Notes on – Observing the cell in its native state: imaging subcellular dynamics in multicellular organisms (Betzig et al.)


No Comments

PDF Version: Notes on – Observing the cell in its native state imaging subcellular dynamics in multicellular organisms (Betzig et al.) – Logan Thrasher Collins

Overview

  • Eric Betzig, a 2014 Nobel laureate in chemistry (for playing a key role in developing super-resolution fluorescence microscopy), has continued to advance biological imaging technologies.
  • In their 2018 paper, “Observing the cell in its native state: imaging subcellular dynamics in multicellular organisms,” Betzig’s group combined lattice light sheet microscopy and adaptive optics to acquire 3-dimensional images and videos of physiological processes in vivo with subcellular resolution, achieving unprecedented detail and clarity.

Fig.0

Image source: (Liu et al., 2018)

Light sheet microscopy

  • Light sheet microscopy uses a laser and a cylindrical lens to project a plane of illumination through a sample.
  • As the sheet only illuminates a single plane of the sample at a time, this technique decreases photodamage relative to other methods which pass light through the entire sample at once. In addition, the single plane illumination decreases Fig.1background noise and so generates images with high contrast.
  • Light sheet microscopy operates rapidly since it scans entire layers of the sample all at once rather than scanning one point of light at a time (the latter is common with other types of microscopy).
  • Each layer of the sample is imaged in this way before the layers are stacked to reconstruct a 3-dimensional image. Betzig’s group used a mathematical algorithm called deconvolution to remove out-of-focus light and enhance more focused light within the Z-stacks generated by his lattice light sheet microscopy technique.

Image source: Jan Krieger, CC BY-SA 3.0, commons.wikimedia.org

Lattice light sheet microscopy

  • In lattice light sheet microscopy, the input laser is first stretched (in the x direction) by a pair of cylindrical lenses and then compressed (in the z direction) into a sheet by another pair of cylindrical lenses oriented perpendicularly to the first pair.
  • Lattice light sheet microscopes combine Bessel beams with 2D optical lattices using an optical element called a spatial light modulator. The spatial light modulator uses a ferroelectric liquid crystal display to create programmable gratings that diffract incoming light into a customized pattern. This is displayed in the figure from Förster et al., where a spatial light modulator displays a customized grid of white squares (light can pass through) and black squares (light is blocked) which forms a diffraction grating.Fig.2
    • Bessel beams are fields of electromagnetic radiation which can be mathematically described by Bessel functions of the first kind. Unlike most electromagnetic radiation, Bessel beams do not spread out as they propagate. As such, they do not form diffraction patterns. Although ideal Bessel beams are physically impossible, but approximate forms of the phenomenon can be created.
    • 2D optical lattices arise from the interference of beams of light that exhibit periodic behavior in two dimensions. They can form the same set of patterns found in 2D Bravais lattices (which are a mathematical formulation for crystal structures).
  • On their own, neither Bessel beams nor 2D optical lattices are useful for light sheet microscopy, but they achieve superior properties for imaging when properly implemented together.
    • Bessel beams contain the same amount of energy in their “side lobes” as they do in their central peaks. For this region, they cause excessive illumination outside of the targeted plane. This is problematic for light sheet microscopy since the technique depends on having a focused plane of light.
    • Despite their name, 2D optical lattices extend into 3D space (since many “copies” of the planar lattice occur along the z direction). This is similarly problematic for light sheet microscopy since the technique depends on the light exhibiting confinement to the xy plane of focus.
    • These issues can be overcome by combining Bessel beams and 2D optical lattices. To achieve this, a ring (or annulus) of illumination is used to destructively interfere with the side lobes of the Bessel beams, creating Bessel-Gauss beams. Then an array of coherent Bessel-Gauss beams is generated (two waves with a constant phase difference, the same frequency, and the same waveform are coherent). This array of Bessel-Gauss beams is suitable as a light sheet for lattice light sheet microscopy.

Image source: (Förster et al., 2014)

Adaptive optics

  • The light used for excitation in lattice light sheet microscopy traverses different regions of the sample relative to the detected light. As such, the light involved in excitation and the light involved in detection are subject to different aberrations.
  • To adjust for such aberrations, Betzig’s group used a two-photon excitation beam from an ultrafast Ti:Sapphire laser. This beam creates a “guide star” (this term comes from a similar technique used in astronomy) which acts as a reference. The guide star is scanned over the entire focal plane so as to compute an average correction since average correction has more accuracy than a correction from a single point in the sample.
  • Next, a switching galvanometer SG1 (a device which rotates a mirror back and forth) facilitates transfer of the two-photon excitation beam to either the excitation or detection arms of the microscope as needed. Fig.3
  • For the detection beam, the light generated from the scanned guide star is collected and sent to a device called a Shack-Hartmann wavefront sensor using another switching galvanometer SG2.
    • The Shack-Hartmann wavefront sensor contains an array of small sensors which measure the “tilts” of the incoming plane waves. By measuring the local tilt of each small wavefront composing the detected light beam, the overall shape of any optical aberration can be approximated.
    • Then a deformable mirror is modified to precisely compensate for the aberration measured by the Shack-Hartmann wavefront sensor. The second switching galvanometer SG2 transfers the light to this deformable mirror. After reflecting from the deformable mirror, the aberration is corrected and the detection objective collects the light to create an image.
  • For the excitation beam, the light from the two-photon excitation laser is scanned over the sample as a guide star and then collected by the excitation objective.
    • The collected light is transferred to another Shack-Hartmann wavefront sensor. Once again, the sensor measures an approximate representation of any optical aberration which occurs.
    • Next, the spatial light modulator used in creating the light sheet itself applies the appropriate correction to the excitation beam. This is highly effective since the spatial light modulator provides exquisite control over the output light sheet. 

Image source: (Liu et al., 2018)

References

Chen, B.-C., Legant, W. R., Wang, K., Shao, L., Milkie, D. E., Davidson, M. W., … Betzig, E. (2014). Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution. Science, 346(6208). Retrieved from http://science.sciencemag.org/content/346/6208/1257998.abstract

Förster, R., Lu-Walther, H.-W., Jost, A., Kielhorn, M., Wicker, K., & Heintzmann, R. (2014). Simple structured illumination microscope setup with high acquisition speed by using a spatial light modulator. Optics Express, 22(17), 20663–20677. http://doi.org/10.1364/OE.22.020663

Liu, T.-L., Upadhyayula, S., Milkie, D. E., Singh, V., Wang, K., Swinburne, I. A., … Betzig, E. (2018). Observing the cell in its native state: Imaging subcellular dynamics in multicellular organisms. Science, 360(6386). Retrieved from http://science.sciencemag.org/content/360/6386/eaaq1392.abstract

 

Only a demon could love the man made from metal (flash fiction)


No Comments

    Fluorescent flowers blossom from the metallic man’s throat and cascade over the dance floor, exploding into blue fireworks and purple lightning and blazing bursts of scarlet stars. He’s arm in arm with a radiant succubus, a pale girl with eyes the color of freshly spilled blood on a hospital napkin. The pair spin together, faster and faster and faster, sapphire flames flaring with every tender touch.

     “Forever and always?” He asks the demon.

     “Forever and always.” She replies and kisses him. They look into each other’s eyes and she opens her mouth, as if to offer another proclamation of love. But a vile horsefly crawls from her throat, latches itself to the metallic man’s neck, and slides a piercing black tubule into his spine. The metallic man looks back at the girl with an expression of shock and betrayal. She smiles at him, ignoring the filthy insect affixed to his throat. He impales the fly on a fingerblade and flicks it away. The gorgeous girl holds the metallic man lovingly. Then dozens of the horseflies emerge and begin a feeding frenzy.  

     As the flies inhale the metallic man’s marrow, they swell to the size of ravens. He staggers cross the dance floor, the horde of engorged insects coating his silvery flesh. The succubus twirls to a garden table in the middle of the ballroom as the metallic man’s bismuth bones crunch audibly. With difficulty, he follows her and sits down. Some flies drop from his warped skin and splatter onto the dark floor like pus-filled balloons. The metallic man pleads with his lover.

     “I need youu. Stop hurrrting me. I love youu. Please. Please. Please stop.” A particularly large and ugly fly attempts to cram itself into his mouthhole. The metallic man grabs the fly and crushes it in his mangled fingers. He spits and raves incoherently for several seconds. The succubus sits across from him and smiles prettily, so the metallic man rises and throws the table aside with inhuman strength, rips open his lover’s chest, and crushes her heart. He feels the ocean of her blood erupt between his mangled digits.

     The metallic man clutches at his chest and looks down. His own heart is exposed, its fatty yellows and lifeblood reds melting into each other like hot gelatin. The flies multiply like pathogenic amoebas and swarm round him while he tries to keep his left ventricle from oozing through his fingers. But he is powerless to stop the feast. The obese horseflies slip proboscises into his thoracic cavity and drain the gelatin away before they die, twitching on their backs.

     The metallic man searches for a door out of the ballroom. But no door can be found and he is trapped with the unbearable stench of rotting blood. The metallic man raves gibberish and flails about and falls to his knees by the remains of his love, weeping tears of molten gold. He cries continuously for nine hundred and ninety seven hours without once falling into sleep. At last, the metallic man gathers the cooling droplets of gold and assembles them, one by one, into a slim rocketship. He opens the hatch and climbs inside. By now, the beautiful succubus has risen. Though her ribs are shattered, she is very much alive. The metallic man gazes at her longingly.

     “I love you forever and always.” He says, anguish clear in his voice. “But if we keep dancing, you will take me apart piece by piece.” The demon snarls like a rabid dog and turns away. Then the metallic man presses a button and his rocketship punches through the ballroom’s ceiling, carrying him to uncharted skies.

Notes on two-photon microscopy


No Comments

PDF version: Notes on two-photon microscopy – Logan Thrasher Collins

Hardware for two-photon microscopy

(Svoboda & Yasuda, 2006)

  • In two-photon microscopy, a rapidly pulsed laser excites a fluorophore via two lower energy photons (which are typically red to infrared range). The fluorophore then emits a higher energy photon which is detected by the microscope.
  • Two-photon microscopy is usually implemented using a laser scanning microscope. The laser is tightly focused on a small focal volume, then scanned over the sample’s area to acquire information from each point within the sample. When the laser overlaps with fluorophores, photons are emitted from the given focal volume. All of these points are mapped to pixels and the full image is reconstructed computationally.
  • Since the focal volumes used in two-photon microscopy are small, high excitation intensities are needed to generate enough emitted signal. This necessitates rapid trains of short pulses from the excitation laser. Mode-locked Ti:sapphire lasers have a frequency of about 100 MHz, a pulse duration of about 100 femtoseconds, and their wavelength can be tuned from about 700-1,000 nm. These properties are nearly ideal for most fluorophores. If the required wavelengths are over 1,000 nm, other types of lasers are available as well.
  • Some examples of fluorophores used with two-photon microscopy include transgenic fluorescent proteins (i.e. XFPs), organic calcium indicator dyes, and other types of fluorescent dye molecules.

Advantages of two-photon microscopy

(Svoboda & Yasuda, 2006)

  • The absorption rate of the fluorophore is given by the intensity of the laser squared. This nonlinear process allows excitation to occur only within the described small focal volumes. When using a high numerical aperture objective, the diameter of an individual focal volume can reach down to just 100 nm. Fig.1
  • Since two-photon microscopy uses long wavelengths, the light penetrates tissue more deeply than with most other techniques.
  • As a result of the nonlinear excitation process which requires two photons in order to excite a fluorophore, single photons scattered by the tissue are too dilute to cause much off-target fluorescence.
  • The only photons which do experience scattering are those which come from the localized focal volume. As such, they still provide useful signal if detected. In traditional fluorescence microscopy, scattered photons are typically lost or they contribute to background fluorescence.
  • It is relatively easy to modify confocal fluorescence microscopes to create two-photon microscopes. Academic laboratories sometimes perform such modifications in-house.

Selected applications of two-photon microscopy

(Ellis-Davies, 2011)

  • Cortical neurons within transgenic mice expressing GFP and transgenic mice expressing YFP were imaged in vivo using two-photon microscopy by the Gan and Svoboda groups respectively. The fluorescent proteins were expressed only by a subset of neurons, allowing for higher contrast against the background. In this way, entire dendritic trees of pyramidal neurons were imaged at depths of up to 500-600 Fig.2 μm. The method established that, under the conditions used, most of the dendritic spines in the mouse neocortex remain fairly stable over a period of one month.
  • Later studies using similar techniques but with deliberate ablations to various sensory systems showed greater destabilization of dendritic spines, indicating that sensory memories might be partly encoded in spine patterns.
  • Two-photon microscopy also helped reveal that spines in the motor cortex show significantly greater stabilization during motor learning tasks than spines elsewhere in the cortex. This was demonstrated in both mice and songbirds.

Two-photon photolysis of caged neurotransmitters

(Matsuzaki, Hayama, Kasai, & Ellis-Davies, 2010)

  • The Ellis-Davies group developed modified glutamate and GABA molecules that include moieties which block their biological function. Such caged neurotransmitters are initially inactive, but can undergo photolysis when exposed to the excitation lasers used in two-photon microscopy, releasing the active molecules. Fig.3
  • Two-photon photolysis was used on caged glutamate within neural tissue samples, causing localized release of glutamate and the induction of action potentials.
  • Similarly, caged GABA was photolyzed by two-photon excitation. By combining this technique with patch-clamp recording, electrical activity was mapped on neural membranes, allowing GABA receptor distributions over the membranes to be estimated.

 

References

Ellis-Davies, G. C. R. (2011). Two-Photon Microscopy for Chemical Neuroscience. ACS Chemical Neuroscience, 2(4), 185–197. http://doi.org/10.1021/cn100111a

Matsuzaki, M., Hayama, T., Kasai, H., & Ellis-Davies, G. C. R. (2010). Two-photon uncaging of γ-aminobutyric acid in intact brain tissue. Nature Chemical Biology, 6, 255. Retrieved from http://dx.doi.org/10.1038/nchembio.321

Svoboda, K., & Yasuda, R. (2006). Principles of Two-Photon Excitation Microscopy and Its Applications to Neuroscience. Neuron, 50(6), 823–839. http://doi.org/10.1016/j.neuron.2006.05.019