1. Modular Fiber Optic System for Intramural Functional Fluorescence Measurement
Dean Tai
Bryan Caldwell
Ian LeGrice
John Harvey
Sally Rutherford
Bruce Smaill
Good day everyone, the topic that I am going to present today is the development and the application of a modular functional fluorescence imaging system that we had developed which can do cellular scale functional imaging. In this particular presentation, we used it for study the cell membrane potential of the rat hearts, intramurally. By intramural, we are looking at the cells deeper under the surface, which was normally sort of a constrain of typical fluorescence imaging studies.

2. Outline Functional fluorescence imaging system Optrode
Modular fiber fluorescence imaging system
Results
Future Development
Here is a quick outline for me presentation. Just in case that if anyone here is unfamiliar with function fluorescence imaging system, I will by giving a short description of what it means. Then I will show you an special optical probing system that we have developed in the University of Auckland, which provides the system to do intramural, 3D mapping. Next, we will see the new modular fiber imaging system, along with some results. And finally, we will see what we can do this this newly developed system.

3. Functional Fluorescent Indicator
Non-ratiometric probes
Data often expressed as relative change in intensity (ΔF/F)
Used for relative comparison
From Molecular Probes Handbook Figure 19.24
Ratiometric probes
Expressed as ratios between wavelengths (eg. F500nm/F400nm)
Used for absolute comparison
From Molecular Probes Handbook Figure 20.4
It is the invention of the functional fluorescent indicators that make the functional fluorescence imaging so popular these days. The special property of these indicators is that, their emission spectra can be modulated by environmental changes. For example, this particular indicator has significantly different emission spectrum when the calcium concentration in the environment changes. The other important issue is that, these functional fluorescent indicators can be categorized into Non-ratiometric and Ratiometric indicators. With the Non-ratiometric probes, the emission spectra undergo intensity variation when the calcium concentration changes, but it can only give relative measurements. The ratiometric probes has quite different spectral responses, we see that the emission spectra sort of going through a spectral shift instead. There is ☼ a point where, so called isosbestic point, the intensity remains constant during the shift. Results are usually expressed as ratios between wavelengths ☼ above and below the isosbestic wavelength. Unlike Non-ratiometric probes, it can give an absolute measurement.

4. Fibre Optics Based Fluorescence Imaging System
Coherent illumination sources
Intramural multi-site recording
Higher spatial resolution
3D mapping
Dual-wavelength recording
>600nm
520~600nm
Now we will move along and take a look of the fluorescence imaging system that we have developed. This fiber optics based imaging system is very similar with conventional fluorescence imaging system, except that it is doing 3D intramural rather than 2D surface imaging. Argon gas laser was used as excitation source ☼ and was led to the optic fibers and eventually to the sampling tissue ☼. An optical probe made with optical fibres was used such that illuminating and fluorescence light can be delivered and picked up from a very small volume deeper under the tissue surface. With the aid of this optical probe, 3D optical mapping is possible intramurally with high spatial resolution. Then, the fluorescence collected by the same optical probe, passed through the first dichroic mirror ☼ , and was split by the second dichroic mirror ☼ to the dual-wavelength detection system. This dual-wavelength recording system enables us the reduce the associated motion artifact in the optical signal, and I will show you an example with this later on. Now ☼, there is a picture of the system in action. Note that we were running heart with some special colourless fluid to keep the heart alive rather than blood, since the presence of the haemoglobin would severely obscured the emission spectra.

5. Optrode
Some of you might be wondering now what does this optical probe look like, so let me show you some pictures of this probe as well. This optical probe, we so called optrode, is made of a bundle of 7 optical fibres and deliberately cleaved and orientated so that it can measure up to 7 sites along the longitudinal direction where the probe is inserted. On the left is a schematic drawing of the design, each fibre terminates in 1mm interval inside a micropipette. On the right, are pictures taken from the side and the end views of the optrode. However, one disadvantage of the design of the optrode is that the coupling efficiency is very low. All 7 optical fibres were packed closely in a hexagonal shape, and were illuminated by a single laser. Because of this coupling mechanism, only the 6 fibres at the surrounding ring are usable while the centre one is disregarded because of the excessive power. And it leaves a coupling efficiency of approximately 10% or less in practice.

6. Modular All-Fibre Imaging System
In order to improve the fibre optics based fluorescence imaging system, the next step we took is to make the previous system into a modular device that is easer and simpler to use, smaller and cheaper and yet, with better performance. Modifications include replacing the ☼ Argon gas laser with solid state laser, replacing ☼ most of the free space optics with fibre coupler. Use of the ☼ 1-to-8 fibre splitter so that light can be coupled into the fibres more efficiently ☼, and now all 7 optical fibres inside the optrode can be used rather than 6. This modular system exhibits the several advantages in comparison with the conventional imaging system. For example, Signal to Noise Ratio, the SNR of the new solid state lasers is better than the gas lasers. Other things like the cost, robustness, compactness, simplicity of system construction and maintenance and, probably most importantly, the scalability. Can you imaging the trouble is takes to set up a hundred channel imaging system with the conventional setup, with all the free space optics and the time it takes to get the alignment right etc.
Solid state laser
Fibre coupler
1-to-8 fibre splitter

7. Laser Noise Peak-to-peak noise ≈ 3.78% Peak-to-peak noise ≈ 0.19%
RMS noise ≈ 1.21%
Peak-to-peak noise ≈ 0.19%
RMS noise ≈ 0.11%
Now, we look at the noises of the 2 illumination sources. It should be fairly obvious why we decided to use solid state lasers instead. In our particular case, it is the peak-to-peak noise that we are interested in and it is improved by nearly 20 folds. Just note that, the RMS noise can be as low as about 0.5% for good gas lasers, but with the solid state lasers, you can get noise level of 0.1% or lower.

8. Photobleaching Rate (for voltage-sensitive dye, di-4-ANEPPS)
Photobleaching rates behave similarly for both wavelengths
Decay rate is significantly higher with 488nm excitation
Initial signal level is ~ 2:1 with 488nm and 532nm excitation respectively
532nm is a more suitable excitation source for long duration or repeated recordings at the same site
Initial Signal Level
532nm
488nm
Mean (a.u.)
18.7
36.8
Standard Deviation (a.u.)
14.9
23.2
However, since the wavelengths from 2 lasers are significantly different, so we also looked at the impact of this on our fluorescent dye. And the most significant difference we have seen initially is the difference in photobleaching rate of the dye. Now, remember that we were doing dual-wavelength recording simultaneously, and it turns out that the photobleaching rates at both wavelength channels are very similar and therefore we will only be looking at the long-wavelength channel. Our data suggested that with the 488nm gas laser, we get higher fluorescence intensity initially, along with very rapid decay. With the 532nm excitation source, although is exhibits less fluorescence intensity but a much slower photobleaching rate. The initial signal intensity is approximately 2-to-1 with 488nm excitation source and 532nm excitation source respectively, but it is a very rough estimation since results are taken from stained tissue and therefore, very scattered. Typically, over a period of approximately 10 seconds, fluorescent signal collected from 532nm excitation source would have higher intensity than that for the 488nm excitation. Therefore, 532nm is a more suitable source for long and repeated recordings.

9. Spectral Shift
Spectral response as a function of membrane potential (-87, -65 and -44mV for 4, 15 and 50mmol/L of potassium concentration)
With 532nm and 488nm excitation sources
Quantitatively expressed as ratio between the intensity of two windows above and below the isosbestic point
If we look at the emission spectrum of our special voltage-sensitive dye, di-4-ANEPPS, surprisingly that, very different emission spectra were recorded when different excitation sources were used. The top diagram shows the emission spectra when it is exposed under 532nm laser, and the bottom was recorded when 488nm excitation source was used. However, although the spectra are quite different, they both demonstrate similar spectral response when the dye was under the influence of varying electrical potential. These spectra were recorded at three levels of different electrical potentials, and with both excitation sources. In all cases, a spectral shift towards the short wavelength direction was observed when the electrical potential increases. This result can be represented by taking the ratio of the intensity at wavelengths above and below the isosbestic point. With the 532nm excitation source, ☼ these were taking from 580nm and 650nm, each a 20nm wide window, and with the 488nm excitation source, ☼ 580nm and 630nm were taken.

10. Quantifying the Spectral Shift
With 532nm excitation, F650/F580 was measured
With 488nm excitation, F630/F580 was measured
Both demonstrate that the spectral shift is a linear function of the cell membrane potential
Now, when we plot these ratios as a function of the membrane potential measured, we can see that, a linear relationship can be observed. Although, different spectra were seen with different excitation sources, similar linear relationship was observed. Therefore, at this point, we are certain that 532nm can be used to replace 488nm as the excitation source in the functional study that we are interested in here.

11. Experimental Data
Ok, now we will move along and take a look of some experimental results. Here, on top left, is the typical signal collected from rat hearts. On the top right, is the ratioed optical recording which was obtained by dividing the 2 wavelength signals one by another. At the bottom left is the intracellular recording. Note what you seen on the top left is when the optical signals are obscured by motion artifact, which is an intrinsic problem due to the fact you are recording optical signals form a moving object. However, with the dual-wavelength recording technique, motional artifact can be minimised by dividing the 2 signals one by another. At the bottom right is an example of typical data from the dual wavelength recording system.

12. Comparisons between Systems
Conventional system
~ 2m x 1m
Water cooled gas laser (5W)
Free space optics
Modular fibre system
~ 20cm x 10cm
Solid state laser (50mW)
Modular fibre construction
Now here are some more pictures, on the top is our old system made with Argon gas laser and free-space optics. And on the bottom ☼ is the picture taken with the new modular system ☼ sitting next to our old system. This modular system is much smaller in size; you can see the dimensions of the two, 2X1m again 20X10cm. All that I am saying is that you can replace this whole lots by this ☼ little black box and yet, it gives better performance.

13. Future Developments High speed spectrophotometer
Possible removal of excitation light with software
Study in the presence of haemoglobin
Other functional fluorescence imaging
Multifunctional study
Now, where we are heading this modular system next is to put it together with high speed spectrophotometer. For example, this is the spectrum recorded with 532nm excitation source, and this is sitting inside the range of the emission spectrum. In order to prevent this leakage into the photodetectors, emission filters are required, which would attenuate the emission intensity as well. With the high speed spectrophotometer, we can remove this excitation leak in software after data acquisition without losing fluorescent light. There are many advantages we can gain with this speed spectrophotometer, for example, it would enable us to do this study in the presence of haemoglobin, which would obscured the spectrum severely, and also, it can be used to many other functional fluorescence imaging as well. Most excitingly, multiple functional studies can also be done with this device, for example, we can measure both the calcium concentration and the cell membrane potential at the same time.

14. Acknowledgement Marsden Fund FoRST University of Auckland
RSNZ R H T Bates Scholarship
At the end of my talk, I would like to thank the following people who helped me out along the way, and the ones who supported my research. And thank you for your patience.