2-Photon Imaging Flashcards
Name drawbacks of fMRI compared to CI; give one advantage
Disadvantage: its not directly neuron activity, just blood flow
We can’t know the interaction between excitatory and inhibitory activity within that area
But we can have whole brain activity
At what neural level can CI answer questions in what time plane?
Almost the level of synapses (subcellular scale) to almost the level of maps; especially appropriate for the neuron and network level. Can measure at a sub second temporal accuracy and can measure over days.
Specifically, what does calcium imaging allow you to do?
Two-photon imaging allows the recording of fluorescence in living tissues at a
subcellular scale
What applications does CI have in neuroscience?
Technical revolution having found many applications in neuroscience:
- Imaging of morphological changes (spine formation, axon/dendrite motility)
- Targeting of specific neuronal subtypes
- Imaging of brain activity at the cellular level
What are the basic principles of 2 photon absorbtion
Fluorophore has a ground state energy level (stable), gets excited by light (blue) and jumps to higher energy level; eventually loses energy to heat and goes back to ground state energy level, emits light. Because of energy drop it can only emit light with longer wavelengths. Longer wavelengths (reddish hues) have a lower ground state, so one photon is not high enough to reach this level, two photons must hit simultaneously.
How can two photons hit simultaeously? i.e could this be done with LED?
You need a special laser; in a continuous wave laser the photons are dispersed throughout the beam, however with a mode locked laser these photons are released in concentrated bursts. This does not equal more power; e.g they may both be 20 photons/ unit time however the CW laser would have these 20 photons spatially and temporally dispersed while the ML laser would e.g release 4 aligned photons in 5 bursts.
Why does this laser allow two photons to hit simultaneously?
Two photon excitation is a very improbable event which will occur only when photons are highly concentrated in time. Two photon excitation can thus only be obtained with mode-locked (pulsed) lasers
What else is required for two photon excitation?
Two photon excitation is a very improbable event which will occur only when photon are highly concentrated in space. Light needs to be focused and fluorescence only occurs at the focal plane
Describe the process from when the light leaves the laser
A laser source provides near-infrared ultrashort pulses; intensity and beam size are adjusted before coupling the laser beam to the xy scanner and microscope. The focal lengths of the scan lens (fS), the tube lens (fT) and the objective (fO) are indicated in docs. Two-photon excited fluorescence (2PEF), which is isotropically emitted (inset), can be collected in epi- and/or trans-collection mode, using whole-area detection by photomultiplier tubes (PMTs). Forward-directed optical-harmonic and Raman signals are detected in transcollection mode in transparent samples. For in vivo experiments epicollection is used exclusively.
What is the purpose of the xy scanner and what happens as the light passes through it?
The laser beam hits a first scan mirror and reflects into a second scan mirror. These mirrors are closely spaced and controlled by two galvanometers (small motors) which can rotate the mirrors. This allows for moving the small laser beams fast with high accuracy and precision.
What is the purpose of the objective lens and what happens as the light passes through it?
At its simplest, it is a very high-powered magnifying glass, with very short focal length. This is brought very close to the specimen being examined so that the light from the specimen comes to a focus inside the microscope tube. For two-photon imaging they collect the scattered fluorescence and diverge the beam onto a focal point
Collectively what does the xy scanner and objective lens set out to achieve?
High concentration in precise space; As fluorescence can only be excited locally the laser beam needs to be focused across an image plan at high speed to reconstruct an image. This can be achieved using a scanner and objective lens.
Where does the light go from the tissue
Light collection is done serially and the image is then reconstructed. This is done either through transcollection or epicollection. With epicollection the light is reflected back into the objective lens and then reflected into a photomultiplier (PMT). In transcollection in small or transparent specimens, the light goes through the tissue and through a high-NA condensor. From there it is also deflected into a PMT.
Why is transcollection only done in vitro?
In most systems a ‘whole-area’ epi-detection scheme is used, with all light that is collected by the objective also guided onto the detector. For small or transparent specimens trans-collection through a high-NA condensor can be used instead or, better, in addition, capturing even more light. Because of the preferentially forward-directed signal, trans-collection is the principal detection mode in optical-harmonic generation and Raman scattering. In deep, scattering specimens, however, no light will penetrate, and epicollection is the only mode that can be used. Epicollection results in surprisingly little signal loss provided the detector’s field of view is large enough because most signal photons eventually leave the tissue surface after multiple scattering events.
How is the image the constructed?
Photomultipliers acquire the light through a glass or quartz window that covers a photosensitive surface, called a photocathode, which then releases photoelectrons that are multiplied by electrodes known as metal channel dynodes (reflected off each other (?)). At the end of the dynode chain is an anode or collection electrode. Over a very large range, the current flowing from the anode to ground is directly proportional to the photoelectron flux generated by the photocathode. This signal is then collected over times and image reconstruction is carried out using software.
To what extent do we need a CCD camera?
No need for a CCD camera. We know precisely where the fluorescence comes from. This allows the use of highly performant photomultipliers (PMTs); it is highly sensitive
(A charge-coupled device (CCD) is a light-sensitive integrated circuit that captures images by converting photons to electrons. A CCD sensor breaks the image elements into pixels. Each pixel is converted into an electrical charge whose intensity is related to the intensity of light captured by that pixel)
What two types of scanning mirror may be used and what are their key characteristics?
Galvanometers: Linearly transform an electrical input into motion (rotating the scan mirrors):
- ScanRate: ~4Hz
- depends on image physical span
- beam can be held to fixed position
- Linear scan path
Resonant scanners: Resonate at very high frequency (4-8 kHz):
- ScanRate: ~30Hz
- independent of image span
- beam cannot be held fixed
- Sinusoidal scan (needs correction)
What is a resonant scanner?
A resonant scanner is a type of galvanometric mirror scanner that allows fast image acquisition with single-point scanning microscopes (true confocal and multiphoton laser scanning). High acquisition speed is required to track fast processes, especially in living samples.
Is 2-photon excitation more or less suited to deep tissue measurements than traditional microscopy? Why?
Loss of power due to absorption and scattering increase exponentially with depth. Localisation of excitation is maintained even in strongly scattering tissue with two photon methods because the density of scattered excitation photons generally is too low to generate significant signal, making nonlinear microscopy (2-photon) far less sensitive to light scattering than traditional microscopy. This is of paramount importance for deep imaging, because it means that all fluorescence photons are known to originate from near the focus and thus can provide useful signal.
From lecture: Photons penetrate better into physical media when their wavelength increases, Two photon excitation uses higher wavelength; better light penetration into biological samples (up to 500 μm below the surface with a good objective).
Is 2-photon optogenetics more or less phototoxic than traditional optogenetics? Why?
Fluorescent molecules emit free radicals in their excited states which are toxic to tissue. In one photon excitation, fluorescence are excited wherever the light goes, photons have high energy. In two photon excitation fluorescence is excited only at the focal spot of the objective, photons have low energy. Therefore there is less phototoxicity and it is applicable in-vivo.
What is relevant about imaging require scanners?
Imaging requires scanning which limits the time resolution (~4Hz with classical galvanometers, ~30Hz with resonant scanners)
What about the cell means it can be imaged?
Fluorescence is not natural; Fluorescent molecules need to be brought to tissues with artificial means
