Intrinsic fluorescence of the parathyroid gland reduces risk of endocrine surgery

Recently, our lab--the Biomedical Photonics Research Group at Vanderbilt University--has discovered that the parathyroid glands (four rice-size glands in the throat) emit an intrinsic fluorescence when excited with near-infrared (NIR) light. Without the application of exogenous chemicals, fluorescence spectroscopy can be used to identify the parathyroid glands during endocrine surgery.

Melanie Gault

Parathyroid detection is one of the biggest challenges in endocrine surgeries. These glands produce a hormone which narrowly regulates blood and bone calcium level. Because of their small size and variable location, inadvertent removal or damage of the parathyroid gland during surgery can have life-long effects on patient health.

Our lab has shown that NIR fluorescence spectroscopy is a unique tool to distinguish the parathyroid gland. The spectroscopy system is made up of a spectrofluorimeter, a 785 nm diode laser coupled through a 7-around-1 fiber-optic probe, and a laptop. It is portable to allow easy transfer into the clinic. When excited with 785 nm light, the fluorescence intensity of the parathyroid gland is consistently two to 11 times greater than that of the thyroid, fat, muscle, and other tissues in the neck.¹ Of the 59 patients measured at the Vanderbilt University Medical Center, the parathyroid emitted a greater fluorescent intensity 100% of the time, regardless of disease state. Each of the spectra measured were compared to the gold standard of visual inspection or histology when available.

This work will continue in several directions. First, we will study the mechanism of the fluorescent signal. There are very few biological fluorophores in the NIR region. Discovering the mechanism will allow us to expand the use of this diagnostic tool. We will also perform additional patient studies to determine whether the fluorescent intensity of the parathyroid varies, depending on disease state. Ultimately, we would like to create an imaging system that provides spatial information to guide the surgeon in real time.

REFERENCE
1. C. Paras, M. Keller, L. White, J. Phay, and A. Mahadevan-Jansen, J. Biomed. Opt., 16, 6, 067012 (2011).

MELANIE GAULT is a PhD candidate in the Biomedical Photonics Lab at Vanderbilt University (Nashville, TN). Her research under Anita Mahadevan-Jansen is related to optical guidance during surgery, specifically using fluorescence spectroscopy.

Optical data analysis looks to clinical applications

Christine Amwake

I am privileged to work with a highly interdisciplinary team where engineers work together with life scientists to solve interesting biological questions and pursue clinical applications of our work. The engineering experts are led by Tom Baer from the Stanford Photonics Research Center, and we provide optical and analytical techniques to collect and analyze data. The life science experts, who determine the pertinent biological questions, are led by Dr. Renee Reijo Pera from the Stanford School of Medicine.

Previous work under the direction of the Dr. Reijo Pera and Dr. Baer correlated dark-field time-lapse imaging with gene expression profiling to conclude that success of the embryo to develop into the blastocyst stage can be reliably predicted within two days of fertilization, when the embryo is made up of only four cells and before embryonic gene activation (EGA), by using time-lapse optical imaging and an algorithm to detect three key features in the development cycle.¹ The three parameters that are used to determine successful development into the blastocyst stage are (i) the duration of the first cytokinesis (the very brief last step in mitosis that physically separates the two daughter cells); (ii) time interval between the first and second mitosis events; and (iii) the time interval between the second and third mitosis events. Software developed by their team of researchers was used to detect the cells via computer vision and to follow the shape of the cells and evaluate the three parameters. The figure shows an example of how the computer vision method detects the cells and their shapes and follows them through this critical stage of development. The outcomes of the algorithm applied to time lapse imaging were correlated to gene expression data, and successful blastocyst formation was found to be predictable with 93% sensitivity and specificity.

The bottom row shows results of the tracking algorithm applied to the top row images of a single embryo. Images were taken at 5-minute intervals, and only a sampling of the images is shown here.¹

In an extension to this study, it was found that the same time lapse imaging parameters can be used within tighter tolerances to predict ploidy versus aneuploidy with 100% sensitivity and 66% specificity.² Aneuploidy is a condition found in 50-80% of cleavage stage embryos, where an abnormal number of chromosomes are present in the embryo which are either not compatible with live birth or can cause conditions such as down syndrome. Fragmentation of the embryo was shown to be correlated with time lapse imaging to predict types of aneuploidies. 

These studies proved promising for applications in successful in vitro fertilization (IVF), since typical IVF methods do not accurately predict healthy embryos and usually multiple embryos are transplanted with negative consequences such as multiple births, the need for fetal reduction, and miscarriage.¹ To this end the PIs and some co-authors of the paper listed in reference 1 have created the company Auxogyn, which brings this time-lapse imaging and computer vision automated analysis to the clinic in their Early Embryo Viability Assessment (EEVA) System.

The current work of our group applies similar imaging and analysis techniques to study other biological phenomena in culture. Optical imaging is an enabling technology for looking at biological samples because it is less invasive than many other imaging techniques. In many of our applications, we image the cells repeatedly over periods of several days, so the imaging system must be designed to support this effort. I look forward to updating you on more of our work soon. Thanks for reading!

REFERENCES
1. C. C. Wong et al., Nat. Biotechnol., 28, 1115-1121 (2010).
2. S. L. Chavez et al., Nat. Comm., 3, 1251 (2012) .

CHRISTINE AMWAKE is a PhD candidate in Electrical Engineering at Stanford University under the advisement of Prof. Olav Solgaard. Her current research interest is applying optical imaging techniques to biomedical applications.

On-chip technologies shine light on human sperms

Ahmet F. Coskun

Human reproduction has been responsible for genetic diversity among offspring, resulting in unique features in human populations. Remarkable factors causing this diversity include the variations in the genome of the sperms or eggs due to recombination and mutation, the journey of the highly specialized sperms to the egg, and the selection of the successful sperm from a population of millions of sperms. There is still much we need to know about the underlying mechanisms of these processes. To this end, recently emerging on-chip technologies shed light on the genetics, movement, and the selection of sperms toward understanding the key dynamics in human reproductive system.

Stanford researchers, led by Prof. Stephen Quake, have sequenced the whole genome of a single sperm cell using a high-throughput microfluidic sequencing device.¹ Based on a genome map extracted from 91 single sperm cells of a 40 year-old man, the genetic diversity of single sex cells is demonstrated, revealing the recombination and mutation in the DNA of an individual. Such an automated high-precision microfluidic system, combining parallelized analysis of single chromosomes, could especially be important to understand the reproductive disorders and its relation to individuals’ condition such as age and health, as well as artificial fertilization applications. This research reveals the genetic diversity of sperm cells extracted even from the same individual (see Fig. 1a).

FIGURE 1. On-chip technologies for sperm analysis are illustrated, where (a) a high-throughput microfluidic platform reveals genetic diversity in sperms, (b) a lensfree imaging system tracks sperms in 3D, and (c) a microfluidic sorter isolates the viable motile sperms from the rest. (Image courtesy of Gulcin Becerik)

Our work at UCLA, led by Prof. Aydogan Ozcan, have developed a lens-free 3D imaging technique that can track the 3D motions of more than 1,500 sperms simultaneously, demonstrating the unique swimming patterns of single sperm cells.² With its large-scale analysis capability, our study revealed the rare 3D pathways of sperm movements, including a helical spiral pattern that is 90 percent of the time in clockwise (right-handed) direction (see Fig. 1b). Such a high-throughput lens-free imaging platform could be useful to understand how the sperm moves in three dimensions. Although these findings may not be helpful to directly explain how the journey of the sperm to the egg occurs in the human body, it is still worthwhile to uncover the swimming patterns of single sperms for further biophysical studies.

Semen sample comprises various types of physiologically different sperm cells, including dead cells, non-motile viable cells and motile viable cells. For human reproduction, the “lucky” sperm reaches the egg and initiates the fertilization in the human body. In the case of male infertility, this process is performed “in vitro”, which refers to the process performed outside the body, and requires careful selection of the “lucky” sperm and injection into an egg. Recently, on-chip devices^3-4 have enabled characterization of the sperm types and then collecting the “viable” and/or “motile” ones in a separate chamber to rapidly perform the in vitro fertilization with a better success rate (see Fig. 1c).

So on-chip technologies may help us understand the human reproductive system, especially for sperm analysis. Such systems, combined with their high-throughput and automated screening capabilities, could especially be very significant to uncover the genetic diversity, 3D motion patterns, as well as the assortment process of the sperms.

REFERENCES
1. J. Wang, H. C. Fan, B. Behr, and S. R. Quake, Cell, 150, 2, 402-12 (July 20, 2012); doi: 10.1016/j.cell.2012.06.030.
2. T-W. Su, L. Xue, and A. Ozcan, Proc. Nat. Acad. Sci., doi: 10.1073/pnas.1212506109 (September 17, 2012).
3. D. Lai, G. D. Smith, and S. Takayama, J. Biophoton., 5, 8-9, 650-660 (August 2012).
4. A. T. Ohta et al., Lab Chip, 10, 23, 3213-7 (December 7, 2010).

AHMET F. COSKUN received his BS degrees in Electrical Engineering and Physics from Koc University, Turkey and MS degrees in Electrical Engineering (Major) and Chemistry and Biochemistry (Minor) from University of California, Los Angeles (UCLA). He is now pursuing his PhD degree in Electrical Engineering at UCLA, where he is conducting research in the Biophotonics Lab at UCLA under the supervision of Prof. Aydogan Ozcan. His main areas of research are widefield on-chip imaging and high-resolution microscopy for biomedical applications. He has authored or co-authored more than 50 peer reviewed research articles in major journals and conferences.

He is also a member of the Optical Society (OSA); SPIE, the International Society for Optics and Photonics; the Institute of Electrical and Electronics Engineering (IEEE), and the Biomedical Engineering Society (BMES). He is also the founder and president of the SPIE Student Chapter and the founder and vice president of the OSA Student Chapter at UCLA.

Femtosecond laser surgery continues to advance

Laura Harris

Since the turn of the 21st century, the femtosecond laser has been gaining momentum in the field of laser surgery. Its high peak intensity and short pulse duration result in an energy-conscious ablation tool that negligibly heats its surroundings. It is widely versatile, and from its debut in LASIK procedures by IntraLase to its role in nerve regeneration studies, plasmonic nanosurgery, endoscopic advancements, and cellular nanoprocessing, the utility of the femtosecond laser is just beginning.^1-8

The Ben-Yakar Laboratory at The University of Texas at Austin is advancing femtosecond laser technologies through a number of novel approaches. Our cutting-edge research of nerve regeneration in C. elegans could lead to advanced understanding of traumatic nerve injuries and degenerative diseases such as Alzheimer’s disease.^2-3 Our breakthroughs in plasmonic laser nanoablation could ultimately impact both cellular and material nanoprocessing.^4-5 Our development of a microsurgical endoscope could lead to a next-generation clinical surgery scalpel, with unprecedented clinical applications that require miniaturization and flexible laser delivery through fibers.^6-8

Now, we are turning our attention to yet another area in which femtosecond lasers can shine. In recent years, the femtosecond laser has been utilized in tissue engineering and live cell processing. We intend to combine our microfluidic and femtosecond laser specialties in order to delve into the realm of cell signaling and gene transfection in mouse embryonic stem cells. The femtosecond laser is becoming an efficient tool for cell membrane optoporation, and in this new project, we will ultimately create a lab-on-chip operated in conjunction with femtosecond laser optoporation in order to investigate the role of Fgf signaling in stem cell fate decisions. The obvious potential of the femtosecond laser is growing, and the Ben-Yakar Laboratory is turning potential into reality.


REFERENCES
1. I. E. Ratkay-Traub, T. Ferincz, R. M. Juhasz, R. M. Kurtz, and R. R. Krueger, J. Refract. Surg., 19, 94–103 (2003).
2. S. X. Guo et al., Nat. Meth., 5, 6, 531–533 (2008).
3. F. Yanik et al, Nature, 432, 822 (2004).
4. A. Ben-Yakar, D. Eversole, and S. X. Guo, "Plasmonic laser nanoablation," US Patent 7834331, issued 11/16/2010.
5. D. S. Eversole, B. Luk’yanchuk, and A. Ben-Yakar, Appl. Phys. A, 89, 283–291 (2007).
6. C. L. Hoy et al., Opt. Exp., 19, 10536–10552 (2011).
7. C. L. Hoy, W. N. Everett, J. Kobler, and A. Ben-Yakar, "Towards endoscopic ultrafast laser microsurgery of vocal folds," Proc. SPIE,  7548, 754831 (2010).
8. C. L. Hoy et al., Opt. Exp., 16, 13, 9996–10005 (2008).


LAURA HARRIS is a graduate research assistant in the Ben-Yakar Laboratory at the University of Texas at Austin, which focuses on femtosecond laser-assisted biophotonics. Her work integrates femtosecond laser nanosurgery and microfluidic techniques to investigate the fate choice of mouse embryonic stem cells.


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Optical treatment planning for interstitial photodynamic therapy

Tim Baran

A number of research groups, including Thomas Foster’s lab at the University of Rochester, have become interested in using photodynamic therapy (PDT) to treat cancers that are located deep within the body.

PDT is an emerging cancer therapy that uses a combination of light-sensitive drugs, known as photosensitizers, and targeted illumination to create photochemical reactions that result in the destruction of cancer cells. Since the treated area is limited by the penetration depth of the treatment light, PDT has typically been used to treat superficial malignancies of the skin and other easily accessible regions.   

To make it work within the body, the photosensitizer is delivered systemically or locally, and allowed to accumulate in the tumor. Under image guidance, cylindrical diffusing fibers are then inserted into the tumor to deliver the treatment light. This treatment light is usually delivered by a laser with wavelengths varying from 630-700 nm (and beyond), depending on the photosensitizer used. As the diffusers used can be as long as 5 cm for large tumors, the required laser power at the source can approach or exceed 1 W.

As interstitial PDT is often performed in regions where there is sensitive healthy tissue nearby, there is a need for careful treatment planning. Towards that end, we have developed a Monte Carlo simulation space that allows for patient optical properties and anatomy to be incorporated into a rigorous treatment plan. 

Unlike radiation therapy, in which radiation doses can be directly computed from CT scan data, calculations of optical dose require knowledge of the patient’s optical properties, which can vary among patients and even within a single patient. Therefore, spectroscopic determination of optical properties is required before a treatment plan can be formulated.

Simulated patient data showing the insertion of four cylindrical diffusing fibers for photodynamic therapy of head and neck cancers

A number of techniques exist to do this. In our case, we use a custom optical probe that is inserted into the treatment region and a Monte Carlo based fitting algorithm in order to extract optical properties. These extracted optical properties are then combined with CT image data from the patient in order to build an optical and anatomical map of the patient in our simulation space. The number of diffusers, and the amount of light delivered by them, can then be optimized using a constrained nonlinear optimization algorithm. This ensures that tumor tissues receive a physician-prescribed light dose, while damage to healthy tissue is minimized.

My work thus far has been preclinical, using simulated data sets and animal models. We are actively seeking to translate interstitial PDT into the clinic at the University of Rochester Medical Center for treatment of cholangiocarcinoma, cancers of the head and neck, and deep-tissue microbial infections.

TIM BARAN is a PhD candidate in the Institute of Optics at the University of Rochester (Rochester, NY).  His research in the Foster lab is related to optical dosimetry and treatment planning for interstitial photodynamic therapy, with an emphasis on the simulation of light propagation in tissue.

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Student researchers discuss biomedical optics' progress and impact

Kellie Chadwick    

Welcome to the BioOptics StudentView blog. Here, you will find posts written by university students studying biomedical optics and photonics and their applications. You will hear about their research and utilization of optical technologies in their studies; that is, advances in both biophotonics tools and the life sciences work they enable.

In a field with technology that continues to rapidly develop, students must remain ahead of the curve. As an intern at BioOptics World  and a student studying biomedical engineering, I've found that as technology continues to progress, developers continue to create devices that are smaller (such as this portable flow cytometer), have higher resolution (like this 29 Megapixel camera), better accuracy and reliability, are more cost-effective, more durable, and sustainable. All of this adds up to more power and flexibility in the hands of researchers.

This blog aims to share the advances and creativity of current students and scholastic laboratories, while looking to the future of biomedical optics and photonics technology. The BioOptics World staff hopes that you enjoy the perspectives offered by students who represent the future of this field!

KELLIE CHADWICK is a senior at Worcester Polytechnic Institute (WPI; Worcester, MA) majoring in biomedical engineering, with a concentration in biomaterials and tissue engineering--the latter of which garnered her interest because of various research possibilities and the advancements in medicine. Her senior year capstone project is to design a biomorphic tissue scaffold that can be fabricated with an inexpensive 3D printer.

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