light microscopy

information sources about cells

• microscopy (light/electron)

• biochemical techniques

• genetic techniques

history of biochemical techniques

• light microscopy was the most important technique until the 1950s

• electron microscopy was dominant from the 1950s to the 1970s

• combination of biochemistry and yeast genetics dominated from 1980s to present

• light microscopy is making a comeback thanks to new abilities to follow dynamics of proteins in living cells

early electron microscopes (Leeuwenhoek)

• only one lens, mounted in a tiny hole in the brass plate (body of the instrument)

• specimen is mounted on the sharp point that sticks up in front of the lens

• position and focus could be adjusted by turning the two screws

origin of “cell”

Robert Hooke observed pores resembling monks’ cubicles in a thin slice of cork

light microscopy

• visualize cells in context, tissues

• can see details in eukaryotic cells, which are large enough to distinguish internal details

types of light microscopes

• compound microscopes (multiple lenses)

• fluorescence microscopes

• confocal microscopes (new kind of fluorescence microscope)

compounds of conventional transmission microscope

• light source

• condenser

• specimen slide

• objective lens

• ocular lens

important types of light microscopy

• transmitted light

• fluorescence

• confocal

• super-resolution

visualization of cells with light microscopy

• cells are transparent, difficult to see with normal illumination

• can be stained to make them visible

• special optics can be used to increase contrast

special optics used to increase contrast in light microscopy

• phase contrast

• differential-interference-contrast (DIC)

• dark field

types of samples

• cells in tissues

• tissue culture cells

  • fixed

  • alive

cells in tissues

• usually fixed, embedded in paraffin or plastic, and sectioned

• freezing and sectioning also used

tissue culture cells

• usually shaped like fried eggs and very flat

• easier to work with, but usually not normal

• growing on glass or plastic

advantages of methods to observe unstained live cells

• allow prolonged observation of live cells

• study movements in cell division and of intracellular structures

• can be filmed (microcinematography)

• often using inverted microscopes

organ culture

• whole organ is cultured

• keeps most of the physiological conditions from a living organism

explant culture

• organ is finely chopped and culture in growth medium

• easier to maintain in culture

• still presents 3-dimensional cell organization

continuous cell lines

• primary cell culture is digested by a protease and divided

• can be immortalized (activated telomerase)

• grow only in monolayer culture due to contact inhibition

primary cell culture

original cells from tissue sample

cell line culture

divided cells, descendants from primary cell culture

non-immortalized cell line

• primary cells are allowed to reproduce in tissue culture

• can be grown for many generations in tissue culture, but not indefinitely

immortalized cell line

• there are mutations (such as expression of telomerase) which allow indefinite growth in tissue culture

• cells otherwise retain good behaviour (contact inhibition)

transformed cell line

• cells lose contact inhibition and have other abnormalities, including abnormal mitosis

• cultured cancer cells are normally transformed

• non-cancerous cell lines can be transformed either through mutations or through the use of viruses or introduced DNA to express oncogenes

contact inhibition

• tissue culture cells obtained from primary cultures will form a single monolayer on a plate and will not grow once the space is filled

• cancer cells (such as HeLa cells) lack contact inhibition and will continue to grow in tissue culture, piling up on each other

requirements for maintaining cells in culture

• artificial medium

  • physiological pH (buffer, indicator)

  • nutrients (amino acids, vitamins, salts)

  • glucose

  • serum (growth factors)

  • antibiotics

• temperature

• sterile environment

fluorescence microscopy

• fluorescent molecules absorb light of high energy and emit light at lower energy

• tissues and cells are irradiated with a blue-violet or UV light

• fluorescent structures appear bright on black background

• sensitivity is very high

fluorescein

fluorescent molecule that absorbs blue light and emits green light

rhodamine

fluorescent molecule that absorbs light of high energy and emits red light

components of early fluorescence microscopes

• carbon arc lamp (emits UV)

• quartz collector lens

• excitation filter

• condenser

• specimen slide

• objective

• eyepiece

epi-fluorescence microscope (modern)

1. light source emits white light

2. first barrier film lets through only blue light

3. beam-splitting mirror reflects blue light towards objective lens, and is absorbed by specimen

4. specimen emits green light which passes through objective lens and beam-splitting mirror

5. second barrier film cuts out unwanted fluorescent signals, passing only specific green fluorescein emission

6. green light passes through eyepiece

autofluorescence

slight natural fluorescence from cells

how to label proteins

• chemically label protein outside cell and add it

• label antibody against protein and stain cell (must be fixed with formaldehyde and permeabilized)

• fuse protein of interest with green fluorescent protein and express (can be done while cell is still alive)