Microscopy III

Question 1

Describe digital wide field microscopy - gen

Answer 1

Digital cam - typically ccd or cmos
Typically attached to fluorescence microscope

Question 2

Describe digital wide field microscopy - camera

Answer 2

Usually v sensitive - accurately measures light
Often cooled to reduce noise bc fluoresce often dim

Question 3

Describe digital wide field microscopy - image

Answer 3

Fluorescence in images can be accurately quantitated
Images similar to what is seen with eyepiece - same for reg micro crops

Question 4

What are digital images made up of

Answer 4

Small elements called pixels
Light intensity represented by a number for each pixel in an image
Allows easy store in computer files, and with modern cams - also accurate quantitation
Add #s together = measure fluorescence

Question 5

Describe greyscale images

Answer 5

Greyscale images or images with one colour = have one number associated with each pixel
(Histology images = have much more illumination and photographed with colour cam)

Question 6

Describe colour images

Answer 6

Multi Channel images - have one number associated with each pixel per colour/channel
Typically 3 = red, green, blue for none microscope digital images

Question 7

Describe microscope cameras

Answer 7

Typically microscope cameras used for fluorescence images are greyscale only
Multiple images can be taken of same field using diff excitation/emission filters = images can be viewed side by side or easily combined to produce colour images

Question 8

Describe fluorescence images

Answer 8

Dyes and filters normally chosen so that each image corresponds cleanly to a diff due
For display- colours typically assigned to red/green/blue, corresponding to 3 colour photoreceptors in eye
Or each of the og greyscale images can be shown side by side
Measure background flourensce and minus out
Combine colours =get full image

Question 9

Describe cds/cmos images - like eye

Answer 9

Show what eye would see through same filters
But camera more sensitive to low levels of light
Camera detects out of focus and in focus light like eye

Question 10

Describe cds/cmos images - depth

Answer 10

Depth of field - in focuse zone is extremely narrow - in light microscopy - especially with high res lens
So light from out of focus structures = considerable problem for thick samples

Question 11

What does confocal microscopy help with

Answer 11

Thick sections

Question 12

What are the limitations of regular traditional microscopy

Answer 12

Traditional microscopes only produce high quality pics at high magnification when specimen thin
Otherwise = sharpness reduced by images from out of focus part fo specimen
3d structure lost when thin section tho

Question 13

How is confocal microscope better

Answer 13

Uses lasers, fluorescence optics and computers to avoid above limitations

Question 14

Describe confocal microscope - how it works

Answer 14

Laser focused onto cell, point of focus strongest, but lots of out of focus light from sample - large field of where fluorescence will be recognized
So used pinhole - computer controls image

Question 15

Describe image - how is confocal better

Answer 15

100 microns drosophila - v thick
See structures way better with confocal

Question 16

How are confocal microscope specimens scanned

Answer 16

Specimen scanned point by point by a laser beam controlled by computer

Question 17

What is a confocal microscope

Answer 17

Fluorescence microscope with highly specialized optics - computer to keep track

Question 18

Name biological uses of confocal micropsies

Answer 18

3d or single slices from fixed samples - including v thick samples
3d or single slices from living cells
Time lapse living cells
Photo bleach techniques
Quantitative information on any of above

Question 19

What is photo bleaching - when is it a problem

Answer 19

Problem with fluorescence microscopy

Question 20

What is photo bleaching - explain

Answer 20

Once excited - fluorophore remains excited for 1-10 nanoseconds - before re emitting
While excited = can react with molecular oxygen = destroy fluorophore

Question 21

Can photo bleaching be useful

Answer 21

When high energy of light - usually laser applied to defined region = fluorescent molecules continuously in excited state
Bleaching can be introduced in controlled manner = useful

Question 22

What is frap

Answer 22

Fluorescence recovery after photobleaching

Question 23

Why is frap used

Answer 23

Can determine whether pops of fluorescent molecules inside cells move or are stationary - live cell

Question 24

Describe frap ex

Answer 24

Can detect diffusion of molecules in pm
Hit bleached area and measure diffusion coefficient

Question 25

What can be measured by frap

Answer 25

Diffusion of moelcuels

Question 26

Describe how frap can measure diffusion of molecules

Answer 26

Ex = protein integral membrane protein of er Region of cell photobleached using laser of confocal microscope Bleached area refills as molecules diffuse in (we already know protein cannot leave eR)

Question 27

Describe diffusion

Answer 27

With a membrane - doesn’t require energy - Brownian motion Diffusion rate = quantitated as diffusion coefficient

Question 28

When is frap harder for seeing diffusion of proteins

Answer 28

Harder for cytoplasmic proteins bc move around v fast compared to proteins stuck in membrane

Question 29

What else can frap see

Answer 29

Movement of proteins between organelles Bleach one organelle and watch it refill over time If proteins moved in membrane bound vesicles = need energy

Question 30

Describe ex of frap seeing proteins move between organelles

Answer 30

See movement Then see organelle refill Can measure how fast coat protein comes on