For those who haven't had the pleasure of listening to me whinge that my experiment is still not working (!), I have been working in Prof. Rubinsztein-Dunlop's optical micromanipulation group since the beginning of the year. I had a look through a paper on micromanipulation within cells yesterday and thought that it probably ties in well with Nelson Ch. 2.
The paper's details are;
Sacconi, L. et al., "Optical micromanipulation inside yeast cells", Applied Optics, 44(11), pp. 2001-2007, April 2005.
The researchers demonstrate that it is possible to optically trap and manipulate organelles inside living yeast cells without (apparently) causing them damage.
How does optical trapping work? You may recall seeing optical tweezers in a problem set last year (concerning DNA stretching- this experiment has been done too!). A focused laser beam does not come exactly to a point because of diffraction: it comes to a focal spot, where the irradiance is quite high. Moreover, since the beam is tightly focused it rapidly widens both above and below this spot.
The upshot of this is that we can exert an optical force on transparent particles.
For a small particle the external (optical) electric field induces polarisation of the material, creating an electric dipole (the higher multipole moments are negligible for a small enough object) which interacts with the time-averaged light field (i.e. essentially the irradiance). We then have migration towards the focal spot, since this decreases the free energy.
Larger particles can be imagined as being small lenses: if the lens is perfectly aligned with the beam it causes no deflection to the sides (transverse to the beam axis), but if it shifts slighlty away it causes light to deflect in the opposite direction. Since light carries momentum (p = E/c), and this must be conserved, there is a restoring force on the particle.
Optical torques can also be used to create rotation, either by using beams with spiral phase-fronts (e.g. Laguerre-Gauss beams) or by passing light through an anisotropic medium (polarisation changes = `spin' angular momentum changes).
Since this focal spot is diffraction limited (i.e. diameters on the order of half the wavelength) it can be made small enough to allow individual organelles to be manipulated. The `only' trick is selecting a wavelength which allows fine enough resolution without frying the cells too! Luckily, cells don't absorb a great deal in the near-infrared (1064nm is common) and the far red (~800nm), whereas water's absorption kicks in around the reds and greens.
Incidentally, when something is killed by light it is called `optocution'!
In this paper Sacconi et al. use an 830nm laser to move organelles within yeast (S.pombe cells with mytotic spindles labelled in GFP) that are affixed to a substrate (so the cell cannot simply pull itself out of the optical trap).
By using a clever laser (pulsed when mode-locking is active, continuous wave when not) they were able to switch between an imaging mode (nonlinear two-photon fluorescence microscopy, another technique which we encountered last year) and a manipulation mode, meaning that they could create a 3D image of the cell, manipulate an organelle and then record the behaviour of the cell in response to this.
They required around ~200mW to trap and move organelles. To give you an idea of how large this is (and hence an idea of how structured the cell interior is), I have trapped 3 micron diameter spheres using as little as 2mW (different wavelength though, 633nm or 1064nm vs. their 830nm, and a different refractive index, which changes the optical force). They estimate the viscosity of the cytosol to be ~20^-1 Pa.s, which is much greater than that of pure water (9*10^-4 Pa.s).
Optocution did not occur for the majority (>95%) of yeast cells trapped with 830nm, but photodamage was clearly observed for 880nm (observed as the formation of bubbles inside the cell). The 830nm-trapped cells survived the experiments even after five minutes exposure, and continued to undergo mitosis essentially as normal. One major difference was the introduction of a delay into the usual mitotic cycle (cell stress?).
The researchers indicate heating due to absorption was minimal and postulate the majority of damage was due to the formation of reactive chemical species. Most importantly, they found that by manipulating small vesicles within the cell they were able to displace the nucleus away from the cell centre, where it is usually located, along the long axis of the cell (a roughly cylindrical shape). The nucleus then tended to move
back into its original position when the trap was removed, indicating involvement of the cellular cytoskeleton and organelles in organising the gross structure of the cell.
This work doesn't really tell us any new physics in and of itself: what it does do is open the way for use of optical tweezers in studies of microtubule remodelling and nuclear positioning. This is believed to be of importance because the nuclear position appears to coincide with the plane of division which appears during cell division, contolling how much of the cytosol and accompanying machinery each daughter cell gets. This is currently a very difficult phenomenon to study,
especially since other methods of displacing the nuclei are not as specific and do not allow the cell to be continuously observed during the process (and may also damage the cells or otherwise effect the mitotic spindle).
It is interesting that such a small environment can display such well-coordinated biochemistry and biophysics! Understanding how cells respond to forces and torques will likely be crucial to understanding processes such as cell differentiation (e.g. differentiation of BMC to osteoblasts is stimulated by application of tension) which have important disease implications.
Thoughts?
James
The paper's details are;
Sacconi, L. et al., "Optical micromanipulation inside yeast cells", Applied Optics, 44(11), pp. 2001-2007, April 2005.
The researchers demonstrate that it is possible to optically trap and manipulate organelles inside living yeast cells without (apparently) causing them damage.
How does optical trapping work? You may recall seeing optical tweezers in a problem set last year (concerning DNA stretching- this experiment has been done too!). A focused laser beam does not come exactly to a point because of diffraction: it comes to a focal spot, where the irradiance is quite high. Moreover, since the beam is tightly focused it rapidly widens both above and below this spot.
The upshot of this is that we can exert an optical force on transparent particles.
For a small particle the external (optical) electric field induces polarisation of the material, creating an electric dipole (the higher multipole moments are negligible for a small enough object) which interacts with the time-averaged light field (i.e. essentially the irradiance). We then have migration towards the focal spot, since this decreases the free energy.
Larger particles can be imagined as being small lenses: if the lens is perfectly aligned with the beam it causes no deflection to the sides (transverse to the beam axis), but if it shifts slighlty away it causes light to deflect in the opposite direction. Since light carries momentum (p = E/c), and this must be conserved, there is a restoring force on the particle.
Optical torques can also be used to create rotation, either by using beams with spiral phase-fronts (e.g. Laguerre-Gauss beams) or by passing light through an anisotropic medium (polarisation changes = `spin' angular momentum changes).
Since this focal spot is diffraction limited (i.e. diameters on the order of half the wavelength) it can be made small enough to allow individual organelles to be manipulated. The `only' trick is selecting a wavelength which allows fine enough resolution without frying the cells too! Luckily, cells don't absorb a great deal in the near-infrared (1064nm is common) and the far red (~800nm), whereas water's absorption kicks in around the reds and greens.
Incidentally, when something is killed by light it is called `optocution'!
In this paper Sacconi et al. use an 830nm laser to move organelles within yeast (S.pombe cells with mytotic spindles labelled in GFP) that are affixed to a substrate (so the cell cannot simply pull itself out of the optical trap).
By using a clever laser (pulsed when mode-locking is active, continuous wave when not) they were able to switch between an imaging mode (nonlinear two-photon fluorescence microscopy, another technique which we encountered last year) and a manipulation mode, meaning that they could create a 3D image of the cell, manipulate an organelle and then record the behaviour of the cell in response to this.
They required around ~200mW to trap and move organelles. To give you an idea of how large this is (and hence an idea of how structured the cell interior is), I have trapped 3 micron diameter spheres using as little as 2mW (different wavelength though, 633nm or 1064nm vs. their 830nm, and a different refractive index, which changes the optical force). They estimate the viscosity of the cytosol to be ~20^-1 Pa.s, which is much greater than that of pure water (9*10^-4 Pa.s).
Optocution did not occur for the majority (>95%) of yeast cells trapped with 830nm, but photodamage was clearly observed for 880nm (observed as the formation of bubbles inside the cell). The 830nm-trapped cells survived the experiments even after five minutes exposure, and continued to undergo mitosis essentially as normal. One major difference was the introduction of a delay into the usual mitotic cycle (cell stress?).
The researchers indicate heating due to absorption was minimal and postulate the majority of damage was due to the formation of reactive chemical species. Most importantly, they found that by manipulating small vesicles within the cell they were able to displace the nucleus away from the cell centre, where it is usually located, along the long axis of the cell (a roughly cylindrical shape). The nucleus then tended to move
back into its original position when the trap was removed, indicating involvement of the cellular cytoskeleton and organelles in organising the gross structure of the cell.
This work doesn't really tell us any new physics in and of itself: what it does do is open the way for use of optical tweezers in studies of microtubule remodelling and nuclear positioning. This is believed to be of importance because the nuclear position appears to coincide with the plane of division which appears during cell division, contolling how much of the cytosol and accompanying machinery each daughter cell gets. This is currently a very difficult phenomenon to study,
especially since other methods of displacing the nuclei are not as specific and do not allow the cell to be continuously observed during the process (and may also damage the cells or otherwise effect the mitotic spindle).
It is interesting that such a small environment can display such well-coordinated biochemistry and biophysics! Understanding how cells respond to forces and torques will likely be crucial to understanding processes such as cell differentiation (e.g. differentiation of BMC to osteoblasts is stimulated by application of tension) which have important disease implications.
Thoughts?
James
Wow, I should whinge more often! Our first correct measurements were made today.
ReplyDeleteInteresting study James and congratulations in producing the first set of accurate results.
ReplyDelete