Solaster-

1. Solaster, commonly known as the sun star, has a flattened, star-shaped body with multiple
arms (typically 8–13). The arms are slender and tapering, radiating symmetrically from a
central disc.
2. The oral surface houses the mouth at the center, surrounded by ambulacral grooves lined
with tube feet.
3. The aboral surface is covered with a rough, spiny texture, often adorned with papulae and
pedicellariae.
4. The mouth opens into a short esophagus leading to a central stomach. Solaster can evert its
stomach through its mouth to externally digest prey, primarily bivalves and other small
invertebrates.
5. Dioecious, with gonads located in pairs within each arm. Reproduction occurs through
external fertilization, releasing gametes into the water.

Astrophyton-
1. Multiple long, slender, and highly branched arms extend from the central disc. These arms
give it a characteristic basket-like appearance, aiding in suspension feeding.
2. The central disc and arms are covered with a rough, leathery skin that is often adorned with
small spines or scales for protection.
3. The tube feet are primarily used for feeding and sensory functions rather than locomotion,
as it’s is mostly sedentary.
4. Dioecious, with gonads located in the central disc. Reproduction occurs via external
fertilization, and the larvae undergo metamorphosis before settling as adults.
5. The mouth, located on the underside of the central disc, leads to a simple sac-like stomach.
Darkfield microscopy- requires a specialized stand containing a reflection mirror and light-shielding plate
to direct an inverted hollow cone of illumination towards the specimen at oblique angles.

1. The darkfield illumination produces an oblique cone of illumination using a specially-designed seven-sided
toroidal mirror that substantially reduces the stray light entering the large common main objective front
lens.
2. The toroidal mirror operates in a manner similar to high numerical aperture reflecting darkfield condensers
that are equipped with internal mirror surfaces having a variety of curvature geometries.
3. The condenser in this case is the paraboloid condenser, which has a curved and mirrored cardioidal internal
surface.
4. Illuminating light passes through the condenser and reflects from a single surface that is made from a
paraboloid truncated by a light stop oriented perpendicular to the condenser and microscope optical axis.

5. A central opaque stop is added to the condenser to block the central rays of light. This ensures only
the peripheral, oblique rays illuminate the specimen, creating the dark field effect.
6. Light from the ring light illuminator is reflected from the internal cylindrical mirror with the central (zeroth
order) rays being blocked by the light stop to form an inverted cone of illumination. Specimens are placed
directly onto a glass plate resting above the stage aperture and can then be visualized with darkfield
illumination.
Applications and limitations-
1. Good candidates for darkfield observation often have refractive indices very close in value to that of
their surroundings and are difficult to image with conventional brightfield techniques. As an example,
small aquatic organisms, oocytes, and cells in tissue culture have a refractive index ranging from 1.2 to
1.4, resulting in a negligible optical difference from the surrounding aqueous medium (refractive index
of 1.3).
2. Often specimens containing very low inherent contrast in brightfield microscopy are readily observable
in darkfield, and this type of illumination is ideal for revealing outlines, edges, boundaries, and
refractive index gradients.
3. Darkfield illumination is less useful for revealing internal details.

Phase contrast microscopy- the phase contrast technique employs an optical mechanism to
translate minute variations in phase into corresponding changes in amplitude, which can be visualized
as differences in image contrast.

1. specialized annulus (condenser annulus) positioned in the substage condenser front focal plane.
Wavefronts passing through the annulus illuminate the specimen and either pass through
undeviated or are diffracted and retarded in phase by structures and phase gradients present in the
specimen.
2. 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 eyepieces.
3. Light waves that are diffracted and shifted in phase by the specimen (termed a phase object) can be
transformed by phase contrast into amplitude differences that are observable in the eyepieces.
Large, extended specimens are also easily visualized with phase contrast optics due to diffraction
and scattering phenomena that occur at the edges of these objects.
4. objectives are available with internal phase plates that have varying levels of absorption and phase
displacement of the surround (undiffracted) illumination to produce a wide spectrum of specimen
contrast and background intensity choices for phase contrast microscopy.
5. A specially designed annular diaphragm, which is matched in diameter and optically conjugate to an
internal phase plate residing in the objective rear focal plane, is placed in the condenser front focal
plane.
6. A phase plate is mounted in or near the objective rear focal plane in order to selectively alter the
phase and amplitude of the surround (or undeviated) light passing through the specimen.

Applications and limitations-

1. One of the major advantages of phase contrast microscopy is that living cells can be examined
in their natural state without previously being killed, fixed, and stained. As a result, the
dynamics of ongoing biological processes can be observed and recorded in high contrast with
sharp clarity of minute specimen detail.
2. Industrial and chemical applications for phase contrast include mineralogy, crystallography, and
polymer morphology investigations.
3. Other commercial products scrutinized by phase contrast optical techniques include clays, fats,
oils, soaps, paints, pigments, foods, drugs, textiles, and other fibers.
4. Reduction in halo and shading-off artifacts remains a primary concern in phase contrast
microscopy.
Fluorescence microscopy-
1. Vertical illuminator- light of a specific wavelength (or defined band of wavelengths), often in
the ultraviolet, blue or green regions of the visible spectrum, is produced by passing
multispectral light from an arc-discharge lamp or other source through a wavelength
selective excitation filter.
2. Wavelengths passed by the excitation filter reflect from the surface of a dichromatic (also
termed a dichroic) mirror or beamsplitter, through the microscope objective to bath the
specimen with intense light. If the specimen fluoresces, the emission light gathered by the
objective passes back through the dichromatic mirror and is subsequently filtered by
a barrier (or emission) filter, which blocks the unwanted excitation wavelengths.
3. In most reflected light illuminators, the excitation filter, dichromatic mirror, and barrier filter
are incorporated into an optical block (often referred to as a cube). Modern fluorescence
microscopes are capable of accommodating between four and six fluorescence cubes
4. the reflected light vertical illuminator comprises an arc-discharge lamphouse at the rear end
(usually a mercury or xenon burner).

Advantages and limitations-

1. The application of an array of fluorochromes has made it possible to identify cells and
sub-microscopic cellular components with a high degree of specificity amid non-
fluorescing material.
2. the fluorescence microscope is capable of revealing the presence of a single molecule.
3. Modifications in fluorescence microscopy such as- FRET (Fluorescence resonance energy
transfer), Total internal reflection fluorescence microscopy (TIRFM) , fluorescence
recovery after photobleaching (FRAP), as well as spectroscopy, are often combined to
achieve additional information.
4. The limited number of fluorophores often pose a challenge on visualising all molecules.
5. The resolution is limited to the resolution of a typical brightfield microscope.

Confocal microscopy-
1. The illumination is achieved by scanning one or more focused beams of light, usually from a
laser, across the specimen. The images produced by scanning the specimen in this way are
called optical sections.
2. a laser is used as a light source, and is combined with a sensitive photomultiplier tube (PMT)
detector, and a computer to control the scanning mirrors or other scanning devices and to
facilitate the collection and display of images.

3. Motorized stages enable precise movement of the specimen for imaging multiple
areas or layers.
4. The aperture is specialised into pinhole aperture, Positioned in front of the detector to
block out-of-focus light. It Ensures only light from the focal plane reaches the
detector, improving image clarity.

Applications and limitations-

1. Confocal microscopy has an advantage in the relative ease with which extremely
high-quality images can be obtained from specimens prepared for conventional
optical microscopy
 2. The image can be zoomed with no loss of resolution by decreasing the area of the
region scanned in the specimen
3. extremely wasteful in their use of light energy from the lasers, and tends to kill
living specimens unless great care is taken to preserve their viability while they
were being imaged.

SEM-
SEM belongs to the family of electron microscopes which produce
images of an object by scanning its surface with highly focused
electron beam. The process involves the interaction of electrons
with atoms of an object, creating signals containing information
of object’s composition and topography.
1. Electron Beam: It has two variables i.e. energy and current.
The voltage is variable from about 1 - 60keV and the current
from 1e-7 to 1e-12 A.
2. Electron Gun: It is used to produce fine electron beam (also
called as electron probe).
3. Lenses- To produce finest beam of electron with desired
crossover diameter therefore two- level lens system used are
condenser and objective lens made of metal cylinders with
cylindrical hole, which operate in vacuum. These lenses are
located beneath the electron gun. Magnetic field is generated
in the inner part of the lenses to focus or de-focus the beam.
4. Sample Stage-It is a motorized plate which has movement in
three directions X, Y and Z controlled by feeding value in
the software. The samples are supported on it and move
smoothly in the required direction. X and Y, the two
horizontal movement are used to change the field of view
whereas Z, the vertical movement is required for image
resolution as well as depth of focus.
5. Secondary Electron Detector: It is consisting of a Scintillator
coating at the detector tip and high voltage of 10 KV applied
to it. The secondary electrons emitted from the specimen
gets magnetized towards this positive voltage, also this
secondary electron collection is supported by supplementary
 electrode (the collector) placed before scintillator by
applying few volts to this collector. When they hit the
scintillator light is produced which is guided to PMT (Photo
multiplier tube) through light guide. Then light is converted
to electrons which are amplified as electric signal.

Applications and limitations-

1. In Material Science for Surface morphology, texture, and
fracture analysis.
2. In Biology and Medicine for High-resolution imaging of
cells, tissues, and microorganisms.
3. In Nanotechnology for Characterization of nanoparticles,
nanostructures, and thin films.
4. Sample Preparation is tedious as it Requires conductive
coating for non-conductive specimens, which may alter
the sample.
5. Vacuum Environment for Biological samples may require
dehydration, leading to potential artifacts.

Isolation of DNA from rat liver extract by spooling-

Principle-
A typical mammalian cell contains about six picograms and 2.2 m
of DNA molecule. Therefore, the isolation of whole DNA from any
human organ consisting of around 200 billion cells will be
challenging and tedious. But the extraction of DNA by a method
called spooling helps to isolate such a large quantity and long
fibers of DNA conveniently. In particular, we can visualize the
white translucent DNA fibers after spooling in this technique. The
basic protocol consists of cell lysis and selective precipitation of
DNA using different biochemicals.
Discussion-
Although spooling method of DNA isolation is being practiced for
a long time now, it is still relevant to spool out DNA in cases
where the entire genome has to be separated out, without
digesting or breaking it into fragments.
In the purification step, since the supernatant is thick from the
cellular contents, carefully pouring the alcohol on top of the
supernatant leaves two distinct layers. DNA is soluble in water,
but not in alcohol; thus, the DNA present at the water-alcohol
interface precipitates out of solution, allowing it to be seen.
The roles of other chemicals used during the extraction process
are as follows-
(If Used) Ribonuclease A is used to degrade the RNA so that it
doesn’t interfere with the separation of the DNA. Chloroform
plays a role in denaturing proteins and dissolving lipids as it’s an
organic solvent. Also since phenol is a strong protein denaturant,
it dissolves the proteins and separates them into the organic layer,
leaving the DNA in the aqueous layer.
The main work of ethanol is to dehydrate the DNA such that it
gets precipitated out of the buffer. EDTA is used to chelate the
divalent cations, which are required by DNAses to function, hence
using EDTA renders the DNAses inactive.
Since the DNA is being extracted from liver, it is essential to keep
the rat on no food diet 24 hours prior to the procedure to ensure
that the sample is not contaminated with glycogen present in the
liver, as the removal of glycogen would be an additional daunting
task.