The present study determines the affinity of cholera toxin for the ganglioside series GM1, GM2, GM3, GD1A, GD1B, GT1B, asialo GM1, globotriosyl ceramide, and lactosyl ceramide using real time biospecific interaction analysis (surface plasmon resonance, SPR). SPR shows that cholera toxin preferably binds to gangliosides in the following sequence: GM1 > GM2 > GD1A > GM3 > GT1B > GD1B > asialo-GM1. The measured binding affinity of cholera toxin for the ganglioside sequence ranges from 4.61 x 10-12 M for GM1 to 1.88 x 10-10 M for asialo GM1. The picomolar values obtained by surface plasmon resonance are similar to Kd values determined with whole-cell binding assays. Both whole-cell assays and SPR measurements on synthetic membranes are higher than free solution measurements by several orders of magnitude. This difference may be caused by the effects of avidity and charged lipid head-groups, which may play a major role in the binding between cholera toxin, the receptor, and the membrane surface. The primary difference between free solution binding studies and surface plasmon resonance studies is that the latter technique is performed on surfaces resembling the cell membrane. Surface plasmon resonance has the further advantage of measuring apparent kinetic association and dissociation rates in real time, providing direct information about binding events at the membrane surface.

A solid tumor is an organ composed of cancer and host cells embedded in an extracellular matrix and nourished by blood vessels. A prerequisite to understanding tumor pathophysiology is the ability to distinguish and monitor each component in dynamic studies. Standard fluorophores hamper simultaneous intravital imaging of these components. Here, we used multiphoton microscopy techniques and transgenic mice that expressed green fluorescent protein, and combined them with the use of quantum dot preparations. We show that these fluorescent semiconductor nanocrystals can be customized to concurrently image and differentiate tumor vessels from both the perivascular cells and the matrix. Moreover, we used them to measure the ability of particles of different sizes to access the tumor. Finally, we successfully monitored the recruitment of quantum dot-labeled bone marrowderived precursor cells to the tumor vasculature. These examples show the versatility of quantum dots for studying tumor pathophysiology and creating avenues for treatment.Intravital microscopy has provided unprecedented molecular, cellular, anatomical and functional insight into tumor biology and response to treatment 1 . This technique captures fluorescence from molecules that are injected into a host or expressed by cells 2,3 . Additionally, intrinsic signals such as second harmonic generation (SHG) emanating from collagen can be imaged using multiphoton microscopy 4,5 . Traditional fluorophores are prone to photobleaching, compromising the ability to image the same region repeatedly, and have relatively narrow excitation and broad emission spectra. Also, several excitation wavelengths may be required to excite all fluorophores and intrinsic signals, and overlapping emissions may obscure the delineation between multiple probes. Quantum dots, colloidal semiconductor nanocrystals 6 , have the potential to overcome these limitations: they are photostable, tunable to a desired narrow emission spectrum, relatively insensitive to the wavelength of excitation Correspondence should be addressed to R.K.J. (E-mail: jain@steele.mgh.harvard.edu). Note: Supplementary information is available on the Nature Medicine website. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. light, and are especially bright fluorophores 7 . Recent studies exploit these optical properties for imaging of cells 8 or whole tumors 9 . The ability of quantum dots to show crucial information at the length scale between these two extremes has yet to be established 10 . Here, we present studies that highlight the synergy of quantum dots and multiphoton intravital microscopy for tumor pathophysiology studies: differentiating tumor vessels from both perivascular cells and matrix, assaying the ability of microparticles to access the tumor, and monitoring the trafficking of precursor cells. NIH Public Access RESULTS Customizing quantum dot emissionBecause quantum dot emissions are tunable by both size and chemical composition 6 , we prepared ...

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