Plasmonic Imaging of Dynamic Interactions between Membrane Receptor Clusters beyond the Diffraction Limit in Live Cells

Throughout the years, fluorescence microscopy has proven to be an extremely versatile tool for cell biologists to study live cells. Its high sensitivity and non-invasiveness, together with the ever-growing spectrum of sophisticated fluorescent indicators, ensure that it will continue to have a prominent role in the future. A drawback of light microscopy is the fundamental limit of the attainable spatial resolution – ∼250 nm – dictated by the laws of diffraction. The challenge to break this diffraction limit has led to the development of several novel imaging techniques. One of them, near-field scanning optical microscopy (NSOM), allows fluorescence imaging at a resolution of only a few tens of nanometers and, because of the extremely small near-field excitation volume, reduces background fluorescence from the cytoplasm to the extent that single-molecule detection sensitivity becomes within reach. NSOM allows detection of individual fluorescent proteins as part of multimolecular complexes on the surface of fixed cells, and similar results should be achievable under physiological conditions in the near future.

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Evidence from lateral mobility studies for dynamic interactions of a mutant influenza hemagglutinin with coated pits.

The Journal of Cell Biology ◽

10.1083/jcb.115.6.1585 ◽

1991 ◽

Vol 115 (6) ◽

pp. 1585-1594 ◽

Cited By ~ 40

Author(s):

E Fire ◽

D E Zwart ◽

M G Roth ◽

Y I Henis

Keyword(s):

Plasma Membrane ◽

Lattice Structure ◽

Lateral Diffusion ◽

Live Cells ◽

Lateral Mobility ◽

Coated Pits ◽

Dynamic Interactions ◽

Intact Cells ◽

Hypertonic Medium ◽

Mobility Measurement

Replacement of cysteine at position 543 by tyrosine in the influenza virus hemagglutinin (HA) protein enables the endocytosis of the mutant protein (Tyr 543) through coated pits (Lazarovits, J., and M. G. Roth. 1988. Cell. 53:743-752). To investigate the interactions between Tyr 543 and the clathrin coats in the plasma membrane of live cells, we performed fluorescence photobleaching recovery measurements comparing the lateral mobilities of Tyr 543 (which enters coated pits) and wild-type HA (HA wt, which is excluded from coated pits), following their expression in CV-1 cells by SV-40 vectors. While both proteins exhibited the same high mobile fractions, the lateral diffusion rate of Tyr 543 was significantly slower than that of HA wt. Incubation of the cells in a sucrose-containing hypertonic medium, a treatment that disperses the membrane-associated coated pits, resulted in similar lateral mobilities for Tyr 543 and HA wt. These findings indicate that the lateral motion of Tyr 543 (but not of HA wt) is inhibited by transient interactions with coated pits (which are essentially immobile on the time scale of the lateral mobility measurements). Acidification of the cytoplasm by prepulsing the cells with NH4Cl (a treatment that arrests the pinching-off of coated vesicles from the plasma membrane and alters the clathrin lattice morphology) led to immobilization of a significant part of the Tyr 543 molecules, presumably due to their entrapment in coated pits for the entire duration of the lateral mobility measurement. Furthermore, in both untreated and cytosol-acidified cells, the restrictions on Tyr 543 mobility were less pronounced in the cold, suggesting that the mobility-restricting interactions are temperature dependent and become weaker at low temperatures. From these studies we conclude the following. (a) Lateral mobility measurements are capable of detecting interactions of transmembrane proteins with coated pits in intact cells. (b) The interactions of Tyr 543 with coated pits are dynamic, involving multiple entries of Tyr 543 molecules into and out of coated pits. (c) Alterations in the clathrin lattice structure can modulate the above interactions.

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4-dimensional, high-resolution imaging of living cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100122484 ◽

1992 ◽

Vol 50 (1) ◽

pp. 416-417

Author(s):

Shinya Inoué

Keyword(s):

Light Microscope ◽

Living Cells ◽

Live Cells ◽

Video Tape ◽

Active Living ◽

High Resolution Imaging ◽

3 Dimensional ◽

Dynamic Architecture ◽

Resolution Imaging ◽

Disk Memory

This paper reports progress of our effort to rapidly capture, and display in time-lapsed mode, the 3-dimensional dynamic architecture of active living cells and developing embryos at the highest resolution of the light microscope. Our approach entails: (A) real-time video tape recording of through-focal, ultrathin optical sections of live cells at the highest resolution of the light microscope; (B) repeat of A at time-lapsed intervals; (C) once each time-lapsed interval, an image at home focus is recorded onto Optical Disk Memory Recorder (OMDR); (D) periods of interest are selected using the OMDR and video tape records; (E) selected stacks of optical sections are converted into plane projections representing different view angles (±4 degrees for stereo view, additional angles when revolving stereos are desired); (F) analysis using A - D.

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Solving the mechanisms responsible for organelle behavior in vivo by same-cell correlative light and electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100120473 ◽

1992 ◽

Vol 50 (1) ◽

pp. 14-15

Author(s):

Conly L. Rieder

Keyword(s):

Electron Microscopy ◽

Quantitative Analysis ◽

Light Microscopy ◽

Living Cell ◽

Light And Electron Microscopy ◽

Time Behavior ◽

Dynamic Interactions ◽

Positive Identification ◽

Cellular Components

The behavior of many cellular components, and their dynamic interactions, can be characterized in the living cell with considerable spatial and temporal resolution by video-enhanced light microscopy (video-LM). Indeed, under the appropriate conditions video-LM can be used to determine the real-time behavior of organelles ≤ 25-nm in diameter (e.g., individual microtubules—see). However, when pushed to its limit the structures and components observed within the cell by video-LM cannot be resolved nor necessarily even identified, only detected. Positive identification and a quantitative analysis often requires the corresponding electron microcopy (EM).

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Confocal Raman mapping

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100132200 ◽

1992 ◽

Vol 50 (2) ◽

pp. 1514-1515

Author(s):

J. Barbillat ◽

M. Delhaye ◽

P. Dhamelincourt

Keyword(s):

Chemical Species ◽

Diffraction Limit ◽

Microscopic Level ◽

Raman Mapping ◽

Complete Spectrum ◽

Laser Illumination ◽

Ccd Detector ◽

Raman Microprobe ◽

Photodiode Array ◽

Raman Image

Raman mapping, with a spatial resolution close to the diffraction limit, can help to reveal the distribution of chemical species at the surface of an heterogeneous sample.As early as 1975,three methods of sample laser illumination and detector configuration have been proposed to perform Raman mapping at the microscopic level (Fig. 1),:- Point illumination:The basic design of the instrument is a classical Raman microprobe equipped with a PM tube or either a linear photodiode array or a two-dimensional CCD detector. A laser beam is focused on a very small area ,close to the diffraction limit.In order to explore the whole surface of the sample,the specimen is moved sequentially beneath the microscope by means of a motorized XY stage. For each point analyzed, a complete spectrum is obtained from which spectral information of interest is extracted for Raman image reconstruction.- Line illuminationA narrow laser line is focused onto the sample either by a cylindrical lens or by a scanning device and is optically conjugated with the entrance slit of the stigmatic spectrograph.

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Multi-mode light microscopy of microtubule assembly dynamics and chromosome movement in vivo and In vitro

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100166117 ◽

1996 ◽

Vol 54 ◽

pp. 730-731

Author(s):

E. D. Salmon ◽

J. C. Waters ◽

C. Waterman-Storer

Keyword(s):

Imaging System ◽

Ccd Camera ◽

Transmitted Light ◽

Microtubule Assembly ◽

Live Cells ◽

Optical Efficiency ◽

Multiple Band ◽

Multi Mode

We have developed a multi-mode digital imaging system which acquires images with a cooled CCD camera (Figure 1). A multiple band pass dichromatic mirror and robotically controlled filter wheels provide wavelength selection for epi-fluorescence. Shutters select illumination either by epi-fluorescence or by transmitted light for phase contrast or DIC. Many of our experiments involve investigations of spindle assembly dynamics and chromosome movements in live cells or unfixed reconstituted preparations in vitro in which photodamage and phototoxicity are major concerns. As a consequence, a major factor in the design was optical efficiency: achieving the highest image quality with the least number of illumination photons. This principle applies to both epi-fluorescence and transmitted light imaging modes. In living cells and extracts, microtubules are visualized using X-rhodamine labeled tubulin. Photoactivation of C2CF-fluorescein labeled tubulin is used to locally mark microtubules in studies of microtubule dynamics and translocation. Chromosomes are labeled with DAPI or Hoechst DNA intercalating dyes.

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Characterization of a Motile Cellular Domain Arising During the Amoebo-Flagellate Transformation of Physarum Polycephalum

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s1431927600002440 ◽

1980 ◽

Vol 38 ◽

pp. 552-553

Author(s):

K.I. Pagh ◽

M.R. Adelman

Keyword(s):

Cell Shape ◽

Physarum Polycephalum ◽

Slime Mold ◽

Live Cells ◽

Cellular Domain

Unicellular amoebae of the slime mold Physarum polycephalum undergo marked changes in cell shape and motility during their conversion into flagellate swimming cells (l). To understand the processes underlying motile activities expressed during the amoebo-flagellate transformation, we have undertaken detailed investigations of the organization, formation and functions of subcellular structures or domains of the cell which are hypothesized to play a role in movement. One focus of our studies is on a structure, termed the “ridge” which appears as a flattened extension of the periphery along the length of transforming cells (Fig. 1). Observations of live cells using Nomarski optics reveal two types of movement in this region:propagation of undulations along the length of the ridge and formation and retraction of filopodial projections from its edge. The differing activities appear to be associated with two characteristic morphologies, illustrated in Fig. 1.

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