2. Fluorescents Flashcards
DAPI
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.
laser line exciation
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
Immunostaining
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.
The direct methods of immunostaining
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
The indirect methods of immunostaining
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.
GFP
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
Stokes fluorescence
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).
Stokes shift
the shift in energy between the excitation and emission, which is represented as heat
Quantum efficiency
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)
Photobleaching
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.
Photobleaching efficiency
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
Super-resolution microscopy
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
pros and cons of a black and white camera
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
Excitation filter
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
Emission filter
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
Dichroic mirror
mirrors the filtered rays down to the specimen and allows the emission rays to pass to the detector
Band pass filter
sets an interval for which wavelengths we want. Can be used both for excitation and emission.
Long pass filter
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.
Filter cube
Holds the excitation filter, emission filter, and dichroic mirror
How to deal with broad emission wavelengths
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.
Mercury lamps as a light source
Typically used (also in Dr. Blum’s lab) and works for 200-400 hours
LED lamps as a light source
The future of light sources, as they don’t burn out
Lasers as a light source
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
Digital cameras in microscopy
CCD and CMOS
The photoelectric effect
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.
photodiode
a semiconductor device that can turn light into an electrical signal. They’re used for quantification of light and its intensity
CCD
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
CMOS
Digital camera that has a local read out of pixels. It more sensitive and faster than CCD
Binning
Creates one pixel out of multiple pixels. Decreases resolution but creates more light
Bit / gray values
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.
