BIOLOGICAL
IMAGING AND
SUBCELLULAR
ORGANISMS
MVDr. Jana Hložková, Ph.D
✓ The main method for obtaining new data is observation, for which it is
possible to use all our senses (primarily sight), the effectiveness can
be multiplied in combination with devices (magnifying glass,
microscope).
✓ Another method of obtaining new data is an experiment,
a research procedure in which the consequences caused by changing
one of the factors affecting the object are determined.
MAGNIFYING DEVICE
• Magnifying glass 4x
• Light microscope 1000x
Improvement - Immersion, Dyeing, Phase
Contrast (Highlighting Contrast Using
Different Sample Rate)
• Electron microscope 1000 000x
Today - computer, analysis, measurement,
calculations, three-dimensional models
ANGULAR RESOLUTION
• The term resolution or minimum resolvable distance is the minimum distance
between distinguishable objects in an image,
• Eye 0.2mm
• Light microscope 0.2 µm
• Electron microscope 0.2 nm
Resolution range of the normal eye 0,2mm
Light microscope 0,2 µm
Electron microscope 0,2 nm
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)
HISTORY OF MICROSCOPY
• Anthoni van Leeuwenhoek.
best known for his pioneering work in the field of
microscopy and for his contributions toward the
establishment of microbiology as a scientific
discipline.
• https://www.youtube.com/watch?v=XgW1HiV9SJs
• Zacharias Janssen (1595)
magnification 9x
• Robert Hook (1665)
https://www.youtube.com/watch?v=TJyOQmdwHhE
Optical part - objectives, eyepieces.
Lighting part - light source, condenser,
diaphragma, filters, mirror (in current
microscopes built into the base).
Mechanical part - base, arm, tube, revolver
lens changer, stage with clamps and crossconductor,
course adjustment knob and fine
adjustment knob
Magnification
Ocular 10× 10× 10× 10×
Objective 4× 10× 40× 100×
Total
Magnification
40× 100× 400× 1000×
Total magnification of the microscope is equal to the
product of lens and eyepiece magnification:
Magnification in the drawings (pictures) can be
written in two ways: as total magnifications (40 ×, 100
×, 400 ×, × 1000), or in a fraction of a zoom eyepiece
lens used to enlarge (10/4, 10/10, 10/40, 10 / 100).
LIGHT MICROSCOPES
• Brightfield
• Darkfield
• Phase contrast
• Nomarski differential contrast (DIC)
• Fluorence
• Confocal
Monocul microscope Binocular microscope
Inverse microscope
➢ observations of cultures of
living cells and microorganisms
at the bottom of culture dishes
or suspended in live soils
➢ examination of tissue cultures,
water quality, sediment
analyses, observation of
crystalline structures
Observation in phase contrast
➢ observation of uncolored objects, and
structures in living cells - nucleus,
nucleus, chromosomes and vacuoles
➢ can significantly increase contrast in
poorly stained and poorly
distinguishable structures of
histological specimens
https://www.youtube.com/watch?v=P2teE17zT4I
Observation in dark field
➢ Microscopes require a special lighting section
(a paraboloid condensor) that passes only
obliquely passing rays
➢ The subject is illuminated only from the
sides and the field of view is dark. This makes
possible to observe objects much smaller
than those that can be observed in the
passing light
➢ for example, to diagnose bacteria
(leptospires, Treponema pallidum) that can
not be seen in the passing light.
Observation in a polarized field
➢ In the eyepiece or just below it, is the
polarizing filter - the analyzer and
under the condensor is the polarizer.
➢ inserting object contains an optically
active substance
➢ The object is placed between the two
polarization systems
Vitamin C
Nomarski differential
contrast (DIC)
➢ Efforts to eliminate "halo" effect around the object
details (Georges Nomarski 1960)
➢ Can differenciate edges of speciments
Ancylostoma duodenale
Chinese Liver Fluke (Clonorchis sinensis) https://www.youtube.com/watch?v=TKTGgAQ2VEs
➢ The sample is exposed to wavelength radiation
that can cause fluorescence.
➢ Areas of the sample in which the fluorescence
did not occur remain dark
➢ Many natural substances, such as chlorophyll or
some vitamins, shine under the influence of UV
rays.
➢ Fluorochromes are fluorescent compounds that
are used to visualize different cellular
components, such as DNA, proteins (proteins).
e.g. in the diagnosis of rabies.
Fluorescence microscopy
Confocal microscopy
➢From the second half of the 1980s
➢Use the fluorescence
➢The source of light is a laser
➢ The microscope creates "optical cuts".
➢ By computerized section processing, threedimensional
models can be created
➢ „Life cell imaging" allows you to monitor
functions of intracellular structures in vivo
ELECTRON MICROSCOPY
• the desire and the need to observe ever-smaller objects
• viruses – in nm of size
• the resolution of the optical microscope is limited by the visible
wavelength (400-700 nm)
• technological advancement - the discovery of electrons and their
properties
• 19th - 20th century - J.J. Thompson, Luis de Broglie
• 1931 Max Knoll and Ernst Russia constructed a transmission electron
microscope - 1986 Nobel Prize
• 1965 scanning electron microscope was constructed
ARMIN DELONG
• Brno professor
• LVEM5 (Low Voltage Transmission Electron
Microscope) – optical resolution 2
nanometres, 100 more resolution than a highquality
optical microscope.
• His microscope could allow detection of
viruses according to their shape
• Rapid diagnosis of the disease (quarantine
shortening)
• No sample modification is needed - use in
biology
• In 2005, the National Czech Republic Award Czech
Head was awarded
ELECTRON MICROSCOPES
TRANSMISSION (TEM) AND RASTER (REM)
➢1 000 000 magnification
➢ resolution of 2-20 nm (viruses)
➢ beam of accelerated electrons as a source of illumination
➢passage through electromagnetic lenses
➢the sample must not contain water
➢ultra-thin cuts - up to 100nm
THE PRINCIPLE OF AN ELECTRON MICROSCOPE
• an electron gun
• a tube through which the electron
passes from the cathode
(tungsten fiber) to the anode
• Vacuum - operating temperature
2 500 ° C
• electromagnetic lenses
DIFERENCE
BETWEEN
LIGHT AND
ELECTRONIC
MICROSCOPY
How a transmission electron microscope (TEM) works
A transmission electron microscope fires a beam of electrons through a specimen to
produce a magnified image of an object.
✓ A high-voltage electricity supply powers the cathode.
✓ The cathode is a heated filament, a bit like the electron gun in an old-fashioned
cathode-ray tube (CRT) TV. It generates a beam of electrons that works in an
analogous way to the beam of light in an optical microscope.
✓ An electromagnetic coil (the first lens) concentrates the electrons into a more
powerful beam.
✓ Another electromagnetic coil (the second lens) focuses the beam onto a certain part
of the specimen.
✓ The specimen sits on a copper grid in the middle of the main microscope tube.The
beam passes through the specimen and "picks up" an image of it.
✓ The projector lens (the third lens) magnifies the image.
✓ The image becomes visible when the electron beam hits a fluorescent screen at the
base of the machine.This is analogous to the phosphor screen at the front of an oldfashioned
TV .
✓ The image can be viewed directly (through a viewing portal), through binoculars at
the side, or on a TV monitor attached to an image intensifier (which makes weak
images easier to see).
How a scanning electron microscope (SEM) works
A scanning electron microscope scans a beam of electrons over a specimen to
produce a magnified image of an object.That's completely different from a
TEM, where the beam of electrons goes right through the specimen.
✓ Electrons are fired into the machine.
✓ The main part of the machine (where the object is scanned) is contained
within a sealed vacuum chamber because precise electron beams can't
travel effectively through air.
✓ A positively charged electrode (anode) attracts the electrons and
accelerates them into an energetic beam.
✓ An electromagnetic coil brings the electron beam to a very precise focus,
much like a lens.
✓ Another coil, lower down, steers the electron beam from side to side.
✓ The beam systematically scans across the object being viewed.
✓ Electrons from the beam hit the surface of the object and bounce off it.
✓ A detector registers these scattered electrons and turns them into a picture.
✓ A hugely magnified image of the object is displayed on a TV screen.
TEM
REM
 scanning on the surface
 creates a 3D image
• https://www.youtube.com/watch?v=cQuX8tfwlYA
A FOLDSCOPE
• is an optical microscope that can be assembled from simple components,
including a sheet of paper and a lens.
• It was created by Manu Prakash and designed to cost less than one USD to
build. It is a part of the "frugal science" movement which aims to make cheap
and easy tools available for scientific use in the developing world
HELA CELLS
• https://www.youtube.com/watch?v=22lGbAVWhr
o
• What are HeLa cells?
• HeLa cell | Cancer Research, Immortal Cells & Tissue
Culture ...
• The designation HeLa is derived from the name of the
patient, Henrietta Lacks.
• HeLa cells were the first human cell line to be
established and have been widely used in laboratory
studies, especially in research on viruses, cancer, and
human genetics
RESEARCH OF THE NEW THROMBOLYTIC
Trombus
světelný mikroskop, HE
Trombus
Elektronový mikroskop
Trombus
Elektronový mikroskop
COMPUTED TOMOGRAPHY, CT
Novobilsky, Scheer, Hložková
Mi CT Skiaskopie (RTG)
X- RAY
MCAO Angio X 15
Scheer, Hložková a kol.
NON-CELLULAR FORMS OF LIFE
NON-CELLULAR FORMS OF LIFE
• Non-cellular life is life that exists without a cellular structure.
• This term presumes the phylogenetic scientific classification
of viruses as lifeforms, which is a controversial issue
• Hypothesized artificial life may or may not be considered
living.
LIVING ORGANISM
• The definition of life is controversial.
The current definition is that organisms:
• maintain homeostasis,
• are composed of cells,
• undergo metabolism,
• can grow,
• adapt to their environment,
• respond to stimuli,
• and reproduce.
Life on Earth:
Non-cellular life Viruses
Viroids
Satelites
Prions
Cellular life Domain Bacteria
Domain Archaea
Domain Eukarya
VIRUS
• a small infectious agent that replicates only
inside the living cells of other organisms
(nm)
• Viruses can infect all types of life forms
• Dmitri Ivanovsky in 1892 described a nonbacterial
pathogen infecting tobacco plants
• about 5,000 virus species have been
described in detail, although there are
millions of types of viruses
• are found in almost every ecosystem on Earth
• The biggest recently discovered virus is the
Mimivirus (the bacterium mimics the virus) found
in the cells of the amoebas, measuring
400 nm
• In 2013 and 2014, even more pandoraviruses
and pithoviruses were discovered - with a
capsid of 1000 and 1500 nm.
• This means that while the smallest virions
resemble their ribosome size, giant viruses are
larger than the smallest bacteria.
TAXONOMY
• Size and morphology – capsid symmetry
• Presence (absence) of the surface coat: enveloped or naked viruses
• Type and molecular structure of genomic NK:
• Type NA: rna-viruses, dna-viruses
• NA: single-stranded ssRNA, dsRNA – double-stranded, ssDNA, dsDNA, linear
(mostly) or circular NK
• NA polarity: (+) RNA (plus RNA, positive RNA, same polarity as mRNA), (-)
RNA (+ RNA), (+) DNA, (-) DNA.
➢ Capside symmetry:
Host Type: animal viruses
human viruses
plant viruses,
fungi viruses,
algae and protozoa,
bacterial viruses (bacteriophages, phages)
phototrophic bacteria (cyanophages)
Affinity to tissues: respiratory, enteral, sexually transmitted, hepatic
VIRION
• Virion the individual particles capable of infecting host cells and multiply
therein.
Capsid: The protein shell, or coat, that encloses
the nucleic acid genome.
Capsomeres: Morphologic units seen in the
electron microscope on the surface of icosahedral
virus particles.
Envelope: A lipid-containing membrane that
surrounds some virus particles. It is acquired
during viral maturation by a budding process
through a cellular membrane.
Nucleocapsid: The protein–nucleic acid complex
representing the packaged form of the viral
genome.
human viruses
AIDS.
Common cold.
Ebola.
Genital herpes.
Influenza.
Measles.
Chickenpox and shingles.
Coronavirus disease 2019
(COVID-19)
ANIMAL VIRAL DISEASE:
• African horse sickness. ...
• African swine fever (ASF) ...
• Bluetongue. ...
• BRSV. ...
• Coronavirus. ...
• Crimean Congo haemorrhagic fever. ...
• Equine infectious anaemia. ...
• Equine viral arteritis.
BACTERIOPHAGES
➢Bacteriophages are viruses that attack bacteria.
➢ The name means "bacteria eaters" and is commonly
shortened to just "phage".
➢Phage particles comprise a "head" that contains DNA
packaged within a protein coat, and a hollow "tail" by
which they attach to the outside membrane of bacteria.
➢The phage DNA is injected into the bacterium, where it
uses the cell's replication machinery to reproduce itself.
➢Production of new phage particles causes the host cell to
rupture ("lyse") and release the phage, which goes on to
infect other bacteria.
REPRODUCTION OF VIRUSES
LYTIC CYCLE
• Attachment: Virus attaches to the host cell.
• Entry: Genetic material is injected into the host
cell.
• Replication: The virus takes over the cell's
metabolism, causing the creation of new proteins
and nucleic acids by the host cell's organelles.
• Assembly: Proteins and nucleic acids are
assembled into new viruses.
• Release: Virus enzymes cause the cell to burst,
and viruses are released from the host cell.These
new viruses can infect other cells.
LYSOGENIC INFECTION
• Attachment: The virus attaches to the host cell.
• Entry: Genetic material is injected into the host cell.
• Integration:Viral DNA integrates into the host cell's genome.
• Replication (lysogenic cycle):When the host cell replicates, viral DNA
is copied along with host cell DNA. Each new daughter cell is infected
with the virus.
• Induction:When the infected cells are exposed to certain
environmental conditions, viral DNA is activated and enters the lytic
cycle.
• Replication (lytic cycle):The virus takes over the cell's metabolism,
causing the creation of new proteins and nucleic acids by the host
cell's organelles.
• Assembly: Proteins and nucleic acids are assembled into new viruses.
• Release:Virus enzymes cause the cell to burst and viruses are
released from the host cell.These new viruses can infect other cells.
SCHEMATIC DIAGRAM OF THE TRANSMISSION PROCESS OF THREE HCOVS. HUMANS
ACQUIRED SARS‐COV AND MERS‐COV FROM BATS THROUGH CIVET CATS AND
DROMEDARY CAMELS, RESPECTIVELY. IT IS UNCLEAR HOW SARS‐COV‐2 SPREAD TO
HUMANS.
The origins of COVID‐19 pandemic: A brief overview
Ying‐Jian Hao, 1 Yu‐Lan Wang, 1 Mei‐Yue Wang, 1 Lan Zhou, 1 Jian‐Yun Shi, 1 Ji‐Min Cao,corresponding author 1 and De‐Ping Wangcorresponding author 1
Virus classification
(unranked): Virus
Realm: Riboviria
Kingdom: Orthornavirae
Phylum: Pisuviricota
Class: Pisoniviricetes
Order: Nidovirales
Family: Coronaviridae
Genus: Betacoronavirus
Subgenus: Sarbecovirus
Species: Severe acute respiratory
syndrome–related coronavirus
Virus: Severe acute respiratory
syndrome coronavirus 2
The origins of COVID‐19 pandemic: A brief overview
Ying‐Jian Hao, 1 Yu‐Lan Wang, 1 Mei‐Yue Wang, 1 Lan Zhou, 1 Jian‐Yun Shi, 1 Ji‐Min Cao,corresponding author 1
and De‐Ping Wangcorresponding author 1
Coronavirus
HIV - HUMAN IMMUNODEFICIENCY VIRUS
• AIDS from English Acquired Immune Deficiency Syndrome, also Acquired
Immunodeficiency Syndrome), also acquired immune failure syndrome.
• an enveloped RNA virus from the lentivirus genus, belonging to the
retroviridae family.
• AIDS is one of the most serious infectious diseases today.
• It occurs en masse, affects entire continents = pandemic.
• Around 1981, the first cases of AIDS in the United States were observed in gay
men, therefore the disease also received the temporary name GRID (GayRelated
Immune Deficiency).
It can spread through
• sexual contact,
• illicit injection drug use or sharing
needles,
• contact with infected blood,
• or from mother to child during
pregnancy,
• childbirth or breastfeeding.
• HIV destroys CD4 T cells — white blood
cells that play a large role in helping your
body fight disease.
Key facts
• HIV remains a major global public health
issue, having claimed 40.4 million [32.9–
51.3 million] lives so far with ongoing
transmission in all countries globally; with
some countries reporting increasing
trends in new infections when previously
on the decline.
• There were an estimated 39.0 million
[33.1–45.7 million] people living with HIV
at the end of 2022, two thirds of whom (25.6
million) are in the WHO African Region.
• In 2022, 630 000 [480 000–880 000] people died from HIVrelated
causes and 1.3 million [1.0–1.7 million] people
acquired HIV.
40 mil of victomes
SMALLPOX
• is considered one of the greatest epidemic
disease scourges in human history.
• Smallpox is an acute infectious disease caused by
the variola virus.The term “smallpox” was initially
introduced in Europe in the 15th century and is
also known by the Latin names “Variola”or
“Variola vera,”
• Edward Jenner
• Karel Raška
• 1980 eradicated
VIROIDS
➢ the smallest infectious pathogens.
➢ They are composed solely of a short strand of circular, singlestranded
RNA without protein coat. All known viroids are
inhabitants of higher plants
• The first recognized viroid, the pathogenic agent of the potato
spindle tuber disease, was discovered, initially molecularly
characterized, and named by Theodor Otto Diener,
SATELITS (VIRUSOIDS)
• are separate short nucleic acid molecules (DNA or RNA)
• cause diseases in plants
• discovered only in 1981
• they can not replicate independently but require a helper
virus in which they are closed (they are, therefore, some
"parasites of other viruses")
• do not encode any protein, replicate in the cytoplasm.
PRIONS
• infectious proteins (without NK) are encoded by the structural hosts of
the host organism
• They result from a conformational change of PrPC prion protein with a
spatial structure of? -Helix (helix) on PrPSc with β-structure (folded leaf)
• Prions are agents of transmissible spongiform encephalopathy
(spongiform appearance of degenerated central nervous system tissue)
• In humans: Creutzfeldt-Jakob disease and Kuru,
• Sheep and goat scrapie,
• Bovine spongiform encephalopathy (BSE)
• Feline spongiform encephalopathy (FSE)
PRIONS
• Prions, named for their description as "proteinaceous and
infectious particles", lack any detectable nucleic acids or
virus-like particles.They resist inactivation procedures that
normally affect nucleic acids.
• Mammalian prions: Agents of spongiform encephalopathies
• Fungal prions
Affected animal(s) Disease
sheep, goat Scrapie[42]
cattle
Bovine spongiform encephalopathy (BSE),
mad cow disease[42]
mink Transmissible mink encephalopathy (TME)
white-tailed deer, elk, mule deer, moose Chronic wasting disease (CWD)
cat Feline spongiform encephalopathy (FSE)
nyala, oryx, greater kudu Exotic ungulate encephalopathy (EUE)
ostrich
Spongiform encephalopathy
(Has not been shown to be transmissible.)
human
Creutzfeldt–Jakob disease (CJD)[42]
Iatrogenic Creutzfeldt–Jakob disease (iCJD)
Variant Creutzfeldt–Jakob disease (vCJD)
Familial Creutzfeldt–Jakob disease (fCJD)
Sporadic Creutzfeldt–Jakob disease (sCJD)
Gerstmann–Sträussler–Scheinker syndrome(GSS)[42]
Fatal familial insomnia (FFI)[44]
Kuru[42]
Familial spongiform encephalopathy[45]
Multiple System Atrophy (MSA): Not a TSE and is not by
typical prions Prp/PrPSc but by a misfolded α-Synuclein.[46]
BOVINE SPONGIFORM ENCEPHALOPATHY (BSE)
MAD COW DISEASE
• transmissible spongiform encephalopathy and fatal neurodegenerative disease in
cattle
• causes a spongiform degeneration of the brain and spinal cord.
• has a long incubation period, of 2.5 to 5 years
• Transmission of prions - cannibalism - MBM
- vertically (mother-fetus) -
no
- horizontally (excretions) – no
• Transmission to humans – new Variant of Creutzfeldt–Jakob disease (vCJD or nvCJD), and by
June 2014 it had killed 177 people in the United Kingdom
BSE
• Precautions - Meat-bone meal must not be fed to
ruminants
Confiscated - the skull, brain, spinal cord, eyes, intestines,
lymph node, spleen
MBM (meal bone meals) - pieces up to 50 mm, 130-140C at 3 bars
20min.
Creutzfeldt-Jakob disease (CJD) is a
very rare and fatal degenerative brain
disorder. CJD is a type of prion
disease, which is caused by an
infectious protein.
KURU DISEASE
• The name kuru means “to shiver” or “trembling in
fear.”
• The symptoms of the disease include muscle
twitching and loss of coordination.
• Other symptoms include difficulty walking,
involuntary movements, behavioral and mood
changes, dementia, and difficulty eating.
• The latter can cause malnutrition.
• Thank you for your attention