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. 2011 Sep 26;30(3):275-299.
doi: 10.1080/0144235X.2011.603237.

Towards controlling molecular motions in fluorescence microscopy and optical trapping: a spatiotemporal approach

Arijit Kumar De  1 , Debabrata Goswami
Affiliations

Towards controlling molecular motions in fluorescence microscopy and optical trapping: a spatiotemporal approach

Arijit Kumar De et al. Int Rev Phys Chem. .

Abstract

This account reviews some recent studies pursued in our group on several control experiments with important applications in (one-photon) confocal and two-photon fluorescence laser-scanning microscopy and optical trapping with laser tweezers. We explore the simultaneous control of internal and external (i.e. centre-of-mass motion) degrees of freedom, which require the coupling of various control parameters to result in the spatiotemporal control. Of particular interest to us is the implementation of such control schemes in living systems. A live cell is a system of a large number of different molecules which combine and interact to generate complex structures and functions. These combinations and interactions of molecules need to be choreographed perfectly in time and space to achieve intended intra-cellular functions. Spatiotemporal control promises to be a versatile tool for dynamical control of spatially manipulated bio-molecules.

Keywords: fluorescence microscopy; optical tweezers; spatiotemporal control.

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Figures

Figure A1
Figure A1
[Colour online] Schematic of the microscope system; the excitation path is shown as red (720-980 nm) or blue (488 nm) arrows while the fluorescence (spanning visible wavelength) collection paths are shown as green arrows.
Figure A2
Figure A2
[Colour online] Schematic of the tweezer set-up.
Figure 1
Figure 1
[Colour online] Confocal (a) and multiphoton (b) fluorescence excitation and detection.
Figure 2
Figure 2
[Colour online] Physics of optical tweezing. The laser beam paths are shown as bent (red) arrows, exerted forces on particle as straight (blue) arrows and the resultant force as dashed (green) arrow and the centre of the propagating beam is shown as dashed (black) line (note that different thickness for lines are used to distinguish relative photon numbers or magnitude of forces.).
Figure 3
Figure 3
[Colour online] Various approaches of spatiotemporal control: the work presented here is marked within the solid circle.
Figure 4
Figure 4
[Colour online] R6G fluorescence enhancement with nanosecond pulsed illumination (a) from the Corona laser (120 ns pulses at 1 and 10 kHz) and (b) using the AOD (150 ns pulses at 500 kHz) compared with that under CW illumination at same average power.
Figure 5
Figure 5
[Colour online] Effective removal of photo-thermal effect by blanked-excitation as evidenced from (a) open-aperture z-scan trace of [Ru(bpy)2]Cl2 in DCM at 770 nm and (b) fluorescence enhancement of R6G at 780 nm.
Figure 6
Figure 6
[Colour online] Excitation scheme for stimulated emission commonly employed (a) in comparison to that used in one-colour pulse-pair and pulse-train methods described here (b)–(d). The excitation and stimulated emission are shown as upward and downward thin arrows, respectively, while fluorescence is shown as broad downward arrow; the colours are chosen to specify the different wavelengths. Little downward black arrows indicate either vibrational relaxation (solid) or internal conversion (broken).
Figure 7
Figure 7
[Colour online] Relative fluorescence intensity modulation of DAPI/Texas Red (a) and DAPI/Mito-tracker Red (b), (c) under one-colour pulse-pair (a, b) and pulse-train (c) excitation.
Figure 8
Figure 8
Fluorescence suppression of Texas Red (a) and Mito-tracker Red (b) relative to DAPI as a function of time delay between pulses under one-colour pulse-pair (a) and pulse-train (b) excitation.
Figure 9
Figure 9
[Colour online] Out-of-phase oscillations of rhodamine 6G (b) and fluorescein (c) fluorescence compared with the interferometric autocorrelation fringe (a) at partial pulse-pulse temporal overlapping region (around −100 fs delay).
Figure 10
Figure 10
[Colour online] Two-photon fluorescence for trapping of (a) 100 nm particles and (b) 10–20 nm Q-dots. The red line in the (a) panel is a guideline for sequential trapping of two particles.
Figure 11
Figure 11
[Colour online] Schematic of the experimental set-up; the excitation path is shown as red (720–980 nm) or green (532 nm) arrows while the fluorescence (550–650 nm) collection paths are shown as orange arrows.
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