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⇒ PART 1 pre-clinical imaging
Case 1: Brightfield and Fluorescence Microscopy
Brightfield
1. What is brightfield microscopy and how does it work?
Light and Matter Interaction: Before delving into brightfield microscopy, it's essential to understand how light
interacts with matter. Light can be transmitted, absorbed, reflected, refracted, diffracted, scattered, or emitted
(fluorescence) when it interacts with different substances.
Brightfield microscopy is an optical microscopy (closely view sample through the magnification of a lens with visible light)
technique used to observe samples that absorb light, thus appearing darker against a bright background.
It relies on the basic principles of light interaction with matter and the use of lenses to magnify the
sample for observation. Prepared on glass slides under a bright background.
Key Principles of brightfield microscopy:
Illumination light is transmitted through the sample and the contrast is generated by the absorption of
light in dense areas of the specimen.
Brightfield microscopy relies on absorption for contrast: When light passes through a sample in
brightfield microscopy, it interacts with the sample's constituents, and some of the light is absorbed while
the rest is transmitted through and collected by the objective lens to form an image. The absorbed light
results in darker regions in the image, creating contrast with the lighter background.
Lens Systems: Magnification in microscopy is achieved through the use of lenses. A compound
microscope typically consists of an objective lens and an ocular lens (eyepiece), which together magnify
the image of the specimen. Objective lenses have different magnification to capture details specimen.
The condenser lens: positioned beneath the specimen and focuses light onto the specimen. F:
controlling the amount and angle of light that reaches the specimen.
The ocular or eyepiece is used for direct observation by the viewer, while a camera can be attached to
capture digital images.
Transillumination: In brightfield microscopy, the sample is illuminated from below with a light source, and
the light transmitted through the sample is observed. A condenser lens helps to focus and improve the
resolution of the microscope.
Resolution: The ability to distinguish two closely spaced objects as separate entities is crucial in
microscopy. The resolution of a microscope depends on factors such as the numerical aperture (NA) of
the objective lens and the wavelength of light used. Higher NA and shorter wavelengths lead to better
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resolution.
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- NA of the objective lens (high NA → )
- Wavelength of light (shorter wavelength → )
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F focus knobs: adjust the focal plane, which determines the position where the specimen being
observed appears sharp and clear; coarse focus (objective lens up and down) and fine focus (smaller scale
adjustments).
(focal plane = specific plane within the specimen that is in focus, appears sharp and clear when viewed through the
microscope. This plane is where the majority of the light rays coming from the specimen converge/ are brought
together to the same point to form a clear image.)
Application brightfield microscopy:
Observing fixed and stained biological specimens, tissue sections, and other transparent materials. Can
use alive and dead (fixed) cells.
Advantages Disadvantages
Easy usage. Low Contrast: relatively low contrast compared
to other microscopy techniques → makes it
challenging to visualize certain structures
within the sample, particularly those with
similar refractive indices.
Cause: few biological samples absorb light to
great extend. Staining often required to
increase contrast → cannot use alive cells.
Alive cells. Low Resolution: resolution may be limited by
the wavelength of light used and the NA of the
objective lens.
Cause: blurry appearance out-of-focus
material.
When detailed visualization of cellular Living cells can be damaged by prolonged
structures is required, fixing and staining the exposure to intense light; phase contrast or
cells can provide enhanced contrast and DIC microscopy is often preferred to maintain
cell viability.
clarity. Fixation involves treating cells with
chemicals to preserve their structure and
prevent decay. Fixed cells can be stained with
dyes or labeled with fluorescent probes to
enhance contrast and visualize specific cellular
structures.
2. What are the different fixation methods for light microscopy? (look at lab protocols)
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Samples prepared for brightfield microscopy include a variety of materials, fixed cells or tissues, live
cells, and polymers or metals.
Fixation is achieved w/ formaldehyde for biological samples or freezing for fresh tissues, and is often
employed to preserve the sample and prevent movement during imaging.
! Live cells can also be observed directly without fixation.
Fixation involves attaching cells to a slide, achieved through heating or chemical treatment, and serves
to immobilize microorganisms within the sample while maintaining the integrity of their cellular
components for observation.
Prior to fixation: consider diffusivity sample, volume, pH and next step is to embed. Embedding involves
embedding the fixed tissue in a solid medium, such as paraffin wax or resin, to provide support and
facilitate the preparation of thin sections for microscopy. The embedding process typically includes:
dehydration (remove excess H2O w/ alcohol), clearing (remove excess alcohol), infiltration (to solidify)
then the dehydrated and cleared sample is embedded in molten-wax or resin.
➢ Physical Fixation:
○ Quick Freezing/Flash Freezing:
■ Rapidly freezes the sample using liquid nitrogen
■ Quick fixation
■ -200 degrees C.
○ Fixation by Heating:
■ Not for histological samples (causes damage, primarily used for microorganisms).
■ Denatures proteins.
○ Cryopreservation/Cryofixation: rapid freezing of the sample using liquid nitrogen or
isopentane → preserves structure of tissues for frozen sections.
➢ Chemical Fixation:
○ Cross-linking Fixation:
■ Formation of covalent bonds between proteins → tissue stiffening → strengthens
tissue structure & prevents degradation.
■ E.g. aldehydes (formaldehyde, glutaraldehyde) → preserve cellular structures.
○ Coagulative Fixation:
■ Embedding samples in paraffin → wax-like consistency.
■ This method is suitable for histological sections.
○ Emersion:
■ Sample is plunged into a fixative solution.
○ Perfusion:
■ Fixative is circulated through the whole animal or organ, commonly done with
formaldehyde.
○ Ethanol/ methanol + acetone fixation:
■ Dehydrated sample, preservation structure, enhance permeability.
■ 10 minutes at -10 degrees C.
■ Fixated > sample washed w/ PBS; removes excess fixative.
○ Ethanol Fixation: Dehydrates the sample to preserve structure.Ethanol replaces H2O
molecules.
○ Methanol Fixation + acetone: Often mixed w/ acetone. Preserve structure & permeability.
○ Cryofixation: Involves rapidly freezing the sample to extremely low temperatures.
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○ Acetone Fixation: Rapid-fixing agent commonly used for frozen sections.
○ Osmium Tetroxide Fixation: Particularly useful for preserving lipid structures.
○ Picric Acid Fixation: Used as a fixative for some tissues, often combined with other
fixatives.
○ Paraformaldehyde-Glutaraldehyde Fixation: Combination provides better preservation of
cellular structures, especially for electron microscopy.
3. What are the different staining methods for light microscopy? (find at least one protocol)
Staining methods in light microscopy → enhancing contrast + visualizing specific structures within
samples.
Brightfield microscopy relies on absorption-based staining to create contrast.
➢ Fixed (dead) cells staining methods: primarily used
○ Hematoxylin and Eosin (H&E): stains nuclei blue-purple with hematoxylin and cytoplasm
and extracellular structures pink or red with eosin.
○ Methylene Blue: imparts a blue color to cell nuclei.
○ Giemsa Stain: versatile stain (blood cells, bacteria).
○ Crystal Violet: Gram staining (bacterial identification).
○ Safranin and Gram's Iodine: differentiation Gram+,- .
○ Wright's Stain: blood smear examination.
○ Periodic Acid-Schiff (PAS) stain: structures rich in carbohydrates.
○ Toluidine Blue: various cellular structures (histology, cytology)
➢ Live cell staining methods: are utilized to visualize specific cellular components or distinguish
between live and dead cells.
○ neutral red: taken up by lysosomes and other acidic cellular structures
○ methylene blue: healthy cell with turn the stain colorless (due to the cell's enzymes, which reduce the
methylene blue causing it to lose its color)
○ eosin Y: stains cytoplasmic structures
○ trypan blue: distinction between live and dead cells (excluded by viable cells but taken up by dead or
damaged cells, allowing for the)
Protocol Hematoxylin and Eosin (H&E) staining → stains nuclei blue-purple with hematoxylin and
cytoplasm and extracellular structures pink or red with eosin:
Interpretation:
- Cytoplasm: Light pink
- Collagen: Pink
- Muscle: Pink/Rose 1.
Protocol:
1. Deparaffinize (before staining with H&E, tissue sections are often embedded in paraffin wax to preserve them. However, paraffin
must be removed prior to staining to allow the dyes to penetrate the tissue effectively) sections if necessary and hydrate
in distilled water (restore moisture to the tissue and prepare it for staining. Hydration helps to prevent tissue damage and
ensures optimal interaction between the tissue and the staining reagents).
2. Apply adequate Hematoxylin, Mayer’s (Lillie’s Modification) to completely cover tissue section
and incubate for 5 mins (en gros c’est mettre le staining dessus).
3. Rinse slide in two changes of distilled water to remove excess stain.
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4. Apply adequate Bluing Reagent to completely cover tissue section and incubate for 10-15 secs
(bluing reagent enhances contrast of stained nuclei by neutralizing any excess red/ pink color that may have been picked up during
the staining process → nuclei more distinct blue color).
5. Rinse slide in two changes of distilled water (remove any excess bluing reagent and residual stains from the tissue
section).
6. Dip slide in absolute alcohol and blot excess of (dehydration process, which removes water from the tissue section
prior to mounting, as excess water can interfere with the mounting medium and cause artifacts in the final stained slide).
7. Apply adequate Eosin Y Solution (Modified Alcoholic) to completely cover tissue section to
excess and incubate for 2-3 min (Eosin Y is a counterstain used in the H&E staining method. It stains cytoplasmic
structures and extracellular components pink or red. This step helps to provide contrast to the blue-purple nuclei stained by
hematoxylin, allowing for better visualization of cellular morphology and tissue architecture under the microscope).
8. Rinse slide using absolute alcohol (removes excess staining).
9. Dehydrate slide in three changes of absolute alcohol.
10.Clear slide and mount in synthetic resin (clearing: removing remaining alcohol and ensuring tissue section is
dehydrated and transparent → optimal visibility of the stained cellular structures.. After clearing > mounted onto a microscope slide
using synthetic resin mounting medium; optically clear substances that permanently affix tissue section to slide & provide support/
protection during microscopy examination. The mounting medium also helps to preserve the stained cellular structures and prevent
them from drying out or deteriorating over time).
11.Erythrocytes: Pink/Red Nuclei: Blue
4. How do you increase contrast in samples? Explain the different mechanisms: altering the
microscope and altering the sample. (Phase contrast and differential interference)
Contrast Enhancement:
● Contrast Enhancement: While brightfield microscopy relies on absorption for contrast, additional
techniques can be employed to enhance contrast further (e.g. phase-contrast microscopy,
differential interference contrast (DIC) microscopy, and staining techniques).
When light passes through a sample in brightfield microscopy, it interacts with the sample's constituents, and some
of the light is absorbed while the rest is transmitted through and collected by the objective lens to form an image.
The absorbed light results in darker regions in the image, creating contrast with the lighter background.
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● Staining: Staining involves the use of specific dyes or stains to enhance the contrast of certain
structures within the sample. Different stains target different cellular components, allowing for
better visualization of specific features.
Two main approaches to enhance contrast involve altering the microscope and altering the sample.
➢ Altering the Microscope:
○ Phase Contrast Microscopy: allows to observe transparent samples (e.g. cells) w/o
need to color them. phase contrast microscopy
■ Exploits differences in the phase of light passing through different parts of a
specimen.
■ Special phase contrast objectives and annular rings in the condenser convert
phase variations into intensity differences
■ Result: transparent structures, which lack contrast in brightfield microscopy,
appear brighter against a darker background.
When destructive interference predominates, samples appear dark against a bright background.
2 small modifications where done compared to brightfield microscopy:
- annular diaphragm placed at the place of the open disc (used in darkfield)
- phase plate placed between the objective and the ocular lens
Light path phase contrast microscopy:
light source > annular diaphragm > condenser lens > specimen > objective lens > phase plate > ocular
lens > eye
- annular diaphragm: blocks most of the light from the illuminator, produces a hollow cone of light
- hollow cone of light is focused on the specimen before reaching the objective lens
- light traveling directly from the illuminator (surrounding wave) passes through one part of the
phase plate, whereas light reflected, refracted or scattered by the specimen (diffracted wave)
passes through another part of the phase plate;
2 types of light are coming:
→ the surrounding wave (the buitenste als je van specimen naar objective len kijkt = undiffracted
light
→ diffracted light that is passing through the specimen, then passes through another part of the
phase plate
- this causes the surrounding wave to be out of phase w/ the diffracted wave; so the surrounding
wave is not going through the sample, that is the out of phase and the diffracted light (diffracted
by the specimen) → both of these waves are producing an out of phase wave ⇒ result:
destructive interference → brings contrast where sample appears dark against bright
background.
- generally structures that differ in refractive index will differ in level of darkness (so cellular
structures that differ in refractive index will differ in levels of darkness)
- in this case, as most of the light is blocked: background will not appear as bright as brightfield
microscopy.
- the surrounding light is also captured by the objective lens, some surrounding light is getting
absorbed by the objective lens → background will not appear dark as in brightfield microscope (in
darkfield microscopy the surrounding light is not absorbed whatsoever by the objective lens so
the background is completely dark)
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Because phase differences are undetectable to the human eye, and are not readily observed in a
microscope under brightfield illumination, the light path through the microscope must be suitably modified
in order to produce satisfactory images of phase specimens.
Advantages Disadvantages
Living cells. Not suitable for thick samples.
No need to fix/ kill cells, do not need to add stain.
High contrast, high resolution images.
Yt video: A condenser annulus modifies the light beam.
When a sample is inserted it scatters a part of the light which is then also refocused to the detector.
To distinguish direct light that is scattered by the sample, a phase plate is inserted.
The scattered light crosses a thicker part of the plate. This shifts its phase compared to the direct light.
Scattered light then interferes w/ direct light → creates phase contrast → results in intensity differences →
visualizes transparent samples.
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++ blue is what want to see in sample, red is the rest, blue and red almost the same, direction is
changed/ phase → blue arrow = red+ yellow; phase shifting, last steps is gray filter –/ relative difference
blue and red is bigger m A→B most important step; change the phase. Green is where the light comes
from. Phase shift → have a bigger contrast.
Do not need to know what happens in the shift ring.
Lecture:
Here, exploit the changes in the phase of light; as light goes through the sample, its phase is
propagation ?? . This induces change in phase and using some components and using special
illumination and detection to convert changes in phase to changes in intensity.
Imagine you're trying to see really small things, like cells, under a microscope. Normally, these tiny things might not
show up very well because they don't have much color or contrast.
Now, phase contrast microscopy: Instead of just looking at color, it pays attention to how light waves change as
they go through the tiny things. When light passes through these small structures, like cells, it can change its phase
(basically, the position of the waves).
What the phase contrast microscope does is use special tricks with the light. It adds some components that help us
notice these changes in the light's phase. It's a bit like magic glasses that make the invisible changes in the light
more visible.
By doing this, we can turn these phase changes into changes in brightness. So, under the microscope, things that
were hard to see before suddenly become much clearer because we're paying attention to how the light waves are
behaving as they pass through the tiny stuff.
Phase contrast microscopy idea: convert these changes to intensity (e.g. see image on the right; can see
interfaces, changes in medium).
○ Differential Interference Contrast (DIC) Microscopy: transparent biological samples
■ DIC microscopy enhances contrast by creating intensity variations based on the
interference of polarized light passing through a specimen.
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■ DIC microscopes use polarizers and Nomarski prisms to split the light beam into
two → resulting in a 3D-like appearance that highlights subtle refractive index
variations within the specimen.
1. Differential Interference Contrast (DIC) microscopy is a technique used to enhance contrast in
transparent specimens by creating intensity variations based on the interference of polarized light
passing through the specimen. Here's how it works:
2. Polarized Light: DIC microscopy begins with the use of polarizers to polarize the light source.
Polarized light consists of waves that oscillate in a specific direction, which helps to control the
orientation of the light waves passing through the specimen.
3. Splitting the Light Beam: The polarized light passes through a series of optical components,
including Nomarski prisms, which split the light beam into two separate paths. These prisms
create two distinct beams of polarized light that travel along slightly different paths through the
specimen.
4. Interaction with Specimen: As the two polarized light beams traverse the specimen, they
encounter variations in the refractive index within the specimen. Differences in refractive index
cause changes in the speed and direction of the light waves passing through different regions of
the specimen.
5. Recombination: After passing through the specimen, the two beams of polarized light are
recombined. Because the beams have traveled along different paths and experienced different
interactions with the specimen, they are now slightly out of phase with each other.
6. Interference Patterns: When the recombined beams of polarized light overlap, they interfere with
each other. This interference produces intensity variations in the resulting image, creating regions
of light and dark that correspond to differences in refractive index within the specimen.
7. 3D-like Appearance: The intensity variations generated by DIC microscopy give the specimen a
three-dimensional appearance. This effect highlights subtle differences in refractive index,
making fine details and structures within the specimen more visible.
8. Overall, DIC microscopy provides high contrast images with a 3D-like appearance, allowing
researchers to visualize transparent specimens and observe intricate details that may be difficult
to discern using other microscopy techniques.
Lecture:
Here we use polarized light and prisms.
Here we exploit the fact that we are creating 2 beams that are going through slightly different parts of the
sample and then we bring them back together and they interact/ interfere.
Bc these beams have experienced different path lengths, when interfere this will come up as an intensity.
So, get an image that gives this pseudo 3D effect. This has to do with changes in the refractive index
and the thickness of the sample.
DIC nice to look at the nucleus bc can clearly see it.
Disadvantage of DIC: do not have specificity; do not know what the organelles/ structures are that we are
looking at there. Can only see the nucleus. To add specificity we stain specifically our sample.
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○ Darkfield Microscopy: Involves illuminating the sample with oblique or scattered light.
Unstained or weakly stained specimens appear bright against a dark background,
enhancing contrast.
■ Light is directed at an angle onto the sample. This results in the specimen
appearing bright against a dark background, making it easier to visualize. Darkfield
microscopy is particularly useful for observing unstained or weakly stained
specimens, as they scatter light and appear bright against the dark background,
enhancing contrast and revealing details that may be difficult to see under
traditional brightfield illumination.
○ Polarized Light Microscopy: Uses polarizers to analyze the birefringence of certain
materials. It enhances contrast by revealing structural details and variations in refractive
indices within the sample.
⇒ These techniques exploit differences in the phase or polarization of light passing through the sample
to generate contrast in the image.
➢ Altering the Sample:
○ Staining: Involves using dyes or fluorescent labels that selectively bind to specific
structures within the sample. Staining introduces contrast by colorizing different cellular
