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Fluorescence Microscopy


Visualisation of subtle changes to cellular environments can be achieved using nanosensors


Nanosensors at a glance

Fluorescent nanosensors are spherical probes composed of an inert matrix with nanometre sized dimensions. They selectively respond to stimuli in their surroundings to transduce fluorescence signals to a detector and 'sense' environmental changes. Due to their small size, high signal-to-noise ratio and versatile inert matrix, fluorescent nanosensors can be thought of as powerful tools that represent an advance in optical sensor based technologies.

Applications of Nanosensing 

Nanoparticles have been developed for sensing:

  • pH
  • Oxygen
  • Temperature
  • Proteins
  • Water

And generating:

  • Reactive Oxygen Species (ROS)

They have been developed with demonstrated application in a range of microenvironments and model organisms, which include mammalian cell lines (hMSCs & HeLa), nematodes (Caenorhabditis elegans, Pristionchus pacificus), Yeast (Saccharomyces cerevisiae), extracellular matrix (collagen) and microelectronics (microelectromechanical systems). 


Figure 1. (A) Fluorescent Nanosensor principles and scanning electron microscopy (SEM) Images of (B) polyacrylamide and (C) silica sol-gel based nanosensors.


pH Sensitive Nanosensors 

Our fluorescent pH-sensitive nanosensors are diagnostic probes synthesised from an inert polyacrylamide matrix and are capable of making accurate ratiometric measurements to:

  • A high spatial (<50 nm) resolution
  • A high temporal (<100 ms) resolution.
  • A high pH resolution (± 0.17 pH units).

We have recently shown how extended dynamic range pH-sensitive nanosensors can be used to monitor real time rhythmic intestinal pH oscillations in the model organisms Caenorhabditis elegans (Figure 2) and P.pacificus (Figure 3) and determined the intracellular pH changes in yeast during glucose metabolism (Figure 2.) 


Fig 2. (A) Intestinal pH map of C. elegans, (Bi) subcellular delivery nanosensors to Saccharomyces cerevisiae and (Bii) corresponding pH distribution. 



170503 Figure 3. P.pacificus gif

Fig 3. Three-dimensional pH map for P. pacificus

Oxygen Measurement 

Oxygen sensitive nanosensors can be embedded in compressed collagen sheets, enabling innovative approaches for real-time resolution of oxygen gradients throughout 3D matrices useful for tissue regeneration.


Figure 4. Methods for determining oxygen levels in compressed collagen using oxygen sensitive nanosensors. 


Temperature Measurement 

A custom designed microelectromechanical systems (MEMS) micro-hotplate, capable of operating at high temperatures (up to 700 °C), was used to thermo-optically characterise fluorescent rhodamine B (RhB) based silica sol-gel temperature-sensitive nanosensors (500 nm diameter).

The MEMS device used for this study could prove to be a reliable, low cost, low power and high temperature micro-hotplate for the thermo-optical characterisation of sub-micron sized particles from 0 – 145 °C, Figure 5. 


Figure 5. (A) Reflected light image of the silicon wafer of the MEMS device (scale bar = 150 μm), (B) temperature-sensitive nanosensors deposited on MEMS micro-hotplate heater (scale bar = 50 μm) and (C) Three and (inset) two dimensional surface plot intensity plots of nanosensors, imaged at 25 ̊C using confocal microscopy (scale bar = 500 nm)



Reactive Oxygen Species Generation 


Fluorescent nanoparticles conjugated to zinc (II) or complexed porphyrins are capable of generating controlled amounts of ROS in human mesenchymal stem cells (hMSCs) upon irradiation with visible light. Control over ROS generation was demonstrated by: (1) attenuating the percentage of porphyrins on the nanoparticle surface and (2) modulating the number of light irradiation doses to the internalised nanoparticles. The degree of ROS production was visualised through use of a newly synthesised dye, which is chemically transformed into a fluorescent entity in the presence of ROS. 

NS_ROSFigure 6. Fluorescence images for untreated hMSCs and hMSCs treated with 5, 10 and 20% Zn(II) functionalized nanoparticles, stained with BPTFMC and MitoTracker® red, irradiated with a single dose of light. BPTFMC, in the presence of H2O2, is converted to fluorescent green HTFMC. Scale bar = 50 μm.



Publications of Interest 

1. Chauhan, V. M.; Burnett, G. R.; Aylott, J. W., Dual-fluorophore ratiometric pH nanosensor with tuneable pK(a) and extended dynamic range. Analyst 2011, 136 (9), 1799-1801.

2. Chauhan, V. M.; Orsi, G.; Brown, A.; I, P. D.; Aylott, J. W., Mapping the Pharyngeal and Intestinal pH of Caenorhabditis elegans and Real-Time Luminal pH Oscillations Using Extended Dynamic Range pH-Sensitive Nanosensors. ACS Nano 2013, 7 (6), 5577-5587.

3. Chauhan, V. M.; Hopper, R. H.; Ali, S. Z.; King, E. M.; Udrea, F.; Oxley, C. H.; Aylott, J. W., Thermo-optical characterization of fluorescent rhodamine B based temperature-sensitive nanosensors using a CMOS MEMS micro-hotplate. Sensors and Actuators B: Chemical 2014, 192, 126-133.

4. Chauhan V. M.; Giuntini F.; Aylott J. W.; Quadruple labelled dual oxygen and pH-sensitive ratiometric nanosensors. Sensing and Bio-Sensing Research 2016, 8 36-42

5. Lightfoot, J. W,: Chauhan V. M,; Aylott J. W,; Rödelsperger C,; Comparative transcriptomics of the nematode gut identifies global shifts in feeding mode and pathogen susceptibility. BMC research notes 2016 9 (1), 142

6. Desai, A. S.; Chauhan, V. M.; Johnston, A. P. R.; Esler, T.; Aylott, J. W., Fluorescent nanosensors for intracellular measurements: synthesis, characterization, calibration, and measurement. Frontiers in Physiology 2014, 4.

7. Giuntini, F.; Chauhan, V. M.; Aylott, J. W.; Rosser, G. A.; Athanasiadis, A.; Beeby, A.; MacRobert, A. J.; Brown, R. A.; Boyle, R. W., Conjugatable water-soluble Pt(II) and Pd(II) porphyrin complexes: novel nano-and molecular probes for optical oxygen tension measurement in tissue engineering. Photochemical & Photobiological Sciences 2014, 13 (7), 1039-1051.

8. Lavado, A. S.; Chauhan, V. M.; Zen, A. A.; Giuntini, F.; Jones, D. R. E.; Boyle, R. W.; Beeby, A.; Chan, W.; Aylott, J. W., Controlled Intracellular Generation of Reactive Oxygen Species in Human Mesenchymal Stem Cells Using Porphyrin Conjugated Nanoparticles. Nanoscale 2015. 

9. Welser, K.; Grilj, J.; Vauthey, E.; Aylott, J. W.; Chan, W. C., Protease responsive nanoprobes with tethered fluorogenic peptidyl 3-arylcoumarin substrates. Chemical Communications 2009,  (6), 671-673.






Interface and Surface Analysis Centre (ISAC)