casting, Freeze fracture replication, freeze etching. It has many applications e.g, for the study of Cancer research, virology, chemical industry, electronic structure etc.
A scanning electron microscope is a type of electron microscope that produces images of a sample by scanning it with a focused beam of electrons. Types of signals produce by SEM include secondary electrons, back scattered electrons, X-rays, light rays. There are many advantages of SEM e.g, Btter resolution, fast imaging easy to operate, work with low voltage etc.
DARK FIELD MICROSCOPY by SIVASANGARI SHANMUGAM
Dark-field microscopy is ideally used to illuminate unstained samples causing them to appear brightly lit against a dark background.
This type of microscope contains a special condenser that scatters light and causes it to reflect off the specimen at an angle
Electron microscope, principle and applicationKAUSHAL SAHU
Introduction
History
Resolution &Magnification of
Electron microscope
Types of electron microscope
1) Transmission electron microscope (TEM)
- Structural parts of TEM
- Principle & Working of TEM
- Sample preparation for TEM
- Advantages & disadvantages of TEM
Scanning electron microscope (SEM)
- Structural parts of SEM
- Principle & Working of SEM
- Sample preparation for SEM
- Advantages & disadvantages of SEM
3) Scanning transmission electron microscope (STEM)
Applications of electron microscope
Conclusion
References
LIGHT MICROSCOPY by SIVASANGARI SHANMUGAM
The optical microscope, The functions of a light microscope is based on its ability to focus a beam of light through, which is very small and transparent, to produce an image.
This presentation include information about electron microscope & types of electron microscope i.e. SEM (Scanning electron microscope) & TEM (Transmission electron microscope).
An electron microscope is a microscope that uses a beam of scattered electrons as a source of illumination. It is used to get information about structure, topology, morphology & composition of materials. It has many advantages. Basically there are 4 types of electron microscope but here we will discuss only 2 types.
Transmission electron microscopy is a microscopy technique in which a beam of electrons is transmitted through an ultra-thin specimen, interacting with the specimen as it passes through it. Its resolution & magnification is about 10,000,000x. There are 5 types of transmission electron microscope i.e. BFTEM (Bright field transmision electron microscope), DFTEM (Dark field transmission electron microscope), HRTEM (High resolution transmission electron microscope), EFTEM (Energy filtered transmission electron microscope), ED (Electron diffraction). there are 4 techniques of TEM i.e. negative staining, shadow casting, Freeze fracture replication, freeze etching. It has many applications e.g, for the study of Cancer research, virology, chemical industry, electronic structure etc.
A scanning electron microscope is a type of electron microscope that produces images of a sample by scanning it with a focused beam of electrons. Types of signals produce by SEM include secondary electrons, back scattered electrons, X-rays, light rays. There are many advantages of SEM e.g, Btter resolution, fast imaging easy to operate, work with low voltage etc.
DARK FIELD MICROSCOPY by SIVASANGARI SHANMUGAM
Dark-field microscopy is ideally used to illuminate unstained samples causing them to appear brightly lit against a dark background.
This type of microscope contains a special condenser that scatters light and causes it to reflect off the specimen at an angle
Electron microscope, principle and applicationKAUSHAL SAHU
Introduction
History
Resolution &Magnification of
Electron microscope
Types of electron microscope
1) Transmission electron microscope (TEM)
- Structural parts of TEM
- Principle & Working of TEM
- Sample preparation for TEM
- Advantages & disadvantages of TEM
Scanning electron microscope (SEM)
- Structural parts of SEM
- Principle & Working of SEM
- Sample preparation for SEM
- Advantages & disadvantages of SEM
3) Scanning transmission electron microscope (STEM)
Applications of electron microscope
Conclusion
References
Microscopy - Magnification, Resolving power, Principles, Types and ApplicationsNethravathi Siri
Magnification, Resolving power, Principles and Applications of Simple, Compound, Stereozoom, Phase contrast, Fluorescent and Electron microscopes (TEM & SEM).
Microscopy is the technical field that uses microscopes to observe samples which are not in the resolution range of the normal-unaided eye.
Microscopy is the technique of using microscopes to observe and analyze objects that are too small to be seen by the naked eye. Microscopes are instruments that magnify and resolve the details of objects, allowing scientists and researchers to study the structure, composition, and behavior of materials and specimens at a microscopic level
Microscopy is the technique of using microscopes to observe and analyze objects that are too small to be seen by the naked eye. Microscopes are instruments that magnify and resolve the details of objects, allowing scientists and researchers to study the structure, composition, and behavior of materials and specimens at a microscopic level
The microscope has evolved a lot from the time of Leeuwenhoek. This presentation gives a brief overview about the types of microscope their principle of function and application.
microscope (1).pdf this is a project for for botany majorarpitakhairwar123
Name - Arpita khairwar
Class - B.sc 1st year
Subject - Botany Major
College - Govt. Jayashankar Trivedi College Balaghat
Guided by - Dr. Pratima bisen
Submitted by - Arpita khairwar
While this ppt given by Dr pratima mam this ppt is a educational institution. My ppt is about microscope
This presentation is about the introduction of microscopy, its history, parts of a microscope and different types of microscopes along with a brief discussion of their working principles.
This presentation is all about Microscope .... The miracle instrument which revolutionised the study of microbiology and Biological science . Be it Cell studies, molecule studies, pathogen studies, virology etc etc ..... All has become possible for this instrument. let us understand the functioning , applications of this instrument .
Types of Light Microscopes used in Histological Studies.pptxssuserab552f
Light microscopes relies on glass lenses and visible light to magnify tissue samples. It was
invented in XVII century, and has been improved over the years, resulting in the powerful
modern light microscopes. As individual cellular structures are too small to be seen by the
human eye, microscopy techniques have played a key role in the development of
histological techniques.
The term isolation refers to the separation of a strain from a natural, mixed population of living microbes, as present in the environment. It becomes necessary to maintain the viability and purity of the microorganism by keeping the pure culture free from contamination.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
The increased availability of biomedical data, particularly in the public domain, offers the opportunity to better understand human health and to develop effective therapeutics for a wide range of unmet medical needs. However, data scientists remain stymied by the fact that data remain hard to find and to productively reuse because data and their metadata i) are wholly inaccessible, ii) are in non-standard or incompatible representations, iii) do not conform to community standards, and iv) have unclear or highly restricted terms and conditions that preclude legitimate reuse. These limitations require a rethink on data can be made machine and AI-ready - the key motivation behind the FAIR Guiding Principles. Concurrently, while recent efforts have explored the use of deep learning to fuse disparate data into predictive models for a wide range of biomedical applications, these models often fail even when the correct answer is already known, and fail to explain individual predictions in terms that data scientists can appreciate. These limitations suggest that new methods to produce practical artificial intelligence are still needed.
In this talk, I will discuss our work in (1) building an integrative knowledge infrastructure to prepare FAIR and "AI-ready" data and services along with (2) neurosymbolic AI methods to improve the quality of predictions and to generate plausible explanations. Attention is given to standards, platforms, and methods to wrangle knowledge into simple, but effective semantic and latent representations, and to make these available into standards-compliant and discoverable interfaces that can be used in model building, validation, and explanation. Our work, and those of others in the field, creates a baseline for building trustworthy and easy to deploy AI models in biomedicine.
Bio
Dr. Michel Dumontier is the Distinguished Professor of Data Science at Maastricht University, founder and executive director of the Institute of Data Science, and co-founder of the FAIR (Findable, Accessible, Interoperable and Reusable) data principles. His research explores socio-technological approaches for responsible discovery science, which includes collaborative multi-modal knowledge graphs, privacy-preserving distributed data mining, and AI methods for drug discovery and personalized medicine. His work is supported through the Dutch National Research Agenda, the Netherlands Organisation for Scientific Research, Horizon Europe, the European Open Science Cloud, the US National Institutes of Health, and a Marie-Curie Innovative Training Network. He is the editor-in-chief for the journal Data Science and is internationally recognized for his contributions in bioinformatics, biomedical informatics, and semantic technologies including ontologies and linked data.
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
Slide 1: Title Slide
Extrachromosomal Inheritance
Slide 2: Introduction to Extrachromosomal Inheritance
Definition: Extrachromosomal inheritance refers to the transmission of genetic material that is not found within the nucleus.
Key Components: Involves genes located in mitochondria, chloroplasts, and plasmids.
Slide 3: Mitochondrial Inheritance
Mitochondria: Organelles responsible for energy production.
Mitochondrial DNA (mtDNA): Circular DNA molecule found in mitochondria.
Inheritance Pattern: Maternally inherited, meaning it is passed from mothers to all their offspring.
Diseases: Examples include Leber’s hereditary optic neuropathy (LHON) and mitochondrial myopathy.
Slide 4: Chloroplast Inheritance
Chloroplasts: Organelles responsible for photosynthesis in plants.
Chloroplast DNA (cpDNA): Circular DNA molecule found in chloroplasts.
Inheritance Pattern: Often maternally inherited in most plants, but can vary in some species.
Examples: Variegation in plants, where leaf color patterns are determined by chloroplast DNA.
Slide 5: Plasmid Inheritance
Plasmids: Small, circular DNA molecules found in bacteria and some eukaryotes.
Features: Can carry antibiotic resistance genes and can be transferred between cells through processes like conjugation.
Significance: Important in biotechnology for gene cloning and genetic engineering.
Slide 6: Mechanisms of Extrachromosomal Inheritance
Non-Mendelian Patterns: Do not follow Mendel’s laws of inheritance.
Cytoplasmic Segregation: During cell division, organelles like mitochondria and chloroplasts are randomly distributed to daughter cells.
Heteroplasmy: Presence of more than one type of organellar genome within a cell, leading to variation in expression.
Slide 7: Examples of Extrachromosomal Inheritance
Four O’clock Plant (Mirabilis jalapa): Shows variegated leaves due to different cpDNA in leaf cells.
Petite Mutants in Yeast: Result from mutations in mitochondrial DNA affecting respiration.
Slide 8: Importance of Extrachromosomal Inheritance
Evolution: Provides insight into the evolution of eukaryotic cells.
Medicine: Understanding mitochondrial inheritance helps in diagnosing and treating mitochondrial diseases.
Agriculture: Chloroplast inheritance can be used in plant breeding and genetic modification.
Slide 9: Recent Research and Advances
Gene Editing: Techniques like CRISPR-Cas9 are being used to edit mitochondrial and chloroplast DNA.
Therapies: Development of mitochondrial replacement therapy (MRT) for preventing mitochondrial diseases.
Slide 10: Conclusion
Summary: Extrachromosomal inheritance involves the transmission of genetic material outside the nucleus and plays a crucial role in genetics, medicine, and biotechnology.
Future Directions: Continued research and technological advancements hold promise for new treatments and applications.
Slide 11: Questions and Discussion
Invite Audience: Open the floor for any questions or further discussion on the topic.
2. MICROSCOPY
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• INTRODUCTION
• HISTORY
• PRINCIPLE
• FACTORS OF MICROSCOPY
• TYPES OF MICROSCOPY
LIGHT MICROSCOPY
A. BRIGHT-FIELD MICROSCOPY
B. DARK-FIELD MICROSCOPY
C. FLUORESCENCE MICROSCOPY
D. PHASE-CONTRAST MICROSCOPY
ELECTRON MICROSCOPY
A. TRANSMISSION MICROSCOPY
B. SCANNING MICROSCOPY
• APPLICATIONS
• CONCLUSION
• REFERENCE
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• Microscopy is the science which deals “with the use of microscope
instrument that magnifies the size of the object & interpretation of
their magnified images”.
• Microscope is the technology of making very small things visible to
the human eye. Therefore, microscope is a major tool of the
microbiologist & biotechnologist.
• At the magnification of 1,000x most of the microorganisms, e.g.:-
Fungi, Bacteria, Mycoplasma, algae, & protozoa can be viewed &
this can be achieved with a light microscope.
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4. MICROSCOPY
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• Antony Van Leeuwenhoek (1676)
discovered the microbial world through
the use of a single microscope containing
a single biconvex lens of shorter focal
length.
• Robert Hooke point the ‘cell’ built
microscopes with two lenses called
compound microscope.
• The Dutch spectacle-maker, Zaccharias
Jansen, is also credited with the
development of compound light
microscope.
• In 1830, many improvements were made
by Joseph Lister which resulted in the
development of many types of
microscopes that are being used now a
days.
Zacharias Jansen
1588-1631
Anthony van Leeuwenhoek
1632-1723
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• Microscopy is the most important instrument of any biological
laboratory. Used to magnifying the size of the object.
• By the help of resolution provided by this instrument very small
organisms or substances are magnified and made visible through
the naked eyes.
• Thus, microscopy is the major tool for microbiologist &
biotechnologist.
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1. Magnification
• The primary of a microscope is magnification that is the power of
enlargement of image of an object.
• Its is the ratio of size of image to the size of object.
Image distance
Magnification = --------------------
object distance
• Three types of objective lens of different magnification are used:
a. Low power- 10x
b. High power- 45x
c. Oil emulsion- 100x
2. Contrast
• It refers to difference in light intensity in order to the perceived to the
microscope, an object must possess a certain degree of contrast with its
surrounding medium.
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3. Resolution
• The resolving power of a microscope is the to distinguish two adjacent
point as separate and distinct rather then a blurred image.
• The greater the resolving power of the microscope the more detailed
can be see in the specimen.
• Resolving power of microscope is determined by three factors:
i. Size of the objective lens.
ii. Wavelength of light passing through the specimen.
iii. The refractive index of the material between the objective lens
and the specimen.
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1. LIGHT MICROSCOPY
A. Bright-Field light microscopy
B. Dark-Field light microscopy
C. Fluorescence light microscopy
D. Phase-contrast light microscopy
2. ELECTRON MICROSCOPY
A. Transmission electron microscopy
B. Scanning electron microscopy
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A. BRIGHT FIELD MICROSCOPY
PRINCIPLE
• Bright field use the visible light as the source of illumination.
• Light microscope with a single lens are called simple microscope.
• Compound microscope with the two lens system the objective lens place near specimen
and the occular lens or eye piece located next to the eye.
Basic part of Bright-field microscope
A broad base
Curved arm
Adjustable light source or mirror
Fine and core adjustable nose
Body tube
Stage or platform
Diaphragm
Condenser
Ocular lens
Eye piece
There are three types of eye piece
a) Huggensian eye piece
b) Hyper –place eye piece
c) Compensating eye piece
Fig-2 bright-field microscopy
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11. MICROSCOPY
The object to be viewed with the compound light microscope is normally
placed on a glass slide and illuminated with a light source. The specimen is
focused by moving the ocular lens & objective lens together relative to the
specimen until the image is clear.
When the specimen has been focused the objective lens magnifies the
specimen & produces earlier image.
The real image is projected to the microscope to the ocular lens which
magnifies the real image and produces an image seen by the observer and
called as virtual image.
The resolving power of the lens system is important in microscopy because it
indicates the size of the smallest object that can be seen clearly.
Resolving power varies for each objective lens and depends on-
1. Wavelength of light used in a optical system,
2. Numerical aperture.
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Conti..A. BRIGHT FIELD MICROSCOPY
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A. BRIGHT FIELD MICROSCOPY
NUMERICALAPPARATURE
It is the light gathering capacity of the objective lens.
It is a measurement of the angle of maximum cone of light that enter the objective.
NA = n Sin𝜽
where, N = refractive index of medium
sin 𝜃 = one and half angle created by light passing through condenser and specimen
& transmitted to object.
o In case of dry air
NA= 1
o In case of oil medium
NA= 1.33
The greater the NA the greater is the resolving power.
LIMIT OF RESOLUTION
The smallest distance by which two points can be separated & distinguished as two separate
objects.
Resolution = 𝝀
-------
2NA
Higher resolution obtained with shorter wavelength & maximum NA.
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B. DARK-FIELDMICROSCOPY
In dark field microscopy the background
remains dark & only the objects
illuminated. It is opposite to that of
Bright-field microscopy in which
specimen appears darker against light
background.
Dark-field microscopy operates on the
principle of scattering which means a ray
of light changes direction or scatters
when it strikes & bounces off a small
object.
In this a special kind of condenser with an
opaque disc or “dark field stop” is
provided. Thus, the light rays reach the
object in the form of the hollow cone.
Fig-3 dark-field microscopy
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B. DARK-FIELD MICROSCOPY
The disc block the light that could enter the objective directly & redirects the light
beam so that it goes to the specimen but misses the objective lens.
The only light rays that enters objective lens & reach the eyes are those that have been
scattered by striking the specimen.
In tis way specimens appears bright against a dark microscopic field.
APPLICATION
Helps in examining movement of motile cells, live microorganisms that are
either invisible in the ordinary light microscope
In diagnostic of microorganisms.
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C. FLUORESCENCE MICROSCOPY
The fluorescence microscopy differs from the Bright-field microscopy as
it uses a mercury vapour arc lamp or a halogen instead of the
incandescent lamp.
PRINCIPLE
The principle of fluorescence microscopy is a diagnostic technique
called the fluorescent-antibody. Antibodies are natural defense
molecule that are produced by humans and many animals in
reaction to a foreign substance or antigen.
TYPES OF FLUORESCENCE
1. Auto fluorescence - collagen fibers (blue green light).
1. Secondary fluorescence - commonly used dye Congo red, eosin.
1. Induced fluorescence – some substances on treatment with some
chemical shows fluorescence.
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COMPONENTS &
INSTRUMENTATION
1. LIGHT SOURCE – Mercury arc lamp,
ultraviolet light of shorter
wavelength.
2. HEAT FILTER – Removes infrared
rays.
3. EXITER FILTER – Allow only
required wavelength to pass through
and block others.
4. DICHRONIC MIRROR – Divide &
divert the beam, reflect light of
certain wavelength but transmit other.
5. CONDENSOR – Dark field condenser
is provide black background against
which the fluorescent object glow.
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Fig-3 fluorescence microscopy
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6. BARRIER FILTER – Remove exited wavelength
APPLICATION
Detection various material e.g. protein can be detected by staining
with rod amine.
Banding pattern of chromosome.
Fluorescent antibody technique or Immuno-fluorescent.
Conti…
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18. MICROSCOPY
D. PHASE CONTRAST MICROSCOPY
• Phase-contrast microscopy is based on the principle that rays of light
move at different speed through materials of different refractive index.
• The phase contrast microscope amplifies the slight difference in
refractive index of the cell and that of its aqueous environment and
converts it to a difference in contrast.
• The phase-contrast microscope consists of special condensers and
objectives that enable one to increase the contrast between the
transparent components in the cell by exploiting differences in their
densities.
PRINCIPLE
Phase contrast microscopy is used for studying living cell to
convert the invisible small phase changes caused by cell
component into visible intensity changes.
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D. PHASE-CONTRAST MICROSOPY
When light rays passed through the living
cells they undergo phase changes due to
different refractive indices & thickness of
cell organelles
When light rays are passed through cell
organelle, they are transmitted at a
velocity inversely proportional to
refractive index of the cell organelle. Cell
organelle are of different refractive
indices.
The light rays emerging would show
variable phase changes.
This invisible phase changes re converted
into visible intensity changes by phase-
contrast microscopy.
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Fig4-phase-contrast microscopy
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D. PHASE-CONTRAST MICROSCOPY
The more the refractive index & thickness the more will be the
change in the phase.
The cells and their component show phase changes value of
phase change is one- fourth of light. This phase change is
imperceptible to the human eye.
The principle behind the phase-contrast microscopy is to convert
the imperceptible phase change.
CONSTRUCTION
It is specially designed light microscope with annular
diaphragm and annular phase plate fitted into it.
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21. MICROSCOPY
D. PHASE-CONTRAST MICROSCOPY
They unable to increase the contrast between the transparent into
the ell by exploiting differences in their density.
Annular diaphragm or annular stock is a disc with a thin
transparent wing at a lower focal plane of the condenser.
It consist of a circular disc with a circular grove through where
light rays are allowed to pass.
WORKING
Light rays pass through the annular group of the annular
diaphragm.
This rays are focused on the on the object.
From the object two types of rays immersed out. one is refracted
rays which under goes a phase change. And other is central rays
which does not under goes any phase –change.
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D. PHASE-CONTRAST MICROSCOPY
The refracted rays are band due to refraction in density and refractive
index with in the specimen and get refracted by about one-fourth
wavelength.
IMAGE FORMATION
Depending on the type of phase plate used image formation takes
place is of two types-
Image formation by positive contrast
• Formed by subtractive super position of central & diffracted rays.
• The object appears dark against the light background also called as
dark positive contrast.
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D. PHASE-CONTRAST MICROSCOPY
Image formation by negative contrast
• Formed by the super position of the central & diffracted rays.
• The specimen appear bright against dark background that may
called as bright phase contrast.
• In this negative plate is used.
ADVANTAGES
We can see living cells and there is no need for staining.
Highly transparent material can be seen.
Intracellular component can be observed, e.g. endospores.
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24. MICROSCOPY
ELECRTON MICROSCOPY
The electron microscope is an optical instrument which utilizes electrons as
a source of illumination for observing objects at a great magnification.
It can achieve a very high power of resolution because it uses electrons of
much shorter wavelength.
Use electromagnetic lenses to focus a beam of electrons onto a
specimen.
This required 10,000x plus magnification.
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26. MICROSCOPY
TRANSMISSION ELECTRON MICROSCOPY
PRINCIPLE
Higher magnification and higher resolution.
The wavelength of electron one lakh time shorter then that of light
rays.
Image is produced on fluorescence on photographic plate.
Instead of mounting the specimen on a glass slide it held on a
proper way.
Instead of using light to that absorb light, to increase contrast
tungsten which absorbs electron are used.
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CONSTRUCTION
1. ELECTRON GUN
It consist of a hot tungsten filament. It is the source
of electrons forming the beam.
2. ELECTROMAGNETIC LENS
The electromagnetic lens corresponds to the
condenser, objective lens and ocular lens.
3. MICROSCOPE COLUMN
It consist of an evacuated metal tube.
4. FLUORESCENCE SCREEN
Since electron are harmful to our eyes magnified
image observed from fluorescence screen.
5. VACUUM PUMP
Electron are reflected by collision air molecule.
6. TRANSFORMERS
It provide high voltage from 220v to 50-100 kV.
7. WATER COOLING SYSTEM
Required to prevent over heating of different part
of microscope.
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TRANSMISSION ELECTRON MICROSCOPY
WORKING
The electron gun generate electron beam , thin tungsten filament.
Electrons are in the form of collimated beam passes to the condenser coil & fall
on the object.
They get scattered & transmitted to the object & pass through the objective coil
which magnifies the image of the object.
The projector coil further magnify the image & thus final image is formed on the
fluorescence screen.
Dense region in the specimen scatter is more and therefore appear darken in the
image where as in contrast, electron transparent regions are brighter.
MAGNIFICATION
1,60,000x - 10,00,000x
APPLICATION
It provides sufficient magnification and resolution to view viruses and the
internal structures of all organisms.
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SCANNING ELECTRON MICROSCOPY
This microscopes gives a typical three-
dimensional appearance.
The illuminating system of SEM is similar
to transmission electron microscopy.
PRINCIPLE
It differs from TEM introducing an image
from electron emitted by object surface
rather than from transmitted electron
microscopy.
It consist of an electron gun which
produces a finely focus beam of electron
called the primary electron beam.
This electron passes through
electromagnetic lens & rapidly scan the
surface of specimen.
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30. MICROSCOPY
B. SCANNING ELECTRON MICROSCOPY
When the beam of electron strikes the specimen secondary electrons are released
and transmitted to the electron collector.
Secondary electrons are collected and use to generate a signal that produces an
image on cathode screen.
It has a resolution of about 50Ȧ.
scanned lamp on the screen
magnification can be given by = ------------------------------------------
scanned lamp on the specimen surface
APPLICATION
The scanning electron microscope has a wide scope in biology for
the study of small specimens, surface scanning of the cells, tissues
and membrane.
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MICROSCOPY
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In medical microbiology for detecting pathogenic bacteria.
For the detection of various types of products of microorganisms.
It helps in examining the movement of motile cells.
Shows greater differentiation of internal structure and clearly shows
the pellicle.
By the use of electron microscopy structures smaller than 0.2
micrometer can be resolved.
By the use of fluorescence microscopy rapidly detection and
identification of microbes can be done.
32. MICROSCOPY
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The microorganisms are so small that their study requires appropriate
methods for observing and culturing them.
Microscopy is the technology of making very small things visible to
the human eye.
Therefore, microscope is a major tool of the microbiologist &
biotechnologist
Historically, it was the microscope that first revealed the secrets of
microbial structure, even today, it remains a powerful tool in
microbiological studies.
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BOOK AUTHORS YEAR EDITION
THE BIOLOGY OF
MICROORGANISM
S
2010 10th
A TEXT BOOK OF
BIOTECNOLOGY
R.C DUBEY 2008
A TEXT BOOK OF
MICROBIOLOGY
R.C DUBEY &
MAHESHWARI
CLASS NOTES
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