cell biology- microscopy light

robert huke (1655)

first person to look at cells

antonie van leeukwenhoek(1674)

looked at protozoa and bacteria

schlien and schwann (1838)

cell theory and industrial revolution

reticular theory

neurons aren’t cells

neuronal theory

entire body are made of cells

resolving power

how much you can see

resolving power of a light microscope

100 um (plant cell), 10 um (animal cell), 1 um (mitochondria)

resolving power for a electron microscope

100 nm (viruses and ribosomes), 10 um ( protein), 1 nm (other molecules)

limit of resolution on light microscope

0.2 um

what are the 2 major decisions for solving a problem using a microscope

1.which microscope to select 2. how do you prepare the specimen (a. fixing + dyes for dead tissue b. living cells)

carl zeiss + ernest abbe (1800s)

worked together to understand resolution

diffraction limited

can’t go beyond a certain point (almost all microscope)

not diffraction limited

super resolution micrcoscopy

what are the 2 types of resolution?

1.theorectical resolution= abbe’s equation (what you should see) and 2. practical limit of resolution (what you actually see)

practical limit of resolution means what

1.little substance/contrast and 2. heat leads to movement

abbes equation

d=0.61(wavelength)/n sin(theta)

how to generate contrast?

dyes, mainipulate light, or computer image enhancement

dyes to stain cells

hematoxylin for nucleus (purple), eosin for everything (pink) and fluorescent dyes

colorimeteric

absorbs certain wavelength but not others (H&E)

flurochromes

fluorescent dyes

brightfield microscope (compound light microscope)

a. pathologist: tissue selection b. fixed & processed

fixed and processed process

1.tissue/ organ 2. fixation (formaldehyde) 3. dehydration 4. replace w/ ethanol 5. “ “ xylene 6. replace w/ molten paraffin 7. repeat step 5 to 2

fibrosis

collegen fibers

is there an alternative for something faster?

cryosection- frozen sections makes it faster but histology is poor

mohs surgery

very serious skin cancer that uses cryosections (melanoma)

phase microscopy

purpose for cell culture biologist, use living cells, difference in refractive indices w/ organelles

differential interference contrast

a 3D image, uses living cells, anf a single cell electrophysiology + patch clamp

patch clamping

monitor ion flow through a single channel

dark field microscopy

useful for microbiologist and increasing contrast

polarizing light microscopy

used by neurobiologist and muscle biologist, purpose to look at highly ordered parallel structures

confocal microscopy

1.1957- original patent 2. 1970-first confocal built 3. 1988-1st commerically available confocal

major components of confocal microscopy

1.lasers (several mocrochromatic) 2. confocal pier hole 3. sums individual spots 4. optical sections 5. stero images 6. image multiple fluorescent dyes

problem with confocal microscopy

imaging takes times causing photobleaching leading to heat generated

the two types of confocal microscope

point scanning confocal microscope and spitting disk confocal microscope

point scanning confocal microscope

fixed fluorescent stained cells but photobleaching can occur

spinning disk confocal microscope

can be used for living cells, faster, less heat, decrease photobleaching, lower laser intensity required