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LAMMP Technology Cores

Microscopy and Microbeam Technologies

Optical tweezers is a notable expertise of LAMMP, wherein mechanical stress can be applied to microscopic particles by a focused microbeam. In optical tweezers, a laser microbeam transfers light momentum to a dielectric particle suspended in solution, within a cell, within a 3-D cell culture scaffold, or to entire cells. Optical tweezers act like a three-dimensional spring with its potential energy minimized when the particle is at the laser focus. Thus the tightly focused laser spot can capture microscopic particles, measure external forces acting on particles with sub-pN resolution, and monitor particle positions with nm resolution. In addition, optical tweezers can oscillate or rotate particles in a substrate, allowing the measurement of local cytoplasmic or extracellular matrix material properties as well as single molecule biophysical parameters such as bond strength, molecular unfolding energy landscapes, and cell generated forces. Taken together, these techniques allow LAMMP investigators and collaborators to explore exciting hypotheses in which physical, and not just chemical cues, likely regulate growth, health and disease.

In the MMT core, focused laser beams are used to achieve high-resolution imaging and to perform precisely controlled micro-manipulations of biological tissues in situ and in vivo. This core includes confocal and nonlinear optical microscopy technologies, optical tweezers and advanced micro-beam techniques. During previous LAMMP funding cycles, the MMT core has made many pivotal contributions to the field of laser-based microscopy, and has broken new ground in applying these technologies to relevant biomedical problems. In the current LAMMP renewal, we continue to push laser microscopy techniques for addressing pertinent questions in cell and tissue biology.

1. Mesoscale imaging with multimodal nonlinear microscopy: Retooling the nonlinear optical microscope for optimized tissue imaging.
2. Optical control: Merging optical tweezers with nonlinear optical contrast for mechanobiology studies.
3. Clinical microscopy: Development and application of clinical nonlinear microscopy

Cartoon showing the interrelation of technical and scientific targets in MMT core. At the center is the tissue, a complex system of cells and extracellular components. Advanced NLO imaging tools and microbeam technologies are used to visualize and interrogate tissue composition, architecture, mechanobiology and pathology. Major goals of our core are to advance technologies that enable better chemical contrast, expand imaging over wider and deeper length scales, reduce image acquisition rates and develop tools for studying interactions of the cells with the extracellular matrix.

Resources and technologies available:

TPEF/SHG/CARS - MDM platform

Zeiss LSM 510 microscopy system

Clinical Tomograph MPTflex

Additional NLO Systems

Optical Tweezers/Scissors
TPEF/SHG/CARS - MDM platform

Vibrational microscopy is an imaging method for visualizing several key molecular compounds in biological materials that neither fluoresce nor have a SHG sensitive non-centrosymmetric configuration. Contrast is derived from the molecular vibrational signatures that manifest themselves in the Raman spectrum. Many vibrational bands are excellent labels for classes of endogenous molecules. The C-H stretching region of the spectrum, for instance, is an excellent indicator of lipids, a crucial tissue component. Figure 1 shows CARS lipid signals taken in our labs from three breast cancer cell lines with increasing malignancy, clearly showing the different lipid distribution as a function of cancerous state.

Figure 1: CARS images (100 x 100 μm) of live nonmalignant (MCF-12A, left), mildly malignant (MCF-7, middle), and malignant (MBA-MB-231, right) breast cancer cells. The bright spots are the lipid droplets. Note that the number of lipid droplet correlates inversely with the malignancy of the cells. This observation is in concert with depletion of lipid pools in breast cancer tissue as seen with NMR and DOS/I. The laser was set to the 2845 cm-1 symmetric CH2stretching vibration of lipids.

CARS microscopy is a nonlinear Raman technique with 3D resolution that enables rapid acquisition of vibrationally sensitive images. It has been demonstrated that CARS clearly resolves lipid structures in cells and in tissue in vivo with high sensitivity. The CARS technique has also been used to measure cellular hydration maps and image protein densities in single cells. Similar to TPEF and SHG, CARS is efficiently generated with ultrashort near-infrared pulses, and can be combined with other nonlinear imaging modalities.

Zeiss LSM 510 microscopy system

This NLO microscopy system is based on a platform of an Axiovert 200M inverted microscope equipped with standard illumination systems for transmitted light and epi-fluorescence detection and a standard set of visible light lasers (an Argon laser 458/477/488/514 nm/ 30 mW, a Helium: Neon laser 543 nm/ 1 mW and a Helium: Neon laser 633 nm, 5 mW) for confocal microscopy. It is equipped with an NLO interface for a femtosecond Titanium: Sapphire laser excitation source (Chameleon-Ultra, Coherent) for multi-photon excitation with tunability from 690 to 1040 nm. The instrument is equipped with two single channel photomultiplier tube detectors, the META polychromatic detector, the NDD detector for SHG detection in reflected mode, and a transmission light channel detector capable of detecting SHG signal in transmitted mode. The microscope platform is equipped with a motorized X-Y scanning stage and long-working distance and high numerical aperture objectives (10, 20, 40, and 100X).

System Specifications:
  • Mode-locked Ti: Sapphire laser (170 fs pulse width, 690-1040 nm tuning range, Chameleon Ultra, Coherent, Inc.)
  • Zeiss Axiovert 200M inverted microscope
  • 6 lines for confocal scanning 458/477/488/514/532/633 nm
  • Up to 4 channels parallel detection
  • Scanning speed of up to 5 frames/second for acquisition of 512x512-pixel images
  • Motorized X-Y stage with mark and find and tile scan functions and fast piezo objective focus for Z drive with 25 nm smallest increment.
  • User-defined ROI.
  • Multiple acquisition modes such as spot, line, frame, Z-stack, lambda stack, or time series.
  • The polychromatic 32-channel detector (META detector) provides spectral separation of fluorophores within the same sample by spectral fingerprinting and linear unmixing algorithms.
Clinical Tomograph MPTflex

In vivo nonlinear microscopy imaging of human skin: optical sections displayed from the skin surface (stratum corneum) to the epidermal-dermal junction and dermis. Green-two photon excited fluorescence (TPEF) from stratum corneum, epidermal cells and elastin fibers; Blue-second harmonic generation (SHG) from collagen fibers and dermal papilla.

Female forearm – skin type III

Moderately pigmented skin (high intensity green fluorescence from epidermal cells) allows for the visualization of the dermal papilla (blue) up to the epidermal-dermal junction of the skin.

Male forearm – skin type I

Low pigmented skin (low intensity green fluorescence from epidermal cells) implies less scattering in the epidermal layer and allowing deeper penetration and imaging of collagen (blue) and elastin fibers (green) in the dermis.

Male forearm – skin type IV

High-pigmented skin (bright green signal from epidermal cells) prevents the light to penetrate in deeper layers of epidermal-dermal junction and dermis.



Elliot Botvinick, Ph.D.
Phone: 949.824.9958
Eric Olaf Potma, Ph.D.
Phone: 949.824.9942
Tatiana Krasieva, Ph.D.
Phone: 949.824.3664

Post-Doctoral Researchers

Mihaela Balu, Ph.D.
Martha Alvarez, Ph.D.
Bhupinder Shergil, Ph.D.

Graduate Students

Richa Mittal (ChemEng),
Jeff Suhalim (BME)
Alba Alfonso Garcia (BME)
Jue Hou (BME)
John Weidling (BME)

B. Shergill; L. Meloty-Kapella; A.A. Musse; G. Weinmaster; E. Botvinick
Optical tweezers studies on notch: single-molecule interaction strength is independent of ligand endocytosis.
Dev Cell. 22 (6), 1313-20.

L. Meloty-Kapella; B. Shergill; J. Kuon; E. Botvinick; G. Weinmaster
Notch ligand endocytosis generates mechanical pulling force dependent on dynamin, epsins, and actin.
Dev Cell. 22 (6), 1299-312.

S.G. Shreim; E. Steward; E.L. Botvinick
Extending vaterite microviscometry to ex vivo blood vessels by serial calibration
Biomedical Optics Express. 3 (1), 37-47.

E. Kniazeva; J.W. Weidling; R. Singh; E.L. Botvinick; M.A. Digman; E. Gratton; A.J. Putnam
Quantification of local matrix deformations and mechanical properties during capillary morphogenesis in 3D
Integrative Biology. 4 (4), 431-439.

Xuejun Liu, Yong Wang, and Eric O. Potma
A dual-color plasmonic focus for surface-selective four-wave mixing
Appl. Phys. Lett. 101, 081116 (2012).

Yong Wang, Xuejun Liu, Aaron R. Halpern, Kyunghee Cho, Robert M. Corn, and Eric O. Potma
Wide-field, surface-sensitive four-wave mixing microscopy of nanostructures
Appl. Opt. 51, 3305-3312 (2012).

Jeffrey L. Suhalim, Chao-Yu Chung, Magnus B. Lilledahl, Ryan S. Lim, Moshe Levi, Bruce J. Tromberg, and Eric O. Potma
Characterization of cholesterol crystals in atherosclerotic plaques using stimulated Raman scattering and second-harmonic generation microscopy
Biophys. J. 102, 1988-1995 (2012).

Jeffrey L. Suhalim, John C. Boik, Bruce J. Tromberg, and Eric O. Potma
The need for speed
J. Biophoton. 5, 387-396 (2012).

Zhang Q, Nguyen AL, Shi S, Hill C, Wilder-Smith P, Krasieva TB, Le AD.
Three-dimensional spheroid culture of human gingiva-derived mesenchymal stem cells enhances mitigation of chemotherapy-induced oral mucositis
Stem Cells Dev. 2012 Apr 10;21(6):937-47. Epub 2011 Jul 28.

Madsen SJ, Baek SK, Makkouk AR, Krasieva T, Hirschberg H.
Macrophages as cell-based delivery systems for nanoshells in photothermal therapy
Ann Biomed Eng. 2012 Feb;40(2):507-15. doi: 10.1007/s10439-011-0415-1. Epub 2011 Oct 7. Review.

Ryan S. Lim, Jeffrey L. Suhalim, Shinobu Miyazaki-Anzai, Makoto Miyazaki, Moshe Levi, Eric O. Potma, and Bruce J. Tromberg
Identification of cholesterol crystals in plaques of atherosclerotic mice using hyperspectral CARS imaging
J. Lipid Res. 52 ,2177-2186 (2011).

J. Zhou; M.B. Alvarez-Elizondo; E. Botvinick; S.C. George
Local small airway epithelial injury induces global smooth muscle contraction and airway constriction.
J Appl Physiol. 112 (4), 627-37.

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  • Hanna Kim
    Resource Coordinator

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