2. Fluorescents

Question 1

DAPI

Answer 1

DAPI is a blue-fluorescent DNA stain that exhibits ~20-fold enhancement of fluorescence upon binding to AT regions of dsDNA. It is excited by the violet (405 nm) laser line.

Question 2

laser line exciation

Answer 2

the wavelength of the laser we use when looking at out specimen in order to have maximum excitation and thereby maximum emission of light in the specimen

Question 3

Immunostaining

Answer 3

Immunostaining is a general term in biochemistry that applies to any use of an antibody-based method to detect a specific protein in a sample. Tagging of a fluorophore to an antibody improves the visualization of the antigens or antigen epitopes where the antibody binds.

Question 4

The direct methods of immunostaining

Answer 4

A directly labeled fluorophore-conjugated primary antibody is used to detect a target protein. The direct method is simpler, more convenient, and less prone to artifacts than the indirect method

Question 5

The indirect methods of immunostaining

Answer 5

A broader species-specific fluorophore-labeled secondary antibody is used to detect and attach to a primary antibody bound to a target protein. This increases signal amplification (several fluorophore-conjugated secondary antibodies binding to a single primary antibody) and is usually cheaper and therefore preferable.

Question 6

GFP

Answer 6

Green Fluorescent Protein (GFP) is a versatile biological marker for monitoring physiological processes, visualizing protein localization, and detecting transgenic expression in vivo. GFP can be excited by the 488 nm laser line and is optimally detected at 510 nm

Question 7

Stokes fluorescence

Answer 7

emission of a longer-wavelength photon (lower frequency or energy) by a molecule
that has absorbed a photon of shorter wavelength (higher frequency or energy).

Question 8

Stokes shift

Answer 8

the shift in energy between the excitation and emission, which is represented as heat

Question 9

Quantum efficiency

Answer 9

photons emitted / photons absorbed. The measure tells you about how much light you need to see your specimen (if the efficiency is low you’re using a lot of light to see little, and it might ruin your specimen)

Question 10

Photobleaching

Answer 10

photobleaching (sometimes termed fading) is the photochemical alteration of a dye or a fluorophore molecule such that it permanently is unable to fluoresce. This is caused by cleaving of covalent bonds or non-specific reactions between the fluorophore and surrounding molecules.

Question 11

Photobleaching efficiency

Answer 11

probability of bleaching / photon absorbed. We want this number to he high, so that there is low alteration in the dye that might make in unable to shine

Question 12

Super-resolution microscopy

Answer 12

Super-resolution microscopy (SRM) bypasses the diffraction limit (max level of resolution for current microscope settings), a physical barrier that restricts the optical resolution to roughly 250 nm and was previously thought to be impenetrable

Question 13

pros and cons of a black and white camera

Answer 13

Pros: we get maximum contrast, Cons: we might not be able to see if any other wavelengths that the ones we are expecting are bleeding through when using multiple stains

Question 14

Excitation filter

Answer 14

Filters away anything that is not within the interval of wavelengths we’re interested in, usually around the laser line for the dye we’re using

Question 15

Emission filter

Answer 15

Filters away anything that is not within the interval of wavelengths we’re interested in, usually around most likely wavelength of the emission of the chosen dye

Question 16

Dichroic mirror

Answer 16

mirrors the filtered rays down to the specimen and allows the emission rays to pass to the detector

Question 17

Band pass filter

Answer 17

sets an interval for which wavelengths we want. Can be used both for excitation and emission.

Question 18

Long pass filter

Answer 18

Used for emission filtering when you want any wavelength above the minimum emission criteria. Can make GFP dyes look more yellow/red due to higher wavelengths passing through. Can make contrasts harder to figure out, but there is in theory more light. Useful for live or calcium imaging where we don’t care about wavelengths but only photons.

Question 19

Filter cube

Answer 19

Holds the excitation filter, emission filter, and dichroic mirror

Question 20

How to deal with broad emission wavelengths

Answer 20

If the interval is too large and spans multiple colors, we use a band pass filter to make sure it doesn’t overlap with the excitation wavelengths on the short-wavelength side. On the long-wavelength side, we make sure it includes the max emission point and is kept to one color, so we could use other stains with it.

Question 21

Mercury lamps as a light source

Answer 21

Typically used (also in Dr. Blum’s lab) and works for 200-400 hours

Question 22

LED lamps as a light source

Answer 22

The future of light sources, as they don’t burn out

Question 23

Lasers as a light source

Answer 23

Gives monochromatic light for 6 - 10K hours.
Pros: has high specificity due to it being monochromatic, creates a light point so we only light up a part of the specimen.
Cons: VERY expensive

Question 24

Digital cameras in microscopy

Answer 24

CCD and CMOS

Question 25

The photoelectric effect

Answer 25

The emission of electrons when electromagnetic radiation, such as light, hits a material. Increasing frequency leads to more negative electrons and increasing the intensity led to more ejected electrons.

Question 26

photodiode

Answer 26

a semiconductor device that can turn light into an electrical signal. They’re used for quantification of light and its intensity

Question 27

CCD

Answer 27

Digital camera with a serial/passive read-out of pixels. Is slower than CMOS due to the analog bottleneck of processing an entire line of pixels in one go

Question 28

CMOS

Answer 28

Digital camera that has a local read out of pixels. It more sensitive and faster than CCD

Question 29

Binning

Answer 29

Creates one pixel out of multiple pixels. Decreases resolution but creates more light

Question 30

Bit / gray values

Answer 30

The ‘bit’ of a picture is how many times 2 should be multiplied with itself (8 bit = 2^8), which gives the amount of gray values available (256). This determines the “steps” of gray we can take. If we reach the max, the pixel is white and it’s saturated.