Optimisation approaches for concurrent transmitted light imaging during confocal microscopy

Plant Methods

David A. Collings¹ 

[1] Biomolecular Interaction Centre, School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand

关键词: Transmitted light detector;    Real colour imaging;    Quantification;    Plant pigments;    Köhler illumination;    Green fluorescent protein;    Confocal microscopy;    Anthocyanin;   

Others  :  1225049
DOI  :  10.1186/s13007-015-0085-3

 received in 2015-06-02, accepted in 2015-08-10,  发布年份 2015

【 摘 要 】

Background

The transmitted light detectors present on most modern confocal microscopes are an under-utilised tool for the live imaging of plant cells. As the light forming the image in this detector is not passed through a pinhole, out-of-focus light is not removed. It is this extended focus that allows the transmitted light image to provide cellular and organismal context for fluorescence optical sections generated confocally. More importantly, the transmitted light detector provides images that have spatial and temporal registration with the fluorescence images, unlike images taken with a separately-mounted camera.

Results

Because plants often provide difficulties for taking transmitted light images, with the presence of pigments and air pockets in leaves, this study documents several approaches to improving transmitted light images beginning with ensuring that the light paths through the microscope are correctly aligned (Köhler illumination). Pigmented samples can be imaged in real colour using sequential scanning with red, green and blue lasers. The resulting transmitted light images can be optimised and merged in ImageJ to generate colour images that maintain registration with concurrent fluorescence images. For faster imaging of pigmented samples, transmitted light images can be formed with non-absorbed wavelengths. Transmitted light images of Arabidopsis leaves expressing GFP can be improved by concurrent illumination with green and blue light. If the blue light used for YFP excitation is blocked from the transmitted light detector with a cheap, coloured glass filters, the non-absorbed green light will form an improved transmitted light image. Changes in sample colour can be quantified by transmitted light imaging. This has been documented in red onion epidermal cells where changes in vacuolar pH triggered by the weak base methylamine result in measurable colour changes in the vacuolar anthocyanin.

Conclusions

Many plant cells contain visible levels of pigment. The transmitted light detector provides a useful tool for documenting and measuring changes in these pigments while maintaining registration with confocal imaging.

【 授权许可】

   
2015 Collings.

【 图 表 】

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【 参考文献 】

• [1]Mathur J. The illuminated plant cell. Trends Plant Sci. 2007; 12:506-513.
• [2]Silverstone AL, Ciampaglio CN, Sun T-P. The Arabidopsis RGA gene encodes a transcriptional regulator repressing the gibberellin signal transduction pathway. Plant Cell. 1998; 10:155-169.
• [3]von Arnim AG, Deng X-W, Stacey MG. Cloning vectors for the expression of green fluorescent protein fusion proteins in transgenic plants. Gene. 1998; 221:35-43.
• [4]Marc J, Granger CL, Brincat J, Fisher DD, Kao T-H, McGrubbin AG, Cyr RJ. A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells. Plant Cell. 1998; 10:1927-1939.
• [5]Baulcombe DC, Chapman S, Santa Cruz S. Jellyfish green fluorescent protein as a reporter for virus infections. Plant J. 1995; 7:1045-1053.
• [6]Heinlein M, Epel BL, Padgett HS, Beachy RN. Interaction of tobamovirus movement proteins with the plant cytoskeleton. Science. 1995; 270:1983-1985.
• [7]Haseloff J, Siemering KR, Prasher DC, Hodge S. Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc Natl Acad Sci USA. 1997; 94:2122-2127.
• [8]Hepler PK, Gunning BES. Confocal fluorescence microscopy of plant cells. Protoplasma. 1998; 201:121-157.
• [9]Blancaflor EB, Gilroy S. Plant cell biology in the new millenium: new tools and new insights. Am J Bot. 2000; 87:1547-1560.
• [10]Feijó JA, Moreno N. Imaging plant cells by two-photon excitation. Protoplasma. 2004; 223:1-32.
• [11]Gutierrez R, Grossmann G, Frommer WB, Ehrhardt DW. Opportunities to explore plant membrane organization with super-resolution microscopy. Plant Physiol. 2010; 154:463-466.
• [12]Fitzgibbon J, Bell K, King E, Oparka K. Super-resolution imaging of plasmodesmata using three-dimensional structured illumination microscopy. Plant Physiol. 2010; 153:1453-1463.
• [13]Liesche J, Ziomkiewicz I, Schulz A. Super-resolution imaging with pontamine fast scarlet 4BS enables direct visualization of cellulose orientation and cell connection architecture in onion epidermis cells. BMC Plant Biol. 2013; 13:226. BioMed Central Full Text
• [14]Maizel A, von Wangenheim D, Federici F, Haseloff J, Stelzer EHK. High resolution live imaging of plant growth in near physiological bright conditions using light sheet fluorescence microscopy. Plant J. 2011; 68:377-385.
• [15]Sena G, Frentz Z, Birnbaum KD, Leibler S. Quantitation of cellular dynamics in growing Arabidopsis roots with light sheet microscopy. PLoS One. 2011; 6:e21303.
• [16]Paddock SW. Confocal laser scanning microscopy. Biotechniques. 1999; 27:992-1004.
• [17]Moreno N, Feijó JA, Cox G. Implementation and evaluation of a detector for forward propagated second harmonic signals. Micron. 2004; 35:721-724.
• [18]Lee DW. Nature’s palette. The science of plant color. University of Chicago Press, Chicago; 2007.
• [19]Tanaka Y, Sasaki N, Ohmiya A. Biosynthesis of plant pigments: anthocyanins, betalains and carotenoids. Plant J. 2008; 54:733-749.
• [20]Sasaki N, Nakayama T. Achievements and perspectives in biochemistry concerning anthocyanin. Plant Cell Physiol. 2015; 56:28-40.
• [21]Wiltshire EJ, Collings DA. New dynamics in an old friend: dynamic tubular vacuoles radiate through the cortical cytoplasm of red onion epidermal cells. Plant Cell Physiol. 2009; 50:1826-1839.
• [22]Inoué S, Spring KR. Video microscopy, the fundamentals. 2nd ed. Plenum Press, New York; 1997.
• [23]Foster B. Optimizing light microscopy for biological and clinical laboratories. Kendall Hunt, Dubuque; 1997.
• [24]Nolte A, Pawley JB, Höring L. Non-laser light sources for three-dimensional microscopy. In: Handbook of biological confocal microscopy. 3rd ed. Pawley JB, editor. Springer, New York; 2006: p.126-144.
• [25]Centonze V, Pawley JB. Tutorial on practical confocal microscopy and use of the confocal test specimen. In: Handbook of biological confocal microscopy. 3rd ed. Pawley JB, editor. Springer, New York; 2006: p.627-649.
• [26]Gratton E, vande Ven MJ. Laser sources for confocal microscopy. In: Handbook of biological confocal microscopy. Pawley JB, editor. Plenum Press, New York; 1995: p.69-97.
• [27]Idris NA, Collings DA. Cell wall development in the velamen layer of the orchid Miltoniopsis investigated by confocal microscopy. Malays J Micro. 2014; 10:20-26.
• [28]Idris NA, Collings DA. The life of phi: the development of phi thickenings in roots of the orchids of the genus Miltoniopsis. Planta. 2015; 241:489-506.
• [29]Russ JC. The image processing handbook. 5th ed. Taylor and Francis, Boca Raton; 2007.
• [30]Thomas J, Ingerfeld M, Nair H, Chauhan SS, Collings DA. Pontamine fast scarlet 4B: a new fluorescent dye for visualising cell wall organisation in radiata pine tracheids. Wood Sci Technol. 2013; 47:59-75.
• [31]Cogswell CJ, Hamilton DK, Sheppard CJR. Colour confocal reflection microscopy using red, green and blue lasers. J Microsc. 1992; 165:103-117.
• [32]Pawley JB. Appendix 2: light paths of current commercial confocal light microscopes for biology. In: Handbook of biological confocal microscopy. 2nd ed. Pawley JB, editor. Plenum Press, New York; 1995: p.581-598.
• [33]Cheng P-C, Lin B-L, Kao F-J, Gu M, Xu M-G, Gan X, Huang M-K, Wang Y-S. Multi-photon fluorescence microscopy—the response of plant cells to high intensity illumination. Micron. 2001; 32:66-669.
• [34]Cheng P-C. Interaction of light with botanical specimens. In: Handbook of biological confocal microscopy. 3rd ed. Pawley JB, editor. Springer, New York; 2006: p.414-441.
• [35]Littlejohn GR, Gouveia JD, Edner C, Smirnoff N, Love J. Perfluorodecalin enhances in vivo confocal microscopy resolution of Arabidopsis thaliana mesophyll. N Phytol. 2010; 186:1018-1025.
• [36]Littlejohn GR, Mansfield JC, Christmas JT, Witterick E, Fricker MD, Grant MR, Smirnoff N, Everson RM, Moger J, Love J. An update: improvements in imaging perfluorocarbon-mounted plant leaves with implications for studies of plant pathology, physiology, development and cell biology. Front Plant Sci. 2014; 5:140.
• [37]Wiltshire EJ, Eady CC, Collings DA (2015) Induction and inhibition of anthocyanin in the inner epidermis of red onion leaves by environmental stimuli, and through transient expression of transcription factors. In preparation
• [38]Collings DA (2015) Anthocyanin in the vacuole of red onion epidermal cells quenches other fluorescent molecules. In preparation
• [39]Brauer D, Uknalis J, Triana R, Tu S-I. Effects of external pH and ammonium on vacuolar pH in maize roothair cells. Plant Physiol Biochem. 1997; 35:31-39.
• [40]Roos W, Evers S, Heike M, Tschöpe M, Schumann B. Shifts in intracellular pH distribution as a part of the signal mechanism leading to the elicitation of benzophenanthridine alkaloids. Plant Physiol. 1998; 118:349-364.
• [41]Gratton E, vande Ven MJ. Laser sources for confocal microscopy. In: Handbook of biological confocal microscopy. 3rd ed. Pawley JB, editor. Springer, New York; 2006: p.80-125.
• [42]Nelson BK, Cai X, Nebenführ A. A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J. 2007; 51:1126-1136.