Lecture 3 Flashcards
Type of camera used is digital wide field microscopy?
CCD or CMOS
How does the camera reduce noise?
By being cooled
What makes up the digital image?
pixels
Light intensity number and the total fluorescence?
-Light intensity for each pixel is represented by the number
-Total fluoresnce is the sum of all light intensity numbers
Digital wide field microscopy
Entire sample is illuminated with light capturing a wide field view
T/F: Greyscale images with one color have one number associated with each pixel
True
T/F: Color images have one number assoociate with each pixel per color?
Ture
Ex. Red-green-blue =3
Microscope camerase used for fluorescence images are greyscale?
True, multiple images can be take of the same field using different excitation filters and these can be combined to produce color images
T/F: camera will detect out of focus light and in focus light?
True
T/F: Depth of field is extremely narrow in LM?
True this means the in focus zone is a tiny part especially for higher resolutions
Confocal Microscope
Allows us to deal with thick samples without physically cutting them into sections
Why do traditional microscopes require thin samples?
In thick samples sharness is reduced due to out of focus parts
Advantages of confocal microscopy?
Do not need to section the sample to get in focus parts and do not lose the 3D structure
How do we get the full image of the cell in confocal microscope?
The laser beam controlled by the computer scan the specimen point by point
Uses of confocal Microscopes?
- Can make a 3D image or single slice from a fixed sample (can be thick)
- Can make a 3D image or single slice of a live ssample
- Can time lapse living cells
- Photobleach
Photobleaching
When a high energy of light (laser) is applied to a fluorescent molecule an causes it to remain in a continuously excited state the molecule reacts with O2 which renders the molecule non fluuorescent
FRAP(fluorescence recovery after bleaching)
Determines whether populations of fluorescent molecules inside cells move or are stationary
What happens when you photobleach an area in the plasma membrane?
Since membrane proteins move around, overtime the bleached area will become fluorescent again
Phtobleaching used to quantify diffusion rate?
The diffusion coeeficient can be quantified by the amouunt of time it takes the photobleached proteins to be replaced
FRAP : movement of fluorescent proteins between organelles?
- Bleach an organelle
- It will refill overtime
