Lecture 4 Reading Chapter 9 visualizing cells

Question 0

Scope of light microscope

Answer 0

0.4-0.7 micrometers

Question 1

Cell doctrine

Answer 1

All plants and animal tissues are aggregated of individual cells. Schleiden and Schwann, 1838

Question 2

Optical diffraction effects

Answer 2

Light waves travel through different routes, and not in a straight line, so they interfere with each other

Question 3

In phase

Answer 3

Interference makes things brighter

Question 4

Out of phase

Answer 4

Light waves interfere with each other in such a way as to cancel each other partly or entirely

Question 5

Micrometer

Answer 5

10^-6 m

Question 6

Nanometer

Answer 6

10^-9 meters

Question 7

Angstrom

Answer 7

10^-10 meters

Question 8

Limit of resolution

Answer 8

The limiting separation at which two objects appear distinct. Depends on wavelength of light and numerical aperture of lens system used.

Question 9

How does numerical aperture work

Answer 9

Affects the light gathering ability of the lens and is related both to the angle of the cone of light that can enter it and to the refractive index of the medium the lens is operating I

Question 10

Refractive index

Answer 10

Ratio of the speed of light in a vacuum to the speed of light in a particular transparent medium

Question 11

How can contrast in a specimen be generated

Answer 11

Light microscopes with special optical systems

Question 12

Bright field microscope

Answer 12

Light passing through a cell in culture forms the image directly

Question 13

Dark field microscopy

Answer 13

Exploits the fact that light rays can be scattered in all directions by small objects in their path. Bright image against a black background

Question 14

How does light change as it passes through a cell

Answer 14

The phase of the light wave is changed according to the cell’s refractive index.

Question 15

How do phase contrast microscope and differential interference contrast microscope work

Answer 15

Increase phase differences caused by cell’s refractive index so that waves are more nearly out of phase, producing amplitude differences when the sets of waves recombine, thereby creating an image of the cell’s structure

Question 16

How do we increase our ability to observe cells

Answer 16

We can attach a sensitive digital camera to a microscope, the camera detects light by means of charged coupled devices(CCDs) or high sensitivity complementary metal-oxide semiconductors (CMOs) sensors. These sensors are ten times more sensitive than naked human eye and can detect 100 times more intensity levels

Question 17

How do we prepare tissue for microscopy

Answer 17

Fix and section tissue, then freeze or embed

Question 18

Sections

Answer 18

Very thin transparent slices

Question 19

Fixative

Answer 19

Forms covalent bonds with the free amino groups of proteins, cross linking the, so that they are stabilized and locked in position

Question 20

Usual embedding medium for tissue

Answer 20

Waxes or resins

Question 21

Three main approaches to working with thin tissue sections that reveal differences in types of molecules that are present

Answer 21

Sections can be stained with organic dyesthat have some specific affinity for particular subcellular components
Sectioned tissues can be used to visualize specific patterns of differential gene expression
Using fluorescent probes and markers

Question 22

How do fluorescent molecules absorb and emit light

Answer 22

Absorb light at one wavelength and emit it at a longer wavelength.

Question 23

Fluorescence microscope

Answer 23

Tool to visualize fluorescent dyes for staining cells. Illuminating light is passed through two sets of filters. One to filter the light before it reaches the specimen and one to filter the light obtained from the specimen,

Question 24

When is fluorescence microscopy used

Answer 24

Ro detect specific proteins or other molecules in cells and tissues

Question 25

How are antibodies used with fluorescent dyes

Answer 25

They are coupled. The. Antibody molecules serve as highly specific and versatile staining reagents that bind selectively to the particular macromolecules they recognize in cells or in the extracellular matrix.

Question 26

What two dyes are used with antibodies frequently

Answer 26

Fluorescein , emits intense green fluorescence when excited with blue light
Rhodamine, emits deep red fluorescence when excited with green-yellow light

Question 27

Quantum dots

Answer 27

Tiny crystals of semiconductor metal that can be excited to fluoresce by a broad spectrum of blue light

Question 28

How are antibodies prepared for microscopy

Answer 28

Either purified from antiserum so as to remove all nonspecific antibodies, or they are specific monoclonal antibodies that only recognize the target molecule

Question 29

Indirect immunocytochemistry

Answer 29

Using an unlabeled primary antibody and then detecting it with a group of labeled secondary antibodies that bind to it

Question 30

Two ways to solve out of focus problem

Answer 30

Give many sectional views of optical sections

Image deconvolution

Question 31

Image deconvolution

Answer 31

Computer calculates what blurring would have done and reverses

Question 32

Point spread function

Answer 32

Blurred image of a point source

Question 33

Confocal microscope

Answer 33

Manipulates light before it is measures. Analog technique.

Generally used with fluorescent optics. Focused on single point not ph whole object

Question 34

Thick specimens are viewed with

Answer 34

Multi photon microscopes, with two-photon effect.

Question 35

Green fluorescent protein (GFP)

Answer 35

Isolated from a jellyfish. Encoded by a single that can be cloned and introduced into cells of other species

Question 36

How is GFP used usually

Answer 36

As a reporter molecule, a fluorescent probe to monitor gene expression

Question 37

How do we use fluorescent proteins to uncover kinetic properties of a cell

Answer 37

Fluorescence resonance energy transfer (FRET)
Photoactivation
Fluorescence recovery after photo bleaching (FRAP)

Question 38

FRET

Answer 38

Teo molecules of interest are each labeled with a different fluorochrome, chosen so that the emission spectrum of one fluorochrome, the donor, overlaps with the absorption spectrum of the other, the acceptor.

Question 39

Photoactivation

Answer 39

Fluorescence tagging technique that allows detailed observations of proteins within cells. Involves synthesizing an inactive form of the fluorescent molecule of interest, introducing it into the cell, and then activating it suddenly at a chosen site in the cell by focusing a spot light on it.

Question 40

Caged molecules

Answer 40

Inactive photosensitive precursors

Question 41

Fluorescence recovery after photo bleaching (FRAP)

Answer 41

One uses a strong beam of light from a laser to extinguish the GFP fluorescence in a specified region of the cell, after which one can analyze the way in which remaining unbleached fluorescent protein molecules move into the bleached area as a function of time.

Question 42

Why are micro electrodes not good at measuring changing ion concentrations

Answer 42

They reveal ion concentration only at one point of cell

Question 43

Ion sensitive indicators

Answer 43

Suited to record rapid and transient changes in ion concentration. Have light emission that reflects local concentration of ion.

Question 44

Aequorin

Answer 44

Luminescent protein. Emits blue light in presence of Ca2+, and responds to changes in concentration in the range of 0.5-10 micrometers.

Question 45

Genetically encoded fluorescent indicators

Answer 45

Bind ion tightly. Are excited by or emit light at slightly different wavelengths when they are free of io than when bound. By measuring the ratio of fluorescence intensity at two excitation or emission wavelengths, one can determine concentration ratio of ion bound indicator to the free indicator and get a measurement of ion.

Question 46

Why can’t single fluorescent molecules be reliably detected in ordinary microscopes

Answer 46

Limitation arises from the strong background due to light emitted or scattered by out-of-focus molecules. This tends to blot out the fluorescence from the particular molecule of interest.

Question 47

Total internal reflection fluorescence (TIRF) microscopy

Answer 47

Because of total internal reflection, light does not enter sample, so majority of fluorescent molecules are not illuminated. Electromagnetic energy does extend, as an evanescence field, for a short distance beyond the surface of the cover slip and into the specimen, allowing just those molecules in the layer closest to the surface to become excited.

Question 48

Atomic force microscopy (AFM)

Answer 48

Provides a method to manipulate individual molecules. With its tip, probe can collect data on the variety of forces it encounters.

Question 49

Approaches in light microscopy that bypass resolution imposed by diffraction of light. Superresolution approaches

Answer 49

Structured illumination microscopy (SIM)
Stimulated emission depletion microscopy (STED)
Photo activated localization microscopy (PALM)

Question 50

Structured illumination microscopy SIM

Answer 50

Fluorescence imaging method with resolution of about 100 nm.uses grated or structured pattern of light to illuminate the sample. Illuminating grid and sample features combine into an interference pattern, from which the original high resolution contributions to the image beyond the classical resolution can be measures. Imaging is repeated several times and then combined mathematically to create an enhanced image.

Question 51

Point spread function

Answer 51

Distribution of light intensity within the three dimensional, blurred image that is formed when a single point source of light is brought to a focus with a lens.

Question 52

Stimulated emission depletion microscopy DTED

Answer 52

Increasing resolution by switching all the fluorescent molecules at the periphery of the blurry excitation spot back to their ground state, where they no longer fluoresce in their normal way. Diffraction limit breached because technique ensures that similar but very closely spaced molecules are in one of two different states, either fluorescing or dark.

Question 53

Photo activated localization microscopy (PALM)

Answer 53

Labels are activated by illumination with near UV light. A small subset of molecules is modified so that they fluoresce when exposed to an excitation beam at another wavelength. These switch and another subset is activates. The photons of each subset given off are recorded and then aggregated into an image.

Question 54

Preparing a specimen for electron microscopy

Answer 54

Living tissue is killed and preserved by fixation, usually with glutaraldehyde and osmium tetroxide, which binds to and stabilizes lipid bilayers as well as proteins. Cut into extremely thin sections.

Question 55

What does image clarity depend on in an electron micrograph?

Answer 55

A range of contrasting electron densities. Impregnate tissues with salts of heavy metals.

Question 56

Immunoglobulins electron microscopy

Answer 56

Incubate a thin section with a specific primary antibody, and then with a secondary antibody to which a colloidal gold particle has been attached.

Question 57

Creating a third dimension view of a specimen

Answer 57

Have views of the same specimen from many different directions

Question 58

Electron microscope tomography

Answer 58

Specimen holder is tilted to arrive at a 3d view

Question 59

Scanning electron microscope (SEM)

Answer 59

Directly produces an image of the three dimensional structure of the surface of a specimen. Uses electrons that are scattered or emitted from the specimen’s surface.

Question 60

Negative staining

Answer 60

Allows finer detail of macromolecules to be seen. Molecules are supported on a thin film of carbon and mixed with a solution of heavy metal salt. Creates reverse or negative image of molecule.