Fluorescence imaging and investigations of directly labelled chromosomes using scanning near-field optical microscopy

Baylis, Richard; Doak, Shareen H.; Holton, Mark D.; Dunstan, P. R.

doi:10.1016/j.ultramic.2006.08.005

Cited by 9 publications

(6 citation statements)

References 21 publications

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“…Where fluorescent features, like microtubules, were separated well enough that their fluorescence signals convoluted with the LM point spread function did not overlap, we could identify individual tubes and correlate them with the cryo-EM image (Figs 3E and F, white arrowheads), providing images at <5 nm resolution of cellular regions chosen in the LM. Granted, there are now various LM techniques that beat the traditional resolution barrier imposed by the diffraction of light to visualize the location of selectively labelled features (Heintzmann & Ficz, 2006), such as nearfield microscopy (Baylis et al, 2007), structured illumination (Gustafsson, 2005) or LM superlenses (Liu et al, 2007;Smolyaninov et al, 2007), however, our correlative method uses commercially available tools and standard methods to provide information about cellular features in a near-native state at molecular resolution.…”

Section: Cryo-fluorescence Light Microscopy Will Be a Powerful Tool Fmentioning

confidence: 99%

Cryo‐fluorescence microscopy facilitates correlations between light and cryo‐electron microscopy and reduces the rate of photobleaching

Schwartz

Sarbash

Ataullakhanov

et al. 2007

Journal of Microscopy

212

152

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SummaryFluorescence light microscopy (LM) has many advantages for the study of cell organization. Specimen preparation is easy and relatively inexpensive, and the use of appropriate tags gives scientists the ability to visualize specific proteins of interest. LM is, however, limited in resolution, so when one is interested in ultrastructure, one must turn to electron microscopy (EM), even though this method presents problems of its own. The biggest difficulty with cellular EM is its limited utility in localizing macromolecules of interest while retaining good structural preservation. We have built a cryo-light microscope stage that allows us to generate LM images of vitreous samples prepared for cryo-EM. Correlative LM and EM allows one to find areas of particular interest by using fluorescent proteins or vital dyes as markers within vitrified samples. Once located, the sample can be placed in the EM for further study at higher resolution. An additional benefit of the cryo-LM stage is that photobleaching is slower at cryogenic temperatures (−140 • C) than at room temperature.

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Section: Cryo-fluorescence Light Microscopy Will Be a Powerful Tool Fmentioning

confidence: 99%

Cryo‐fluorescence microscopy facilitates correlations between light and cryo‐electron microscopy and reduces the rate of photobleaching

Schwartz

Sarbash

Ataullakhanov

et al. 2007

Journal of Microscopy

212

152

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“…Accessing information beyond the diffraction limit from a wide range of sample types has been made possible following the emergence of scanning probe microscopy (SPM) techniques. SPM enables examination of a sample's metrology with nanoscale resolution and scanning near-field optical microscopy (SNOM) is one such example which is particularly suited to interrogate the interactions and functions of biological materials [13]–[15]. SNOM has the capacity to simultaneously probe topography and examine optical features on scales that can not normally be achieved using conventional fluorescence microscopy by exploiting the properties of evanescent waves.…”

Section: Introductionmentioning

confidence: 99%

Quantum Dots for Multiplexed Detection and Characterisation of Prostate Cancer Cells Using a Scanning Near-Field Optical Microscope

et al. 2012

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In this study scanning near-field optical microscopy (SNOM) has been utilised in conjunction with quantum dot labelling to interrogate the biomolecular composition of cell membranes. The technique overcomes the limits of optical diffraction found in standard fluorescence microscopy and also yields vital topographic information. The technique has been applied to investigate cell-cell adhesion in human epithelial cells. This has been realised through immunofluorescence labelling of the cell-cell adhesion protein E-cadherin. Moreover, a dual labelling protocol has been optimised to facilitate a comparative study of the adhesion mechanisms and the effect of aberrant adhesion protein expression in both healthy and cancerous epithelial cells. This study reports clear differences in the morphology and phenotype of healthy and cancerous cells. In healthy prostate epithelial cells (PNT2), E-cadherin was predominantly located around the cell periphery and within filopodial extensions. The presence of E-cadherin appeared to be enhanced when cell-cell contact was established. In contrast, examination of metastatic prostate adenocarcinoma cells (PC-3) revealed no E-cadherin labelling around the periphery of the cells. This lack of functional E-cadherin in PC-3 cells coincided with a markedly different morphology and PC-3 cells were not found to form close cell-cell associations with their neighbours. We have demonstrated that with a fully optimised sample preparation methodology, multiplexed quantum dot labelling in conjunction with SNOM imaging can be successfully applied to interrogate biomolecular localisation within delicate cellular membranes.

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“…Near field Scanning Optical Microscopy (NSOM) has taken a more important role as it combines the high resolution of Scanning force Microscopy with the ease and high resolution of FISH (see for an early example M.H.P. Moers, 1996 andBaylis et al, 2007;Fukushi et al, 2003, for later examples).…”

Section: Afm In Cytogeneticsmentioning

confidence: 99%

Atomic force microscopy on chromosomes, chromatin and DNA: A review

Kalle¹,

Strappe²

2012

Micron

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Fluorescence imaging and investigations of directly labelled chromosomes using scanning near-field optical microscopy

Cited by 9 publications

References 21 publications

Cryo‐fluorescence microscopy facilitates correlations between light and cryo‐electron microscopy and reduces the rate of photobleaching

Cryo‐fluorescence microscopy facilitates correlations between light and cryo‐electron microscopy and reduces the rate of photobleaching

Quantum Dots for Multiplexed Detection and Characterisation of Prostate Cancer Cells Using a Scanning Near-Field Optical Microscope

Atomic force microscopy on chromosomes, chromatin and DNA: A review

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