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Optics clustered to output unique solutions: A multi-laser facility for combined single molecule and ensemble microscopy
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Image of FIG. 1.
FIG. 1.

Layout of the OCTOPUS facility. (a) Laser hub and multiphoton confocal microscopy; (b) three-colour single molecule tracking and eight-channel single molecule combined spFRET and smFP; (c) data processing area; (d) one-photon multicolour confocal FLIM; (e) one-photon multicolour confocal FLIM, five-colour single molecule tracking, and TIRF tweezers; and (f) development laboratory. The boxes on walls mark the position of the laser patch panels.

Image of FIG. 2.
FIG. 2.

(a) SMA optical fibre patch panel; (b) Magelis Human Machine Interface control panel used to control lasers in the OCTOPUS facility; and (c) Cerberus information display system showing the status of the facility (illuminated boxes indicate lasers that are switched on).

Image of FIG. 3.
FIG. 3.

(a) Optical layout of the three-colour single molecule tracking microscope showing TIRF excitation beam (blue-green), three colour fluorescence emission (brown), and individual fluorescence emission channels (green, orange, and red). (b) Optical layout of TIRF-tweezers system showing optical trapping beam (red), TIRF excitation beam (green), and fluorescence emission (orange).

Image of FIG. 4.
FIG. 4.

Simultaneous three-colour single molecule TIRF imaging of HeLa cells labelled with (a) EGF-Atto 647N; (b) EGF-Alexa 546; and (c) EGF-Atto 488.

Image of FIG. 5.
FIG. 5.

OCTOPUS TIRF tweezers for manipulation of fluorescently labelled 250 nm size polystyrene bead (488 nm excitation) in live A431 mammalian cells (DiI membrane labelled). (a)–(d) shows approach of trapped beads in less than 2 μm steps. Arrow indicates position of trapped bead (scale bars 20 μm). The TIRF provides video rate background-free imaging of sub-wavelength resolution.

Image of FIG. 6.
FIG. 6.

(a) 3D distribution of EGFR in A431 epithelial carcinoma cells. The image was obtained from cells exposed to 100 nM solution of EGF labelled with Alexa 488 dye from a stack of optical sections obtained by one photon confocal microscopy (scale bar 20 μm). (b) High magnification confocal images showing the initial stages of receptor internalisation in A431 cells (sample preparation same as (a), scale bar 5 μm). (c) Confocal image showing the distribution of EGFP-Rheb in live A431 cells. (d) Confocal image showing the location of Alexa 546-labelled EGF/EGFR after internalisation, and (e) merged image, scale bars 10 μm). (f) Donor fluorescence lifetime image of the same cells shown in (c)–(e) (scale bar 10 μm). The inset shows the distribution of lifetimes in the image. (g) Single molecule TIRF image of EGFR molecules in the plasma membrane of A431 cells exposed to Alexa 488-conjugated Fab fragments of monoclonal antibody mAb 2E9. (h) Tracks of molecules imaged in (g), which describe the paths of individual receptors or receptor complexes.

Image of FIG. 7.
FIG. 7.

(a) FRET efficiency as a function of acceptor density measured A431 cells treated with Ouabain to induce membrane depolarisation, loaded with DiD and labelled with 100 nM EGF-Alexa 546. (b) Cells treated instead with 2-deoxyglucose and sodium azide (DSA) to inhibit ATP production. The best fit of Monte-Carlo simulation results to the data was calculated as previously shown (Ref. 24) and are labelled with the corresponding distance of closest approach. (c) Examples of single-pair FRET traces. Fluorescence intensity versus time traces of donor (green) and acceptor (red) fluorophores. Examples are shown of long ligand-ligand separation (left; low FRET, high donor emission and low acceptor emission), medium ligand-ligand separation (centre; moderate FRET, moderate emission from both donor and acceptor), and short ligand-ligand separation (right; high FRET, low donor emission and high acceptor emission).

Image of FIG. 8.
FIG. 8.

Two-colour single molecule tracking of T47D cells. (a) White light transmission image; (b) cells labelled with 0.1 nM anti-ErbB2 affibody-Atto647N; (c) cells labelled with 2 nM anti-EGFR Affibody-Alexa 488; (d) and (e) single molecule tracks (white) from the spots located within the boxes marked in (b) and (c).


Generic image for table
Table I.

Lasers installed in the OCTOPUS cluster.


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752b84549af89a08dbdd7fdb8b9568b5 journal.articlezxybnytfddd
Scitation: Optics clustered to output unique solutions: A multi-laser facility for combined single molecule and ensemble microscopy