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  • Techniques in Biophotonic Imaging

Mar 21, 2013
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Sponsored by: Hamamatsu & Applied Scientific Instrumentation

Additional Questions & Answers from the webinar are below:

Dr. Kimani C. Toussaint Jr.
Quantitative Imaging of Collagen Fibers Using Second-Harmonic Generation
Assistant Professor, Department of Mechanical Science & Engineering
(Photonics Research of Bio/Nano Environments lab group)

University of Illinois, Urbana-Champaign

Advances in nonlinear microscopy, e.g., multiphoton fluorescence microscopy and second-harmonic generation (SHG) microscopy, have permitted both noninvasive and high-resolution imaging of biological specimens. In recent years, there has been increasing effort to use these techniques to perform quantitative inspection of specimens under study. In his talk, "Quantitative Imaging of Collagen Fibers Using Second-Harmonic Generation," Toussaint will focus on two particular techniques that the PROBE group is researching: Fourier transform-second-harmonic generation (FT-SHG) and polarization-second-harmonic generation (P-SHG). Information pertaining to collagen fiber structural organization is obtained using FT-SHG, which combines SHG with spatial harmonic analysis, while P-SHG carries information that is sensitive to molecular-level changes to collagen fibers, by exploiting the intrinsic coherence in the SHG process to determine the polarization-dependent normalized tensor components of the 2nd-order scattering coefficient. Potential biomedical applications will be discussed.

Kimani C. Toussaint Jr. leads the PROBE lab group which is part of the Department of Mechanical Science and Engineering at the University of Illinois, and has the mission of noninvasively investigating a variety of inorganic and organic systems, at mesoscopic scales, through the development of advanced optical instrumentation. He earned his BA from the University of Pennsylvania in 1996, and MS and PhD degrees in Electrical Engineering from Boston University in 1999 and 2004, respectively. Prior to starting at Illinois, Toussaint was an NSF Minority Postdoctoral Fellow in Biology at the University of Chicago, where he worked on interference microscopy, exotic polarization states, and optical trapping. In 2006, he was one of 100 top American scientists in the United States (under 45) to be selected for the National Academy of Science's 18th Annual Kavli Frontiers of Science Symposium. Dr. Toussaint's group pursues interdisciplinary research in optical physics and engineering that has applications in many disciplines, including biology and chemistry. Currently, he is developing an advanced optical microscopy platform for characterizing and manipulating both nano- and biological systems.

Question & Answer

1. What is the minimum resolution required to resolve subcellular structures?

The smallest feature that is resolvable using our microscope system is about 400nm with a 1.4 NA oil immersion objective and incident light at 800 nm wavelength. Therefore, cellular structures such as cells and collagen fibers can be readily seen using our system. \ In order to be sensitive to molecular level changes in collagen fibers using SHG, we make use of the polarization dependence of SHG. From our previous experiments, we noticed that it appears that changes in the molecular structure of collagen alter the SHG polarization response in SHG, and the alteration is sensitive enough that we may utilize polarization SHG \. Although P-SHG does not provide any spatially resolved information in the traditional imaging sense, but we believe that it provides us with information related to the crystalline symmetry of collagen molecules measured.

2. What is good enough in fluorescence microscopy or endoscopy?

I’m not sure that I understand the question. Two-photon fluorescence microscopy provides approximately the same spatial resolution as SHG microscopy. Over the past decade, many researchers have developed a variety of superresolution techniques based on fluorescence microscopy, e.g., SIM, STORM/PALM, and STED. The power of SHG microscopy is particularly  its specificity to (fibrillar) collagen-based tissue specimens. My lab does not work with endoscopy. However, it’s my understanding that generally the spatial resolution is relatively worse compare to traditional microscopy because of the limited types of optics and materials that can be employed. However, the ability to perform in vivo imaging and their compact designs make endoscopy a worthwhile technological pursuit.

3. Can I know a few details about the microscope setup that was shown by Professor Kimani?

The microscope setup consisted of an IX81 Olympus microscope that is modified to incorporate both backward and forward collection geometries. The light source is a tunable Ti:Sapphire laser (Spectra-Physics Mai-Tai HP DeepSee) that produces 100 femtosecond-duration pulses at 80-MHz repetition rate. The beam is linearly polarized and spectrally centered at 800 nm, and is spatially filtered and collimated before it is scanned by a pair of galvo-mirrors (Cambridge Technology). The beam is then directed through a combination of relay lenses (scan and tube lens). The input polarization is controlled by a polarizer and half-wave plate. The beam is subsequently focused onto the sample using an objective lens. The SHG signal is filtered through a laser blocking filter (Semrock FF01-680/SP-25) and a 390/20 nm bandpass filter (Semrock FF01-390/18-25), and collected by an EMCCD camera (Hamamatsu C9100-13).

4. Using SHG, what is the maximum depth of field?

From our experience, we have been able to achieve depths of 40 um in cortical bone (e.g., of a highly scattering tissue) and 600 um in cornea (e.g., of a least scattering tissue). However, remember high resolution images require high NA (1.2-1.4) objectives, wherein the depth is limited by working distance.

Dr. Melissa Skala
Photothermal Optical Coherence Tomography of Nanoparticle Contrast Agents
Optical Imaging Laboratory, Vanderbilt University School of Engineering
Assistant Professor of Biomedical Engineering, Vanderbilt-Ingram Cancer Center

Molecular imaging is a powerful tool for studying disease progression and potential therapies. Optical coherence tomography (OCT) is an important biomedical imaging modality, filling the spatial niche (resolution and imaging depth) between ultrasound and microscopy. However, OCT suffers from an inherent lack of molecular contrast. This is because the scattering cross-section, the source of contrast in OCT, does not vary widely between molecular species. We and others have demonstrated that photothermal detection of highly absorptive nanoparticles can be achieved by incorporating an amplitude-modulated laser into the sample arm of a standard OCT system. In photothermal imaging, strong optical absorption by a target of interest — such as a nanoparticle — results in a change in temperature around the particle (i.e., the photothermal effect). This temperature change causes index-of-refraction changes and thermoelastic expansion in the microenvironment of the nanoparticle that can be detected with phase-sensitive OCT. This photothermal OCT approach has the advantage of allowing for highly sensitive in vivo molecular imaging in a unique spatial niche. This talk will cover the principles of photothermal OCT, its characterization, and in vitro and in vivo molecular imaging applications using gold nanorods contrast agents.

Optical technologies are attractive for diagnosing cancer and for monitoring cancer therapy because these techniques are low cost, portable, fast and provide a wealth of information on tissue structure and function. Dr. Skala's research focuses on the development of optical imaging tools that monitor biological markers such as cellular metabolic rate, molecular expression, blood oxygenation and blood flow in vivo. These tools are applied to pre-clinical models for the design and development of effective therapeutic strategies, and in clinical studies to provide early detection and individualized treatment to cancer patients. Specific technologies under development include optical coherence tomography to monitor blood flow and microvessel morphology, multiphoton and fluorescence lifetime microscopy to image cellular metabolism, photothermal microscopy with nanoparticle contrast agents for molecular imaging, and optical spectroscopy to quantify tissue blood content and oxygenation.

Question & Answer

1. Is it safe to inject nanoparticles into the living tissue?

Yes, gold nanoparticles are safe to inject into living tissues. Gold nanoshells are currently in clinical trials, and gold is already used as a therapeutic for arthritis.

Dr. Ofer Levi
Multimodal optical neural imaging with VCSEL light sources Assistant Professor at the Institute of Biomaterials and Biomedical Engineering and the Edward S. Rogers Sr. Department of Electrical and Computer Engineering at the University of Toronto

We present the development of a multi-modality optical neural imaging system, to image blood flow velocity and oxygenation in a rat brain, using a fast CCD camera and miniature VCSEL illumination. We combined two techniques of laser speckle contrast imaging (LCSI) and intrinsic optical signal imaging (IOSI) simultaneously, using these compact laser sources, to monitor induced cortical ischemia in a full field format with high temporal acquisition rates. Simultaneous imaging is based on fast coherence reduction techniques applied to Vertical Cavity Surface Emitting Lasers (VCSELs) operating at 680, 795 and 850 nm. We have demonstrated the use of this system in tracking ischemia and with adding a fluorescence modality, in evaluating the disruption of a blood-brain barrier and tracking seizure activity in the brain. Finally, we will present our initial design and system analysis for a low-cost CMOS-based portable imaging system as a minimally invasive method for long-term neurological studies in un-anesthetized animals. This system will provide a better understanding of the progression and treatment efficacy of various neurological disorders, in freely behaving animals.

Ofer Levi is an Assistant Professor at the Institute of Biomaterials and Biomedical Engineering and the Edward S. Rogers Sr. Department of Electrical and Computer Engineering at the University of Toronto since 2007. In 2000-2007 Dr. Levi worked as a Post Doctoral Fellow and a Research Associate at the Departments of Applied Physics and Electrical Engineering, Stanford University, CA. He is a member of OSA, IEEE-Photonics, and SPIE. His recent research areas include biomedical imaging systems and optical bio-sensors based on semiconductor devices and nano-structures, and their application to bio-medical diagnostics, in vivo imaging, and study of bio-molecular interactions. More details can be found at

Questions & Answers

1. What EMCCD camera did you choose to work with and what benefits did it provide?

We chose to work with the QImaging EMCCD, model EM-C2. We operate it as a fast, high dynamic range camera with EM gain of 1 (traditionally, for low light applications, people use much higher EM gain). The benefits it provided were a high dynamic range (14 bit), large well size (7.4 micron) and fast operation (> 60 frames/second in a full 512x 512 frame speed). It also has a convenient hardware/software interface that allowed us to create a real time control of the laser illumination schemes and for each frame of the camera, a match unique illumination pattern that included a specific VCSEL wavelength and a specific operating mode (i.e. single mode, high coherence  or current sweep, low coherence ).

2. How do you assure registration of LSCI and spectroscopic modality? (How can you tell which vessels are oxygenated)?

We use the SAME camera to capture both images, and the VCSEL laser diode sequence is synchronized to the frames of the camera, so we know that in space we see the same image, while in time, it is 1-3 frames away between the flow velocity image and the oxygenation image. In a speed of ~ 60 frames/second from a fast EMCCD that we are using, there is a full spatial registration and a ~60 msec. registration between these two maps. We can tell which vessels are oxygenated by evaluating the reflection image in a multi-mode illumination at three pre-determined wavelengths (680 nm, 795 nm, 850 nm) that allow us to separate the contributions of Oxy-Hb and Deoxy-Hb to the image and therefore establish the oxygenation change map. We described this calculation in our paper in Biomedical Optics Express from 2012 by Hart Levy et al.

3. Can we deduce the speed of blood flow from the captured image in single mode case or multimode case? If yes, can that be done as an in-vivo measurement?

You can conduct a calibration experiment where you use a controlled blood flow velocity in a micro-fluidic tube (using a syringe pump) or use "time of flight" image recording of the velocity in a real vessel where you measure the transverse movement of the red blood cells within a given exposure time to know what is the average blood velocity. Then you can compare it to the relative velocity you obtain in LCSI, using the single mode (high coherence) VCSEL illumination and deduce the velocity of the blood flow. The second technique can be used as an in vivo measurement. We described this process in our paper in Biomedical Optics Express from 2012 by Hart Levy et al.


ASI manufactures top-of-the-line hardware for sub-micron micropositioning and microscopy applications. Our products include closed-loop XYZ stages for ultra-precise positioning, stages with piezos for high resolution/speed positioning, autofocus systems, optics & illumination, systems & components for inverted selective plane illumination microscopy (iSPIM), as well as turnkey systems built to user specifications.

Hamamatsu is an Importer and distributor of photonic components and subsystems for use in instrumentation. Products include photomultiplier tubes, solid-state photodetectors, IR detectors, image sensors, scientific-grade CCD cameras, light sources and x-ray solutions.
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