View source for Cell Imaging Using Picosecond Ultrasonics

==Motivation==
This project is to develop an instrument for imaging cells using their mechanical properties as a contrast mechanism. The goal is to produce a system that can be used as a general microscopy tool for cell biologists.

This is very challenging as to obtain high resolution imaging necessitates using high frequency ultrasound and at high frequencies the waves do not travel very far.  Picosecond laser ultrasound has been shown (link to bordeaux) to be a promising candidate.

==Detection mechanism - Brillouin Oscillations==
[[Image:Brillouin simple.png|150px|right]]
The  probe light is reflected from the sample surface to the detector, and if the sample is transparent there is also a reflection from the acoustic wave packet traveling in  the sample. This happens because the acoustic wave causes a change in  the local refractive index and so acts as a weak mirror.  These reflections interfere at the detector and as the phase of the  reflection from the acoustic wave is changing with time it leads to an oscillating  signal. These oscillations are termed Brillouin Oscillations.

The frequency ''F'' of the oscillation is given by the simple equation:
''F = 2*Va*n/λ''

So  if we can measure this signal we can measure the acoustic velocity ''Va'', as  long as we know the laser wavelength ''λ'' and the refractive index ''n''.

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==Substrate Design ==

{|class="wikitable" align="right"
|-
|[[Image:Pump_Transmittance.png |150px|alt=alt text|Pump Transmittance]]||[[Image:Transmittance_Probe.png |150px|alt=alt text|Probe Transmittance]]
|-
|[[Image:Transmitance_sensitivity.png |150px|alt=alt text|Sensitivity]]||[[Image:Resonance_Freqs.png |150px|alt=alt text|Resonant Frequency]]
|}

The cells are grown on a transducer substrate similar to those described  [[Nanoscale_Ultrasonic_Transducers|here]]. The optimization of the  transducer layer is different in this case. Here we are much more concerned with optimizing the optical properties of the transducers. The  goal is to reduce the amount of blue light reaching the cell and either  maximize the reflected or transmitted light for the probe beam. We still have to ensure that the acoustic  performance of the transducer is ok and that the acoustic bandwidth is  sufficient to generate waves in the Brillouin frequency range of the sample.

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==Instrumentation advancements to enable imaging==
[[file:BNC_ASOPS_Layout.jpg|150px|right|alt=alt text|ASOPS pump probe instrument]]

Measurements are made using a pump/probe picosecond laser ultrasound instrument utilizing ASOPS lasers. These laser control the delay between the pulse electronically removing the need to mechanically scan a delay line. this allows the very small signals present in these typos of experiments to be obtained quickly. 

The pump beam is frequency doubled in a non linear crystal to have a wavelength of 390nm, the probe beam is at 780nm and both beams can be directed to the sample through and objective lens normal to the sample. optionally the pump beam can also be directed to the underside of the sample to generate the acoustic waves from below.

The instrument also has optical paths for wide field phase contrast imaging to allow the cells being studied to be visualized. This allows to assess the cells health and locate the scan region and register the acoustic image with the cell of interest.

The acoustic signals are captured by recording the probe beam power with a photo-diode, this signal is then filtered and amplified before being digitized in a digital oscilloscope. To build up an image the sample is raster scanned.

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==Ultrasonic imaging of cells==
An example trace is shown below, there are three main components present in the signal. 
A signal from the transducer substrate, once from Brillouin oscialltions in the glass, and one from the cell or media. These signals are all clearly separated in the the frequency domain as shown by the FFT.
{|class="wikitable" align=""
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|[[Image:PLU_sample_trace_filtered.png  |300px|alt=alt text|Typical signal]]
|[[Image:PLU_sample_trace_fft.png  |300px|alt=alt text|Cell outline]]
|}

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===Fixed Cells===
By recording the Brillouin frequency while scanning the sample images can be taken. The below shows a Brillouin frequency map of a fixed fibroblast cell, this image shows the location of the nucleus (the redder region).
{|class="wikitable" align=""
|-
|[[Image:PLU_fixedCell1.png   |300px|alt=alt text|image of cell]]
|[[Image:PLU_fixedBrillouin1.png   |300px|alt=alt text|Brillouin image of cell]]
|}
A different fibroblast cell is shown below, here we can see much more detail, with the filopodia clearly visible.
{|class="wikitable" align=""
|-
|[[Image:PLU_fixedCell2.png   |300px|alt=alt text|image of cell]]
|[[Image:PLU_fixedBrillouin2.png   |300px|alt=alt text|Brillouin image of cell]]
|}
The image below shows a scan of a fixed cardiac cell, the striations of the cell are clearly visible, these repeating structures are how the cells contract, and have spacings on the order of a few microns.
{|class="wikitable" align=""
|-
|[[Image:PLU_cardiacOptical.png  |300px|alt=alt text|image of cell]]
|[[Image:PLU_cardiacBrill.png   |300px|alt=alt text|Brillouin image of cardiac cell]]
|}

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===Live Cells===
Measuring live cells is much more challenging, this is because the amount of light they can tolerate without being effected is much lower than the amount that would damage fixed cells. Secondly the cells can move around and so the scans need to be taken as quickly as possible. 

The pictures below show a series of scans of live cells, their optical image and a corresponding fluorescence image which shows which cells are dead. if the cell is alive they do not fluoresce if they have died they light up.

==3D imaging==
It is possible to use signal processing techniques to extract more information from the data we obtain during the scans. By analyzing the frequency of the signal vs time we can extract changes in frequency with depth. This enables the mapping of internal structures in the cells, or should the acoustic waves leave the cell and enter the medium it allows us to obtain the profile.

There are a number of different ways to do this, from relatively fast and simple methods (zero cross analysis) to more complex approaches (wavelets).

Below is an example of the process for a phantom cell made from polystyrene beads. In the time trace the transition from the phantom back to the media is clearly visible. 

[[Image:PLU_transition_samplett.png  |300px|alt=alt text|trace of phantom cell]]

If we take the Brillouin frequency map and a map of the refractive index  we can produce a map of the acoustic velocity of the material. 

{|class="wikitable" align=""
|-
|[[Image:PLU_phantom2brillTrans.png  |300px|alt=alt text|Brillouin Freq]]
|[[Image:PLU_phantomNMap.png  |300px|alt=alt text|index map]]
|[[Image:PLU_phantomSpeedMap.png |300px|alt=alt text|velocity map]]
|}
Combining this map with the transition time information gives us the profile of the cell phantom.
[[Image:PLU_phantom_profile.png  |400px|alt=alt text|trace of phantom cell]]

==Related Publications and Talks==
<bibtex>@article{smith_optically_2015,	title = {Optically excited nanoscale ultrasonic transducers},	volume = {137},	issn = {0001-4966},	url = {http://scitation.aip.org/content/asa/journal/jasa/137/1/10.1121/1.4904487},	doi = {10.1121/1.4904487},},	number = {1},	urldate = {2015-02-09},	journal = {The Journal of the Acoustical Society of America},	author = {Smith Richard J. and Cota Fernando Perez and Marques Leonel and Chen Xuesheng and Arca Ahmet and Webb Kevin and Aylott Jonathon and Somekh Micheal G. and Clark Matt},	month = {jan},	year = {2015},	keywords = {Acoustic sensing, Optical resonators, Proteins, Ultrasonic transducers, Ultrasonography},	pages = {219--227},
</bibtex>

Richard Smith, Fernando Perez, LeonelMarques, Matt Clark,Design and application of nano-scaled transducers,Institute of Physics Optics + Ultrasound One Day Meeting, May 2013, Nottingham, UK

Fernando Perez Cota, M. Clark, K. F. Webb, and R.J. Smith, Nanoscale transducers for picosecond laser ultrasonics in cells,3rd symposium of laser ultrasonics, June 2013, Yokohamma, Japan,