Topic 2A - Visualizing cells Flashcards
How small of an object is it possible to resolve with a typical light microscope?
200 nm
The naked eye is able to see objects as small as…
About 0.3 mm
The very best electron microscopes are able to resolve images as small as…
Just over 0.1 nm (a little over 1A)
Who was the first person to build a compound microscope in 1590?
Janssen
Who coined the term microscope?
Giovanni Faber
The first people to build commercial microscopes with known magnifications were…
Zeiss and Abbe
The eyepiece of a typical compound light microscope magnifies this much…
10 times
Light waves travelling at slightly different phases which interact with each other can create visual artifacts in a magnified image observed through a microscope. This effect is called…
Optical diffraction
Is it possible with current technologies to overcome optical diffraction?
No
Two waves IN phase will overlap to create an image that is (bright/dim)
Bright
Two waves OUT OF phase will overlap to create an image that is (bright/dim)
Dim
What factors influence the limit of resolution?
Wavelength of light
Numerical aperture
Medium of the sample
What is the difference between resolution and detection
Resolution = being able to make out a shape Detection = seeing that something is there
Just because you can see it does not mean you can determine what it is
What are the 4 kinds of light microscopy
- Bright field
- Dark field
- Phase contrast
- Differential interference
What is the main problem with bright field microscopy?
SInce biological tissues are largely uncoloured it is difficult to see any contrast
Describe how dark field microscopy works
Add an opaque disc to bright field microscopy, only scattered rays enter the objective
Phase contrast microscopy makes what feature of a sample visible?
Varying degrees of thickness
What is used in a differential interference microscope to obtain a 3D looking image?
Polarizer = makes all the light rays go in the same orientation
What are 2 methods we can use to detect small objects below the resolution of light microscopes?
- Electron microscopes
2. Fluorescent microscopes
Describe the absorption and emission of fluorescent molecules
Absorb light at one wavelength, emit it at a longer wavelength
Describe autofluorescence
Particles/cells that fluoresce naturally without the addition of fluorescent probes
What are the 3 kinds of probes we can fluorescently label?
- Dyes
- Antibodies
- Proteins
Briefly, how does a fluorescent microscope work?
Light comes in, reflected by a dichroic mirror, to point at a sample, sample emits light which is allowed to pass through the dichroic mirror, reaches the eye
Describe the outcome of confocal microscopy
A stack of images which can be pasted together to create a 3D “scan” of the sample
The basis of confocal microscopy is…
The 2-pinhole setup to focus light on a very specific plane
What are the three methods we can use to detect specific molecules?
- Dyes detect a class of molecules
- Antibodies can detect specific proteins
- Genetically engineered proteins detect specific molecules
What class of animals produces antibodies?
Mammals
Why do we use secondary antibodies in biological research?
The signal produced by secondary antibodies is 4x stronger than that of the primary antibodies
What dye stains genomic DNA?
DAPI
How can antibodies bind to DNA?
Can bind to the histone proteins at the centromere
GFP is originally derived from this animal
Jellyfish
What can be determined from data using genetically engineered fusion proteins?
Expression patterns within the cell, can determine what regulatory sequences of a gene have which role
At the N-terminus of a protein, an “address” exists, called the…
Peptide localization signal
What do we detect from using signal peptide marker gene fusions?
Intracellular localization of a protein - where it goes in the cell
What is the problems with using GFP-like fusion proteins in some settings?
Fusion protein can be large which might hinder its function, needs to be translated/transcribed, amplified with PCR… can be tedious
Photoactivation experiments typically yield data pertaining to…
Protein mobility
