light microscope

what type of image is produced by a light microscope

an absorption image

what is the Dmin?

the resolution limit, the smallest distance at which two points can still be distinguished

what does resolution of the lens mean

the ability of the lens to distinguish between two close together points as separate

what is the equation for Dmin 

1/R = Dmin 

R = the resolution (unitless or in mm / lines) 

Dmin = the resolution limit 

is the dmin and resolution proportional or inversely proportional?

inversely proportional

• better resolution (smaller Dmin), lower resolution (higher Dmin)

what is abbe’s equation?

dmin = 0.5 * λ / NA

λ = wavelength of light in nm or micrometers

NA = numerical apperture

What’s Abbe’s equation when numerical aperture is not given directly 

dmin = 0.5  λ / n * sina 

n = refractive index of medium between object and lens 

a = semiangle of the maximum cone of light entering the lens 

does a higher NA result in a better or worse dmin?

a better dmin, as a higher NA due to the equation means a smaller dmin

e.g. if the top part of the equation was 10 and the bottom part 5 dmin would be 2 which is better if the top part of the equation was 10 and the bottom part 2 then the dmin would be 5 which would be worse.

why would immersion oil be used 

to match the refractive index of the medium to the glass slide of the specimen and the lens of the microscope → minimises light refraction and maximises light capturing resulting in a clear image 

e.g. air has a refractive index of 1.003, and the glass slides 

what is NA

How much light a lens can collect from a specimen and how finely it can resolve detail 

does resolution depend on NA

yes

why is using blue light better for resolution over using red light?

due to Abbe’s equation; a smaller wavelength → smaller λ -→ smaller dmin → better resolution

• blue light has a smaller wavelength (470nm) than red light (700nm)

what is the equation for total magnification? 

magnification of objective lens (4,10,40) x magnification of occular lens (which is always x10) 

does increased magnification mean better resolution?

not all the time, it depends on the image produced

what is a useful magnification and what is an empty magnification

useful magnification is when increased magnification provides an image that is detailed and clearer

empty magnification is when increased magnification leads to an image that is blurred / provides no new information

light passes through the specimen and as a result some wavelengths are absorbed differently depending on tissue density and stain,

how do more dense areas look compared to less dense areas / clear areas and why?

dense areas absorb more transmitted light → appear darker

clear areas absorb less transmitted light → appear brighter

describe the image from the objective lens

real, inverted and magnified

describe the image from the eyepiece

virtual, inverted, magnified

which part of the microscope is responsible for the resolution

objective lens

which part of the microscope is responsible for the contrast

condenser

which parts of the microscope is responsible for the magnification

objective lens and ocular lens

what is the resolving power of a microscope?

(same as dmin) the smallest distance between two points that a microscope can distinguish as separate

why do we apply immersion oil outside of it improving resolution at a higher magnification

to increase and match the refractive index of the medium to the refractive index of the glass slide of specimen and objective lens to minimise light refraction to prevent blurring in the image produced 

how do we apply immersion oil

apply small drop of immersion oil on the cover slip after focusing at a low power, rotate the 100x objective oil immersion lens so that it contacts the immersion oil. 

the condenser (or maybe light source) isn’t centred properly → uneven illumination 

name the different behaviours and state which one is essential for light microscopy

• transmission

• refraction (light bends when passing through materials of different RIs like from glass slide to air and then lens)

• absorption (diff parts of the specimen absorb light differently → contrast)