Exam 1 Euk

Cell

Simplest form of life that is capable of living and reproducing in a non-living environment

What are all cells surroded by?

A plasma membrane

Non-living environment

Any environment outside of the cell

What can curved glass surfaces (lens) do?

bend light and form images

two hisotorical scientists who made their own microscope

• Robert Hooke (1665)

• Anton van Leeuwenhoek (1665-1675)

compound microscope

• built by Robert Hooke

• two lenses

• looked at a wine cork

single-lens microscope

• built by Leeuwnhoek

• looked at stuff in ponds

three tenets of cell theory

created by Matthias Schleiden (1838) and Theodor Schwann (1839)

• the cell is the structural unit of life for all organisms

• all organisms are composed of one or more cells

• these two scientists thought cells could arise from non-living cell material, but that is not true

created by Rudolf Virchow

• cells can arise only by division from a preexisting cell

9 basic properties of cells

1. highly complex and organized

2. possess a genetic program and the means to use it

3. capable of producing more of themselves

4. acquire and utilize energy

5. carry out a variety of chemical reactions

6. engage in mechanical activities

7. able to respond to stimuli

8. capable of self-regulation

9. evolve

three domains of life

• bacteria

• archaea

• eukarya

what is the phylogenetic tree of domains of life based off of?

• rRNA

• RNA is conserved

two fundamental classes of cells

• prokaryotic cells

  • archaea (archaebacteria)

  • bacteria (eubacteria)

• eukaryotic

  • protists

  • fungi

  • plants

  • animals

archaea’s relatedness to eukarya and bacteria

• archaea and eukarya share similar genes for informational processes

• archaea is metabolically and strucurally more like bacteria, that is why they are both prokaryotic

9 features that prokaryotic and eukaryotic cells share

• plasma membrane of similar construction

• genetic information is encoded in DNA using identical gene code

• similar mechs for transcription & translation, including ribosomes

• shared metabolic pathways

• similar apparatus for conservation of chemical energy

  • plasma membrane of prokaryotes

  • mitochondria of eukaryotes

• similar mech of photosynthesis

• similar mech for synthesizing and inserting membrane proteins

• proteasomes of similar construction (archaea and eukarya)

• cytoskeletal filaents built of proteins similar to actin and tubulin

12 features in eukaryotic but not prokaryotic cells

• division of cells into nucleus and cytoplasm, separated by a nuclear envelope containing complex core structures

• complex chromosomes composed of DNA and associated proteins that are capable of compacting into mitotic structures

• complex membranous cytoplasmic organelles

• specialized cytoplasmic organelles for aerobic respiration and photosynthesis

• complex cytoskeletal system (actin filaments, intermediate filaments, and microtubules) and associated motor proteins

• complex flagella and cilia

• ability to ingest particulate material by enclosure within the plasma membrane vesicles

• cellulose-containing cell walls (plants)

• cell division using a microtubulue-containing mitotic spingle that separates chromosomes

• presence of two copies of genes per cell, one from each parent

• presene of three different RNA synthesizing enzymes (RNA polymerases)

• sexual reproduction requiring meiosis and fertilization

3 major differences in plant vs animal cells

plants have a cell wall, (central) vacuole, and chloroplast

model organisms

• organisms we use to study and experiment on because they are easy to maintain and breed in a lab setting

• less ethical to experiment on a human first

• experiemntal advantages come with model organisms

viruses

microscopic particles that can infect the cells of biological organisms

viral structure

genetic material surrounded by a protein coat (capsid) and sometimes has a lipid envelope

why is a virus considered non-living?

needs a host to perform function

5 common virus properties

• all are obligatory intracellular parasites

• outside of a living cell, it exists as a particle or virion, essentially a macromolecular package

• genetic material is surrounded by a protein capsule (capsid) usually made up of a specific number of subunits

• viruses have surface proteins that bind to particular host cell surface components (specificity)

• most viruses have relative narrow host range

two basic types of viral infection

• lytic infection: the infecting virus kills the host cell

  • infects, propogates, kills by breaking membrane to release more virus

• lysogenic infection: the infecting virus does not kill the host cell

  • integrate genetic information into host genome

provirus

integrated viral DNA is called a provirus

3 provirus properties

1. upon a stimulus, becomes lytic

2. produces new viral progeny without lysing the host cell

3. host cell becomes malignant

viroids

• small circular RNA infectious agent that lacks a protein coat

• 240 to 600 nucleotides (10% size of smaller viruses)

• do not code for proteins

two technqiues in cell and molecular biology (for this class)

1. pulse-chase experiment

2. microscopy

pulse-chase experiment- use of radioisotope

• an experiment in which cells are growing radioactive medium for a brief period (the pulse) and then transferred to nonradioactive medium/unlabeled chemicals for a longer period (the chase)

• during chase period, samples are taken for analysis

• put cells in radioactive medium, wash cells, put them in unlabeled chemical or nonradioactive medium, and take sample during a chosen time interval, then analyze

• can follow protein synthesis or other dynamics

• purpose is to track the synthesis, movement, processing, and degradation of molecules (like proteins or RNA) inside a cell over time.

light microscope

• a microscope that uses visible light and lenses to magnify and resolve small objects, such as cells and microorganisms.

• hard to see living organisms

type of microscope, where the light comes from, and parts labeled

• light microscope

• external light (mirror reflects light from surrounding)

1. coarse focus knob

2. eye piece

3. fine focus knob

4. body tube

5. low power objective lens

6. high power objective lens

7. stage dip

8. iris diaphragm lever

9. condensor

10. condensor knob

11. mirror

12. inclination point (allows the stage to tilt)

type of microscope, where the light comes from, and parts labeled

• light microscope

• produces internal light

1. ocular lens

2. objective lens

3. specimen

4. condensor lens

5. light source

pathway of light for a light microscope

1. light is concentrated to the condensor lens

2. light travels from condensor lens to specimen

3. light travels to objective lens

4. light travels to ocular lens

ocular lens function

• amplifies image under the microscope

• the image inside is reflected by a mirror inside of the microscope, located between the ocular lens and objective lens

what determines the quality of a microscope?

the objective lens

objective lens

• amplifies the object/specimen itself

• forms the image on the mirror

light path of the objective lens to form the image

distance between objective lens and specimen

• lower power objective lens, the distance is bigger

• higher power objective lens, the distance is smaller

• lens is specific to the specimen: only at a certain distance will you get a clear image

2 alpha angle, objective lens, and speciment

• increasing distance between objective lens and specimen decreases 2 alpha angle

• decreasing distance between objective lens and speciment increases 2 alpha angle

• higher power objective lens will create a larger angle than a lower power lens

empty magnification

• a magnification that does not tell you any new details than you already saw

  • making an image where you can see everything clearly bigger and still seeing everything clearly

• an effective magnification will tell you more information about an image

resolution

• how small the distance (d) that a lens can distinguish between two neighboring points as two distinct entities

• used to measure the optical quality of an objective lens

equation for resolution

• lambda is the wavelength of light used

• alpha is the angle created between objective lens and specimen

• n is refractive index of the medium between the objective lens and the speciemen (air)

  • determines how much the light bends

resolution and alpha

• larger alpha, d will decrease, better resolution

  • higher power lens

• smaller alpha, d will increase, not as good resolution

  • lower power lens

lens and “n”

each lens is designed for a specific medium/refractive index. you can’t use the same medium for every lens because different lenses require different refractive indices to work properly.

numerical aperture (N.A)

• for a particular lens, the refractive index and the angles involved are fixed, so the amount of light the lens/fiber can accept is also fixed.

• tells you how much light a lens or optical fiber can collect/accept.

• quality of a lens is determined by the numerical aperture of the lens

diffraction

• the bending and spreading of waves when they pass through an opening or around an obstacle.

• caused by the waves overlapping and interfering with each other

• if two spots overlap, it will be seen as one spot instead of two since the waves overlap

  • no resolution

• diffraction limits resolution

visibility and contrast

• essentially the same thing

• a part of a specimen is visible when it interacts with light differently than the areas around it or the background.

• if contrast is too small, then visibility is too small and we cannot see it

• visibility is determined by the reflective index of the objective and of the specimen and its surroundings

• if the refractive indices of the specimen and its surrounding medium are very similar, there is little difference in how light interacts with them, so the specimen has low contrast and is difficult to see.

• if the refractive indices of the specimen and its surrounding medium are very different, visibility and contrast is better

Why is oil used with a high-power objective?

Oil has a refractive index (~1.5) similar to glass (~1.5). This reduces the bending/refraction of light between the slide and objective, allowing more light to enter the objective, increasing numerical aperture (NA) and improving resolution.

reflective index of air, water, oil

• air: 1

• water: 1.33

• oil: 1.5

oil should have best quality, then water, then air

staining

• increases visibility and contrast

• very difficult to see internal structure of chromosome and cells because all parts have similar refractive indexes

• dyes can specifically stain the target structure , giving it a different refractive index

phase-contrast microscope

• converting differences in refractive index into differences in intensity

  • relative brightness and darkness (increase contrast)

• allows us to avoid killing a cell by staining it

function of a phase-contrast microscope

1. separating the direct light that enters the objective lens from the diffracted light emanating from the specimen

2. causing light rays from these two sources to interfere with one another (brings them back together after separated to create differences in brightness)

summary: separate the two light sources (direct and diffracted) and let them interfere with each other

fluorescence microscope

• light enters a filter that only allows a certain wavelength of light to go through

• light reaches beam splitting mirror

• beam splitting mirror reflects the incoming light through the objective lens

• objective lens focuses on the specimen

• light comes back from the specimen, go to the objective lens, which will amplify the image

• the light will pass through the beam splitting mirror, go through the filter to the ocular lens to form an image

how does light hit the specimen and then come back

• a specimen has some atoms or something which can absorb light, called excitation light

• after absorption, it will emit another light for a short period of time

• the shorter remnant light will become a longer remnant light called emission light

• excitation light is shorter remnant light (shorter wavelength) while emission light is longer remnant light (longer wavelength)

fluorochrome/fluorophore

• a fluorescent chemical molecule that absorbs light at a specific wavelength and re-emits it at a longer wavelength

1. energy is absorbed by the atom, which becomes excited

2. electron jumps to higher energy level

3. electron will drop back to its ground state, emitting a photon, causing the atom to fluoresce

• this is what creates shorter remnant light to longer remnant light

• light absorbed has higher energy than when emitted later because energy is lost

• the energy lost is light called fluorescence

• uses dyes or the fluorochromes/fluoropheres

• 
  

what kind of light is excitation light? why is this bad?

• UV light

• looking at a specimen for a long time using fluorescence microscope can damage it, making it difficult to get a detailed structure

laser scanning confocal microscope

• scans sample so it will not damage the specimen

• uses laser as the light source

  • laser uses different wavelengths

• light can go over specimen in any different directions so you can observe the whole area rather than just one space

• stack images together to create a 3D image

why is it advantageous to change the wavelength/energy of light with a laser scanning confocal microscope?

• fluorochrome in the specimens have a requirement for what kind of light they can emit

• wavelengths are fixed for a specific fluorescence dye, so we need to be able to change the wavelength of light to best view the specimen

laser scanning confocal microscope structure/light pathway

• dichroic mirror is the same as the beam splitting mirror in a fluoroscence microscope

• works similar to fluorescence microscope

• laser, dichomic mirror, scanner, objective lens, specimen, objective lens, pinhole, computer

how images are created using laser scanning confocal microscope

• light comes from focal plane and will go through the pinhole to form image on the computer

• light that comes from the below or above the focal plane will be blocked by the pinhole aperture, creating a clear image

• without the pinhole, light from the lower or higher plane will go through to form the image, making it blurred

dichromic mirror/beam splitting mirror

• reflects incoming shorter wavelengths light

• lets longer wavelengths of light to pass through

focal plane

• illuminated plane

• the imaginary flat surface where a lens or mirror bends light rays to form a sharp, clear image

• specimen does not have to be very thin

pinhole aperture

• confocal with the focal plane

  • light coming from the focal plane will be fixed in the pinhole, (will go through the pinhole) to form an image

relative resolution of our eyes and different microscopes

• naked eye is about 100 micrometers

• light microscope is 0.2 micrometers, which is sufficient enough to see larger cellular organelles like nuclei and mitochondira

  • the fine details of the interior structures of organic cells cannot be resolved by light microscope

• electron microscopes have better resolution and were created so we can see smaller structures

  • the amplification of electron microscope to a light one is significantly better

transmission electron microscope vs light microscope pathway of light

• light microscopes use light as a light source, while TEM uses a beam of electrons

• light microscopes use glass lenses, but electron microscopes cannot because glass woud block the electrons.

  • electron microscopes use electromagnetic lenses because it can change the path of the electrons, just like glass changes the path of light

• light microscopes use different wavelengths of light, beam of electrons use voltage to calculate their wavelength

  • lambda (wavelength of electrons) is the square root of 150/V (voltage)

  • increase in voltage makes electron wavelength shorter

• condensor lens condenses the electron to the specimen, then objective lens, then ocular lens/projector lens to further amplify the image

preparing specimens for electron microscopes

• electrons will damage specimen and dyes

• negative stain: stain speciment with heavy metal (uranial acetate or lead citrate).the heavy metal stain interfere or block the electrons so when an electron goes there, that part of the specimen higher exposure (brighter). the parts not stained will appear darker when the electron goes there

• shadow casting: evaporate heavy metal (platinum wire) so it becomes vapor. the vapor will deposit onto the specimen

  • the less deposition, the more electrons will go through, making it brighter

  • the more deposition, the less electrons will go through, making it darker

• freeze-fracture replication/freeze etching: freeze sample, use a sharp knife to heat the sample

  • a preparation method that splits frozen biological samples and evaporates surface ice to expose internal cell structures in three dimensions

TEM vs SEM

• TEM looks at internal structures of cell, SEM looks at surfaces of objects

• TEM specimens need to be sliced and stained with solutions of heavy metals while SEM requires critical-point drying and coating with metals

• TEM involves an electron beam that is focused to simultaneously illuminate the entire viewing field

• SEM involves electrons that are accelerated as a fine beam to scan the specimen

• TEM functions by electrons passing through the specimen to form the image

• SEM functions by forming the image by electrons reflecting back from the specimen or by secondary electrons given off by the specimen after being struck by the primary electron beam

comparison of microscope (bright-field, fluorescence, confocal, electron)