Catalog  |  Cart  |  Log In

Focus: Cell Imaging

Innovations in Fluorescence Imaging of Live Cells

We review some of the advances in fluorescent live-cell imaging. We discuss improvements in calcium ion imaging, the development of the green fluorescent protein and its derivatives, and new technologies for labeling live cells including FlAsH and ReAsH labeling and the HaloTag™ Interchangeable Labeling Technology.

By Michele Arduengo, Ph.D.
Promega Corporation

Published in February 2006


Introduction

Some of the most remarkable images in biology during the 20th century were dramatically labeled fluorescent images of fixed cells. Micrographs of bright green mitotic spindles garnished with red centrioles graced the covers of many biological journals and graduate school recruitment brochures. As dramatic and instructive as these snapshots of cellular activity were, they still did not capture the dynamic nature of cellular processes. In 1953, Shinya Inoue designed a polarizing light microscope in which he was able to resolve the mitotic spindle in healthy cells, giving scientists a look at one of most important dynamic processes of cell biology and beginning a revolution in live-cell imaging (1).

Imaging with Ca2+-Sensitive Dyes

Fluorescent markers have been indispensable for labeling specific proteins, enabling scientists to localize proteins to subcellular structures. However many fluorescent molecules are not cell-permeant, requiring lysis and fixation of the cells, allowing only snapshots of an entire cellular process. Ca2+ imaging of cells was one of the first areas in which fluorescent dyes or luminescent proteins were used to follow cellular processes. In 1978, the calcium-sensitive luminescent protein, aqueorin, was used to visualize the Ca2+ wave that occurs during medaka egg fertilization (2). Researchers also used a cell-permeant ester derivative of the fluorescent molecule, quin-2, to look at Ca2+ fluxes in cells; however quin-2 had several disadvantages including an excitation wavelength of 339nm that caused significant cellular autofluorescence (3). Ca2+ imaging was improved by a second generation of calcium dyes based on the Ca2+ chelator, BAPTA. Of these, the cell-permeant methoxyester of fura-2 has become the most widely used for live-cell Ca2+ imaging (4).

The Green Fluorescent Revolution

Although the Ca2+-sensitive dyes enabled scientists to visualize a dynamic process such as fertilization, activation, and even slow Ca2+ waves at cleavage furrows (2), these dyes did not allow resolution of a specific protein or significant subcellular localization. The discovery, cloning (5) and successful expression of the green fluorescent protein from Aequorea victoria in the nematode (6) provided biologists an invaluable tool for labeling and following a specific protein in living cells. Since that initial work, the green fluorescent protein has been engineered to improve folding, expression and light production and modify the emission spectra (7–9). In addition, intrinsically fluorescent proteins from other marine organisms have added the flexibility of labeling a protein with a red tag as well as green, yellow and cyan, enabling multicolor imaging studies (10).

Green Fluorescent Protein (GFP) and its related proteins continue to revolutionize live-cell imaging. GFP is most often expressed as a fusion with a particular protein of interest. One of the most dramatic processes visualized using GFP was the movement of Listeria monocytogenes inside MDCK cells via an actin tail (11). GFP is also becoming an important tool in cancer research where it has been used to tag cancer cells and to detect, in vivo, single metastases (12). Several tumor cell types including brain, breast, colon, lung skin, pancreas, prostate and stomach can be followed from primary tumor to metastases using GFP (12).

Fluorescence Speckle Microscopy

New techniques, such as fluorescence speckle microscopy (FSM), are also being developed. In FSM the dynamics of a polymeric structure such as the mitotic spindle can be studied by introducing a labeled protein in small amounts either by microjection or low-level expression of a fusion protein and following the “speckles” as the structure changes (13,14). Using FSM one group has suggested that microtubule treadmilling results from dynamic instability resulting from differences in polymerization rates between the plus and minus ends rather than unidirectional growth from the minus to the plus ends (15). In this study however, instead of using GFP fusion proteins, the authors used X-rhodamine-labeled tubulin to provide the “speckles” (15). GFP-labeled tubulin has been used to visualize microtubule dynamics in live Saccharomyces cerevisiae (14).

Fluorescence Resonance Energy Transfer Microscopy

Fluorescence Resonance Energy Transfer Microscopy (FRET) uses energy transfer from an excited fluorophore (donor) to another fluorophore (acceptor). The energy transfer is distance dependent and can be highly efficient when the donor and acceptor are positioned 3–6nm of each other. Engineered GFP proteins, the red-shifted yellow fluorescent proteins (YFP) and the cyan fluorescent protein (CFP) are often paired for FRET studies (10). FRET studies using the YFP-CFP pairs have allowed investigators to study macromolecular complexes that are associated with events such as gene transcription or signal transduction (16). For instance, Shaw and Burack used FRET to describe the interaction of ERK2 and MEK and the translocation of ERK2 from the cytoplasm to the nucleus (17).

FlAsH and ReAsH Fluorophores

One approach to labeling live cells is to use cell-permeant, biarsenical compounds, FlAsH (fluorescein arsenical helix binder) and ReAsH (red arsenical helix binder), to covalently label proteins containing tetracysteine tags (13,18). Because of the available red and green fluorophores, this technique allows differential labeling of protein pools in a pulse-chase manner (19); this technique was used to demonstrate that newly synthesized connexin43 was incorporated at periphery of gap junctions, while older molecules were removed from the interior (19). Also, because ReAsH can cause diaminobenzidine to form an insoluble precipitate it allows researchers to correlate fluorescence microscopy with electron microscopy. At a conference organized to honor Shinya Inoue, Tsien and colleagues described using these techniques to image fragmentation of the Golgi apparatus and show that during mitosis the Golgi fragments remain almost completely separate from the ER in cells (1). As promising as the tetracysteine labeling is, the technique does have some limitations. First, the biarsenical compounds can bind to tetracysteine motifs in any protein in cells, so endogenous background is a concern (13) and the interaction requires a cellular environment in which cysteine residues are reduced, introducing potentially toxic conditions for the cells (13).

HaloTag™ Interchangeable Labeling Technology

The HaloTag™ Interchangeable Labeling Technology expands the field of multicolor, live-cell fluorescence imaging (See an animation of the HaloTag™ Technology). Like the FlAsH and ReAsH labeling methods, HaloTag™ technology covalently labels fusion proteins, and the availability of multicolored ligands for the protein gives the researcher the ability to perform multicolor dual labeling experiments, multiplex with immunocytochemistry experiments and differentially label protein pools (pulse-chase labeling; 20). HaloTag™ Ligands are available with blue, green and red fluorophores as well as biotin tags. The HaloTag™ Protein is engineered from a protein that is not endogenous to eukaryotic cells, and therefore labeling of the HaloTag™ fusion is highly specific, avoiding the background staining problems associated with FlAsH and ReAsH labeling. Also, the ligands have shown no detectable toxicity at the recommended labeling conditions. The covalent bond between the HaloTag™ Protein and the ligands forms rapidly at physiological conditions and is stable even under denaturing conditions, allowing labeling of fixed cells as well. Additionally, the same construct that was used to create the fusion protein for labeling can be used to express the protein in vitro or in cells for protein capture and analysis.

Summary

New labeling technologies that allow scientists to visualize dynamic cellular processes will greatly enhance our understanding of fundamental biological questions. The future looks bright as we find new ways to maintain healthy cells on the microscope stage, improve our optics and detection systems, and develop new, nontoxic and easily detectable reagents for labeling.

References

  1. Dell, K.R. and Vale, R.D. (2004) A tribute to Shinya Inoue and innovation in light microscopy. J. Cell Biol. 165, 21–5.
  2. Brownlee, C. (2000) Cellular calcium imaging: So, what’s new? Trends Cell Biol. 10, 451–7.
  3. Grynkiewicz, G., Poenie, M. and Tsien, R.Y. (1985) A new generation of Ca2+ with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440–50.
  4. Knot, H.J. et al. (2005) Twenty years of calcium imaging: Cell physiology to dye for. Mol. Interv. 5, 112–27.
  5. Prasher, D.C. (1992) Primary structure of the Aequorea victoria green fluorescent protein. Gene 111, 229–33.
  6. Chalfie, M. et al. (1994) Green fluorescent protein as a marker for gene expression. Science 263, 802–5.
  7. Heim, R., Cubitt, A.B. and Tsien, R.Y. (1995) Improved green fluorescence. Nature 373, 663–4.
  8. Delagrave, S. et al. (1995) Red-shifted excitation mutants of the green fluorescent protein. BioTechnology (N.Y.) 13, 151–4.
  9. Almond, B.D. (2003) Monster Green® Protein: A brighter, longer-expressing green fluorescent protein. Promega Notes 84, 12–15. http://www.promega.com/pnotes/84/
  10. Lippincott-Schwartz, J. and Patterson, G.H. (2003) Development and use of fluorescent protein markers in living cells. Science 300, 87–91.
  11. Robbins, J.R. et al. (1999) Listeria monocytogenes exploits normal host cell processes to spread from cell to cells. J. Cell Biol. 146, 1333–50.
  12. Paris, S. and Sesboüé, R. (2004) Metastasis models: The green fluorescent revolution? Carcinogenesis 25, 2285–92.
  13. Stephens, D.J. and Allan, V.J. (2003) Light microscopy techniques for live cell imaging. Science 300, 82–6.
  14. Waterman-Storer, C.M. and Salmon, E.D. (1999) Fluorescent speckle microscopy: how low can you go? FASEB J. 13, S225–S230.
  15. Grego, S., Cantillana,V. and Salmon, E.D. (2001) Microtubule treadmilling in vitro investigated by fluorescence speckle and confocal microscopy. Biophysical J. 81, 66–78.
  16. Sekar, R.B. and Perasamy, A. (2003) Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations. J. Cell Biol. 160, 629–33.
  17. Burack, W.R. and Shaw, A.S. (2005) Live cell imaging of ERK and MEK. J. Biol. Chem. 280, 3832–7.
  18. Griffin, B.A. et al. (1998) Specific covalent labeling of recombinant protein molecules inside live cells. Science 281, 269–72.
  19. Gaietta, G. et al. (2002) Multicolor and electron microscopic imaging of connexin trafficking. Science 296, 503–7.
  20. Los, G. et al. (2006) HaloTag™ Technology: Cell imaging and protein analysis. Cell Notes 14, 10–14.