3. Confocal microscopy

Question 1

photoswitch imaging

Answer 1

dSTORM or STROM, method where we make our fluorescence blink by them bleaching and recovering. The blinking is stochastic, so they blink at different timepoints. This makes it easier to separate the objects. This is a super resolution method, meaning that the resolution extends what is possible based on the wavelength, but it dependent on the size of the fluorescent antibody.

Question 2

super resolution imaging

Answer 2

the resolution extends what is possible based on the wavelength, but it dependent on the size of the fluorescent antibody

Question 3

Epifluorescence microscopy

Answer 3

Our “normal” microscopy, where the light is hitting the whole specimen and is reflected back, so we get a lot of blur

Question 4

two-photon microscopy

Answer 4

afluorescenceimaging technique that allows imaging ofliving tissueup to about one millimeter in thickness. Unlike traditionalfluorescence microscopy, in which the excitation wavelength is shorter than the emission wavelength,two-photon excitationrequires simultaneous excitation by two photons with longer wavelength than the emitted light. Two-photon excitation microscopy typically usesnear-infrared(NIR) excitation light which can also excitefluorescent dyes

Question 5

Confocal imaging

Answer 5

a technique where the light source is limited by a pinhole at the light source and at the detector. This makes the light hit the specimen more precisely, and the light that is mirrored back only researches the detector if it’s reflected from the exact point in the specimen that we’re interested in.

Question 6

Pros and cons of confocal imaging

Answer 6

It gives us less blur and therefore a picture with higher contrast (not necessarily higher resolution). However, it’s expensive and takes a lot of time

Question 7

common misconception about confocal imaging

Answer 7

That it increases resolution. This could in theory be true, but as this only occurs when the pinhole is infinitely small, this is not something we see, as the pinhole usually around the size of one airy disk. At this size, the resolution is the same as for epifluorescence microscopy, but we have a better contrast.

Question 8

Nyquist sampling rate

Answer 8

Nyquist sampling (f) = d/2, where d=the smallest object, or highest frequency, you wish to record. The Nyquist Theorem states that in order to adequately reproduce a signal it should be periodically sampled at a rate that is 2X the highest frequency you wish to record.

Question 9

Formula to calculate the width of that halfway point at a peak for the HWFM criterion

Answer 9

d = 0.51λ / NA