lec 2- light and electron microscopy methods

Question 1

who made the first light microscope via modifying older microscopes?

Answer 1

Hooke in 1660

Question 2

how did Hooke make the light microscope?

Answer 2

used lenses and a flame together with mirrors to illuminate the sample

Question 3

how did Hooke discover cells?

Answer 3

looked at a cork, which is made of bark of a tree, under a microscope and saw boxes that reminded him of the rooms monks live in

Question 4

what is considered a compound microscope?

Answer 4

a microscope that has more than one lense

Question 5

how did Hooke’s design of the light microscope improve over the years?

Answer 5

-better lenses
-better light sources, like light bulbs
-from light bulbs, to mercury arc lamps which use electricity to excite mercury vapor to generate light
-magnification
-resolution

Question 6

what is the smallest res a light microscope can see?

Answer 6

0.2 micro m

Question 7

what is the “ limit of resolution” also called?

Answer 7

the Abbe limit

Question 8

did Abbe actually make the formula for resolution ?

Answer 8

no, it was made 61 years prior but it didn’t matter cause he was a co-owner of zeiss (microscope company)

Question 9

what does the dichroic mirrors do in a microscope?

Answer 9

allows a certain wavelength to pass through, while reflecting others

Question 10

what is used in the microscope to magnify?

Answer 10

the objective lenses which are usually maxed out at 100x mag.

Question 11

what is the magnificaiton of the projection/eyepiece lens and what is the overall magnification of a microscope?

Answer 11

-10x
-10x multiplied by 100x = 1000x

Question 12

what is bright field microscopy, explain what occurs?

Answer 12

-unstained sample, uses few lenses and 1 light source
-light passes through condenser lens, which results in it being concentrated on the sample, then it goes through the objective lens, then to the projection lens, and finally to the eye
-small amount of detail seen

Question 13

what is phase contrast microscopy, what image does it produce?

Answer 13

-unstained sample, takes advantage of the refractive index
-produces an image with dark and light areas based on the refractive index of those zones of the sample or cell

Question 14

explain what happens in phase contrast microscopy?

Answer 14

-light moves slower in areas with a high refractive index
-light is refracted/bent when it passes through the object
-these microscopes use an annular diaphragm that allows some light to pass through, then some light passes through the phase plate, and then to the eye

Question 15

what is DIC microscopy and what does it do?

Answer 15

-unstained sample, differential interference contrast (AKA Nomarski interference contrast)
-breaks light into 2 perpendicular components
-light is then recombined, interference pattern is then observed
-useful for seeing small details in cells
-uses the refractive index of the sample and the surrounding material to generate what looks like shadows

Question 16

what does fluorescence microscopy help with?

Answer 16

-helps identify the location of specific molecules
-can use those molecules to label structures

Question 17

what does fluorescence microscopy use?

Answer 17

-bright light
-lenses
-dichroic mirrors
-detection devices

Question 18

when does fluorescence happen?

Answer 18

when a molecule absorbs light at a specific wavelength and emits it at a longer wavelength

Question 19

what do you not want when labelling something?

Answer 19

don’t want the emission Spectra from 2 colours to overlap, because it will be harder to tell which is which

Question 20

what occurs during fluorescence microscopy?

Answer 20

-light passes through excitation filter, which allows certain wavelengths to pass through it , then the dichroic mirror allows a certain wavelength through while reflecting the rest
-the light that reflects activates the fluorochrome attached to the molecule to be visible in the sample
-the fluorochrome then emits light at a different wavelength which passes through the dichroic mirrors all the way to the eye

Question 21

what is the difference between direct and indirect fluorescence microscopy?

Answer 21

direct: fluorochrome binds directly to molecule
indirect: primary antibody binds to sample, secondary antibody with flurochrome attached then binds to the primary antibody

Question 22

how is out of focus light generated?

Answer 22

when looking at 1 plane of the sample, the whole sample must be excited, causing out of focus light

Question 23

how is out of focus light removed?

Answer 23

-using computers to remove it
-change the light source

Question 24

what is phototoxicity?

Answer 24

during live imaging, high powered light sources (arc-lamps or lasers) are used to generate free radicals in the cells which can ultimately kill the cell

Question 25

how is phototoxicity fixed?

Answer 25

-lower the intensity of the light hitting the sample -decrease exposure time -put things in the light path

Question 26

what does a confocal microscope do?

Answer 26

-uses lasers to excite the sample -takes images from only 1 specific focal plane resulting in very little out of focus light -collects images from the entire sample 1 focal plane at a time

Question 27

what extra parts does the confocal microscope have compared to the regular light microscope?

Answer 27

-uses a pinhole to further ensure out of focus light is eliminated -uses a photomultiplier tube to build the image from the area of the sample being illuminated from the scanning laser

Question 28

what are the 2 problems with a confocal microscope?

Answer 28

1. its really slow, causing live events to be missed 2. because its slow and the laser is intense, the fluorescence molecules can bleach out

Question 29

how can the confocal microscope be fixed to solve the problems?

Answer 29

-the laser is shot through the spinning confocal which splits the light into many lenses and pinholes -this enables the scanning of the entire sample in milliseconds -this decreases the exposure time and the intensity of the laser -commonly used for live imaging

Question 30

how can un-needed damage on the sample from the light of a confocal microscope be avoided?

Answer 30

via two-photon/multiphoton microscopy

Question 31

what does two-photon/multiphoton microscopy do? why is it good and what is its one problem?

Answer 31

-uses multiple lasers at half of the intensity ( or 1/3 or 1/4) to excite the fluorescent molecule -generates less phototoxicity -ideal for live imaging -costs a lot of money