microscopy Flashcards
Who created the microscope that allowed you to see bacteria?
Leeuwenhoek
what magnification was the microscope Leeuwenhoek made?
300x
What are the two main functions of the microscope?
magnification and resolution
What is the ocular lens responsible for?
inverts what you are seeing
What is the parfocal responsible for?
ensures you don’t have to adjust your focus when switching between objectives
how many lenses are there on a microscope? what are they?
2, objective lense, ocular lense
what are the 4 objectives lenses?
4x, 10x, 40x, 100x
what magnification does the ocular lens have?
10x
how do you determine total magnification?
objective lens x ocular lens
what is resolution?
defined as the ability to distinguish two adjacent objects as distinct and separate
Resolution was mathematically done by whom? when?
ernst abbe, 1870’s
what are the two things resolution depends on?
physical property of light (wavelength) and numerical aperture
what does numerical aperture (NA) refer to?
light gathering capabilities of a lens
what is the equation for resolution?
R= 0.61λ / NA
how do you enhance resolution?
you have a better image when R is small
how do you make R small?
decreased the wavelength of light used and increase numerical aperture
what is the equation of numerical aperture?
NA= n x sin(theta)
why can you see better on 40x and 100x objectives?
they allow for more light so you can see your image more clearly
As you move through objectives you are increasing ________ and thus enhancing the ________ through the ocular.
numerical aperture, resolution
what is the 100x referred to as?
oil emergent objective
why does oil help enhance the image?
because oil has a higher refractor index of 1.5 than opposed to air 1.0
why doe you start with the 4x objective when looking at a sample?
it has the widest field of view
how is dark field microscopy different?
uses the opaque light stop, and uses a dark background
T/F you cannot use staining procedures when using dark field microscopy.
true
when do you use dark field microscopy? example?
when samples are hard to stain, spirochetes
what would you use phase contrast microscopy for?
use to look at unstained organisms that are ALIVE. Gives you the ability to watch motility and inclusion bodies of cells
why cant you use stains on bacteria you want alive?
stains kill bacteria
immunofluorescence microscopy typically requires a ______ attached to an _____.
flourophore, antibody
confocal microscopy
when you concentrate the excitation light to a specific focal point. this can create 3D images of the cells
z series
allows you to have that 3D image of the cells
what is the advantage of using electron microscopy?
electrons have a much smaller wavelength of light which equals much higher resolution
what level of magnification can electron microscopes have?
100,000x - 300,000x
T/F electrons can go through glass
false, they cannot so we use electromagnetic lenses
what do electromagnetic lenses do? what do they require when using them?
Control the beam of electrons, require a vacuum which protects them form vibrations
what are the two types of electron microscopes?
transmission, scanning
what kind of sample does the transmission electron microscope require?
extremely thin specimen (20-100nm)
are you able to see some internal structures using the transmission electron microscope?
yes, but in 2D
scanning electron microscope
electrons don’t pass through the sample, they just hit the sample.
the microscope collects what comes back at us (back scattered electrons and secondary electrons)
back scattered electrons
electrons that originate from the beam
secondary electrons
electrons from atoms within your sample
