Phase Contrast Microscopy Flashcards
Phase contrast microscopy was first described in 1934 by Dutch physicist
Frits Zernike
was first described in 1934 by Dutch physicist Frits Zernike
phase contrast microscopy
a contrast-enhancing optical technique to produce high-contrast images of transparent
specimens
phase contrast microscope
phase contrast microscope is used for transparent specimens such as
living cells (usually in culture)
microorganisms
thin tissue slices
subcellular particles (nuclei, other organelles)
Phase-contrast microscopy converts ___shifts in the light passing through a transparent
specimen to brightness changes in the image, which is barely, or not at all, possible in a
brightfield microscope. T
phase shifts
The phase shifts can be observed as differences in ____
image contrast
why do phase shifts happen
undeviated and diffracted light collected by the objective is segregated at
the rear focal plane by a phase plate
collects the undeviated and diffracted light by the objective
phase plate
focuses the collected light from the phase plate and focused at the
intermediate image plane
The phase shifts can be observed as differences in image contrast.
This happens when undeviated and diffracted light collected by the objective is segregated at
the rear focal plane by a phase plate and focused at the intermediate image plane to form the
final phase-contrast image observed in the ___.
eye pieces
Phase-contrast microscopy is basically a specially designed light microscope
with all the basic parts, in addition to which are the
annular phase plate
annular diaphragm
m is made up of a
circular disc having a circular annular groove and is situated below the
condenser
annular diaphragm
is a transparent disc that is
either a negative phase plate having a thick circular area or a positive phase
plate having a thin circular groove.
phase plate
how does phase contrast achieves its results with annular phase plate and annular diaphragm
This is by separating the direct rays from the
diffracted rays, the former passing through the annular groove, and the latter
through the region outside the groove
phase contrast is achieved by separating the ___ rays from the ___ rays
direct rays
diffracted rays
rays that passe sthrough the annular groove
direct rays
rays that passes through the region outside the annular groove
diffracted rays
has a corresponding match in lens
phase contrast
view the illumination pathway
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live microbial specimen include
yeast
e. coli
hay infusion
aspergillus niger
. Place one to two loopfuls of ___ ___ and ___ on
separate glass slides. Put cover slip and set aside for microscopy.
bacterial
yeast suspension
hay infusion
results in extremely small differences in the intensity distribution in the image.
poor light absorption
is an optical microscopy technique that converts phase shifts in the light passing through a transparent specimen to brightness changes in the image.
phase contrast microscope
When light passes through cells, small phase shifts occur, which are invisible to the human eye. In a phase-contrast microscope, these phase shifts are converted into changes in ___, which can be observed as differences in image contrast.
amplitude
Partially coherent illumination produced by the ___ lamp is directed through a collector lens and focused on a specialized annulus (labeled condenser annulus) positioned in the substage condenser front focal plane.
tungsten halogen
Partially coherent illumination produced by the tungsten-halogen lamp is directed through a collector lens and focused on a specialized ___(labeled condenser annulus) positioned in the substage condenser front focal plane.
annulus
Wavefronts passing through the annulus illuminate the specimen and either pass through ___ or are ___and retarded in phase by structures and phase gradients present in the specimen.
undeviated
diffracted
Undeviated and diffracted light collected by the objective is segregated at the rear focal plane by a
phase plate
Undeviated and diffracted light collected by the objective is segregated at the rear focal plane by a phase plate and focused at the
intermediate image plane
It is situated below the condenser.
annular diaphragm
It is made up of a circular disc having a circular annular groove.
annular diaphragm
The light rays are allowed to pass through the annular groove.
annular diaphragm
Through the ___groove of the annular diaphragm, the light rays fall on the specimen or object to be studied.
annular
At the back focal plane of the objective develops an image.
annular diaphragm
The annular phase plate is placed at
back focal plane of the objective
It is either a negative phase plate having a thick circular area or a positive phase plate having a thin circular groove.
phase plate
This thick or thin area in the phase plate is called the
conjugate area
the phase plate is a ___disc.
transparent
advantages of phase contrast microscopy
Living cells can be observed in their natural state without previous fixation or labeling.
It makes a highly transparent object more visible.
No special preparation of fixation or staining etc. is needed to study an object under a phase-contrast microscope which saves a lot of time.
Examining intracellular components of living cells at relatively high resolution. eg: The dynamic motility of mitochondria, mitotic chromosomes & vacuoles.
It made it possible for biologists to study living cells and how they proliferate through cell division.
Phase-contrast optical components can be added to virtually any brightfield microscope, provided the specialized phase objectives conform to the tube length parameters, and the condenser will accept an annular phase ring of the correct size.
limitations of phase contrast microscopy
Phase-contrast condensers and objective lenses add considerable cost to a microscope, and so phase contrast is often not used in teaching labs except perhaps in classes in the health professions.
To use phase-contrast the light path must be aligned.
Generally, more light is needed for phase contrast than for corresponding bright-field viewing, since the technique is based on the diminishment of the brightness of most objects.
What are the main differences between phase contrast and bright-field microscope?
The main difference between phase contrast and bright-field microscopes lies in
their method of enhancing contrast in transparent specimens. Phase contrast microscopy
uses optical manipulation to translate subtle differences in refractive index and specimen
thickness into variations in image intensity, making unstained structures visible
e. In contrast,
bright-field microscopy illuminates the sample with a direct beam of white light, often
requiring staining to enhance contrast, which can obscure details and may be toxic to living
cells. Phase contrast microscopy is better for observing live, transparent specimens, while
bright-field suits stained samples, offering color representation.
- Why is phase contrast microscopy particularly useful for observing living cells?
Phase contrast microscopy is ideal for observing living cells because it doesn’t
require staining, which could possibly harm or alter cells. It converts differences in how light
passes through different parts of the cell (refractive index) into variations in brightness,
making transparent structures visible. This allows observation of cell processes real time
(e.g. motility, movement, or chemotaxis) real time without disturbing the cells,
- Which cell structures are more clearly visible using phase contrast microscopy?
Phase contrast microscopy helps visualize cell structures that are transparent or
low contrast under brightfield microscopy. This technique makes the nucleus, cell
membrane, and organelles such as mitochondria, Golgi, and granules more apparent.
view phase contrast results
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- What is the advantage of using phase contrast microscope in ecological studies?
Phase contrast microscopy (PCM) offers advantages in studying ecology because
natural status of living microorganisms can be observed without staining. This feature is
crucial in understanding the ecological niche and interaction within the environment, as
aforementioned staining can possibly disturb the according study of behavior
