Wang et al., (2020). Near-infrared nerve-binding fluorophores for buried nerve tissue imaging. Science Translational Medicine. Retrieved from https://www.science.org/doi/10.1126/scitranslmed.aay0712
Wang, L. G., Montaño, A. R., Combs, J. R., McMahon, N. P., Solanki, A., Gomes, M. M., Tao, K., Bisson, W. H., Szafran, D. A., Samkoe, K. S., Tichauer, K. M., & Gibbs, S. L. (2023). OregonFluor enables quantitative intracellular paired agent imaging to assess drug target availability in live cells and tissues. Nature chemistry, 15(5), 729–739. https://doi.org/10.1038/s41557-023-01173-6
Roopa, Swetha. “What Is the Difference between a Fluorophore and Fluorochrome.” Www.tutorialspoint.com, Tutorials Point, 18 May 2023, www.tutorialspoint.com/what-is-the-difference-between-a-fluorophore-and-fluorochrome
Warwick, Heath. “Urodacus Manicatus, Black Rock Scorpion. Grampians National Park, Victoria.,” Museums Victoria, 2012, https://collections.museumsvictoria.com.au/specimens/2468843. Accessed 16 Aug. 2023.
Bilovitskiy, Maxim. “On This Picture You Can See Fluorescence of Different Substances under UV Light. Green Is a Fluorescein, Red Is Rhodamine B, Yellow Is Rhodamine 6G, Blue Is Quinine, Purple Is a Mixture of Quinine and Rhodamine 6g. Solutions Are about 0.001% Concentration in Water.,” Wikimedia Commons, 3 Jan. 2014, https://commons.wikimedia.org/wiki/File:Fluorescence_rainbow.JPG.Accessed 17 Aug. 2023.
Created from BioRender: Figure 3, 4 (Lab mouse with syringe)
Photos from Karibou B.: Fig 6, Fig 5, Fig 4,
Fluorescent light can be found easily in our day-to-day lives along with some animals naturally producing chemicals or proteins that are fluorescent like the [Black Rock] Scorpion; under UV light, their exoskeleton glows a neon blue as a showcase of biofluorescence.
What is a Fluorophore? A molecule that emits light after excitation, it does this whenever this chemical compound is exposed to light from a range of UV to blue (visible) lights to even near infrared– it will then release some of this energy stored as a different higher frequency wavelength of light. This proportity/reaction of light emission is called fluorescence, and is what makes compounds a fluorophore or called a fluorescent dye
Wet Materials and Methods and Techniques
Narratively speaking: majority of my time was spent with expansion and contractions of the concentration of chemicals within solvents, the other parts were learning how to use machinery and the data analysis/results they produced and how to analysis the data figures for what I was doing.
Following synthesis procedures, for the reactions I was present for took about ⅓ of my work time there, once the reaction was quenched it was time again to expand and contract it down to satisfactory purity concentrations for the compound to be a clean enough intermediate or final product
Results: Wet Chemistry Lab
When fluorescent dyes are ready to be shipped off from Gibbs wet lab, one of their destinations are into a rodent, either through injection or orally taken. In Gibbs Lab there’s a project on: In Vivo nerve staining for near-infrared fluorescence guided surgery.
Some fluorophores created in this lab are specially designed to be nerve-binding and also cross the blood brain barrier. Mice had either direct application of the dye or were injected with the dye for systemic application and operated on hours post-injection.
- Rodent In Vivo Studies -
Fluorescent-Guided Surgery
Discussions + Limitations
A limitation is that the light emission from different fluorophores can be widely variable in intensity and/or duration from even a couple small changes in structure or composition, along with some unpredictability in which structure will shine the brightest and that difference of brightness between different applications (Systemic vs direct)
Future goals: Doing fluorescence swine nerve imaging studies to witness how fluorophores respond and react in larger animals– this will bring FGS closer to human trials as pigs are a closer analogue compared to rodent for humans
Future hopes: Following a fluorophore line from it’s intal synthesis in the wet lab to in vivo studies
Figures + Literature Citations
Acknowledgments
This poster was created based on the experiences I’ve had and witnessed over 6 weeks spent in the Gibbs laboratory in this Summer of 2023. It has sparked my interest into organic chemistry even more.
I hold many thanks to my mentors both within the lab, from my Knight Scholars Program, and all advocates who supported me throughout my education– if it were not for all of them, my path in life would not be as beautiful as it is.
Hundreds of chemicals all with different groups of purpose and capabilities,, many machines that makes different sized, and shaped glassware containers spin, shake, gyrate and/or vibrate and possibly with magnets that make the “stir bars” spin. Along with hot plates, ovens, and freezers at -80 celsius or just a cold room set at -4 degrees celsius
Synthesis of intermediates occurred less than I anticipated walking into the lab.
Oregon Health & Science University,Department of BioMedical Engineering, Knight Cancer Institute, Knight Scholar Program, Portland OR.
Karibou B., Antonio M., Summer G., Lei G. Wang
All of these techniques to machines are the data needed to show what is being done in the laboratory is successful or not. Such as the LC-MS being an analytical machine to determine characteristics of our compound and analysing if the data displayed is desired or not, and hopefully what can be done about it. Organic chemistry as I experienced it in the Gibbs lab is collecting and purifying compound yield from different chemistry procedures– we would spend our time running purification experiments along with synthesizing intermediates, then collecting the yield to then use that yield as the next synthesize produces’ starting material, and purify that done once
finished. At the end, it can be stored and used for later; it may end
up being used further in cells for fluorescence microscopy or…
Above Figure 2: Black rock scorpion shown under UV light, Museums Victoria
Wet Materials and Methods and Techniques, cont.
Fluorophores: Wet Chemistry Laboratory synthesis to In Vivo Nerve imaging studies towards Fluorescence Guided Surgery Applications
Shown below (Fig. 6) are a row of Thin Layer Chromatography plates I spotted of racks that were 5x7, I’d spot 1,7,14,21,28, and 35, and if needed between those margins of 7 to spot when there was no determinable mixed product or multiple compound coming from a single spot. This is the larger of flash chromatography racks compared to all others i’ve assisted with. The leftmost TLC plate is spotted with this compounds starting material as a reference plate for mixed tubes
Figure 5: BioTage Flash column Chromatography in progress for purification of a blue fluorescent dye. The saturation of the colors in the tubes give a visual indicator of how concentrated the dye is in the solvent system.
To the Left
Figure 4: 10g silica Biotage flash column (used) Above
Above, Figure 1: Fluorescence of different substances under UV light. Green is a fluorescein, red is Rhodamine B, yellow is Rhodamine 6G, blue is quinine, purple is a mixture of quinine and rhodamine 6g. Solutions are about 0.001% concentration in water.
Figure 3: BioRender created depiction of a Liquid Chromatography - Mass Spectrometry, machine equipment + MS graph
- Rodent In Vivo Studies -
Fluorescent-Guided Surgery, cont.
This parent compound created derivatives can be seen in vivo imaging usage on the mice nerve images all starting LGW
Across all surgeries is the concern for nerve damage to inadvertently occur, and is a major source of morbidity or chronic pain. By having the nerves easier to image– it can reduce iatrogenic (in hospital) nerve injuries due to heightened visibility.
For this FGS study, the fluorescent compounds were derived from Oxazine 1 (Below), A library of fluorophores was created based off this model,
Figure 8: In vivo brightness and nerve specificity of lead probe candidates. Representative color and fluorescence images of parent LGW03-76 and lead candidate derivatives LGW14-46, LGW14-63, LGW03-07, LGW05-65, LGW05-73, LGW13-79, LGW14-76 and a noninjected/non-stained control group following (A top) systemic administration at 7.5 µmol/kg and (A bottom) direct application (125 µM) to exposed brachial plexus nerves in mice. All images represent data collected across either n = 4 mice for systemic administration or n = 6 nerve sites for direct application per fluorophore. Intensity values provided boxed in the lower left corner indicate the fractional intensity value of each imaged compound relative to the brightest fluorophore (LGW14-76). Average nerve tissue fluorescence intensities of the fluorophore library following systemic administration plotted (B) against average nerve tissue fluorescence intensities following direct application. Quantified N:M and nerve-to-adipose (N:A) Signal-to-background ratios were calculated for comparison of parent LGW03-76, lead candidates and control groups following (C) systemic administration and (D) direct application as in (A). All quantified values are presented as the mean ± Standard deviation.
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