Fluorescence Microscopy Flashcards
Cellular structures are visualized based on what? and through which the of microscope?
on light emission by the specimen and through a fluorescence microscopy
How do you produce a fluorescent image
the specimen is illuminated with light at a wavelength that excites natural fluorescent or synthetic fluorescent dyes
What can the natural fluorescent and synthetic fluorescent dyes do?
bind directly to the structures in the specimen or can attach to antibodies that specifically recognize antigen in cells or tissues
What is epifluorescence?
excitation light directed onto the specimen from the viewing side
What is the purpose of a dichroic mirror?
To keep reflected excitation light from entering the optical system
How does the dichroic mirrors work?
allows emitted wavelength but not excitation wavelengths to pass is placed in the light path
How is sensitivity enhanced?
By using emission filters
How does emission filters work?
allows emitted light of the desired wavelength range to pass through
What how many image can traditional fluorescence microscopes have?
a single fluorophore at a time
When is two or more images of a fluorophore acquired?
When a specimen contains multiple fluorophore with different excitation and emission wavelength
What is the collected images called?
Channels
Channels combined digitally are use to create
A composite image
The light emitted by the fluorophores is often
very weak or at a wavelength that is difficult for the human eyes to detect
Digital cameras used with fluorescence microscopes usually record images in
grayscale
Why do the digital cameras used with fluorescence microscopes usually record images in grayscale?
because the color doctors are less sensitive
What happens before the channels are combined
They are assigned a false color that matches the color of the emitted light
What do newer microscopes use
single-color LED excitation sources and multi-bandpass filters
True or False: Are there sometimes “bleed-through” between channels
True
What is one limitation epifluorescence microscopy?
Its difficult to determine if a combined signal tells us if two fluorophores are in very close proximity within the specimen
What solves the limitation of epifluorescence microscopy?
confocal microscopy
Structures that are truly co-localized ____
appear in the same virtual section
What’s another benefits of the confocal microscopy?
Sequential sections can be stacked on top of one another to create a three-dimensional image(a “Z stack”)
Another difference between epifluorescence microscopy and confocal microscopy?
confocal microscopy images are more clear
