high resolution fluorescence microscopy
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  The structure of membrane components in the z-direction, i.e., the direction perpendicular to the plane of the membrane, plays a major role in signal transduction at  membrane interfaces. Nanometer-resolution z-structural information at these surfaces could elucidate the mechanisms underlying processes such as formation of focal contact between adherent cells and vesicle fusion at a neurological synapse. Additionally, such detailed information is key for assessing self-organized and hierarchical structures at surfaces.

  I further developed two existing techniques, variable incidence angle total internal reflection fluorescence microscopy (VIA-TIRFM) and fluorescence interference contrast microscopy (FLIC), and used them to characterize membrane structures. I also introduced the novel technique of variable incidence angle fluorescence interference contrast microscopy (VIA-FLIC), which combines advantages of the versatility of VIA-TIRFM and the experimental simplicity of FLIC.

  VIA-TIRFM, FLIC, and VIA-FLIC are all structured illumination techniques. To obtain the z-positions of fluorescent objects with high resolution, structured illumination techniques rely on the creation of an excitation intensity gradient in z and modulation of this excitation gradient. The gradient encodes distance information of a fluorescent object in its fluorescent intensity; modulation of the gradient varies the manner in which fluorescence intensity depends on distance. The z-position is then determined by fitting the fluorescence intensity as a function of the excitation gradient and its modulation to an appropriate model.



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   synthetic networks in budding yeast
   high resolution fluorescence microscopy
   supported lipid membranes
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Variable Incidence Angle TIRFM

 Via TIRFM Web Pic
In VIA-TIRFM the excitation gradient is caused by an exponentially decaying evanescent field, and varying the incidence angle modulates the 1/e distance of the evanescent field (see cartoon to right).


The fabrication of hybrid surfaces consisting of nm layers of SiO2 on lithium niobate (LiNbO3, n = 2.3) permits assembly of supported membranes and lowers the minimum 1/e distance such that contrast between fluorescently labeled structures that differ in vertical position by only tens of nm can be created. However, VIA-TIRFM imaging and analysis of a well-defined model system provided only qualitative agreement between the data and predictions. Moreover, despite its applicability to many biological systems, the nature of VIA-TIRFM imaging makes it a difficult and non-routine method.
FLIC Web Pic

Fluorescence Interference Contrast Microscopy (FLIC)

  In FLIC (originally developed by Peter Fromherz and coworkers), the sample is assembled on a series of SiO2 steps of different thickness on a highly reflective Si surface, a FLIC chip. When a sample is imaged using a conventional epi-fluorescence microscope, interference between incoming and reflected light creates a well-defined variation in light intensity in the z direction, and the different SiO2 step thicknesses modulate the position of the fluorescent molecules relative to this overall interference pattern (see cartoon to the left). My work with FLIC consists of some straightforward modifications to improve its resolution and using FLIC to characterize the water layer underlying supported bilayers and membrane-tethered double stranded oligonucleotides.

Variable Incidence Angle - FLIC (VIA-FLIC)

 VIA FLIC Web Pic

  Although FLIC is impressive in its experimental simplicity and ability to resolve nanometer distances, it cannot image many objects of biological interest such as individual vesicles at a neuronal synapse or focal contacts of an anchorage-dependent cell.This limitation exists because in order to z-image a structure with FLIC, one must be able to replicate it over many SiO2 steps laterally separated by tens of microns.

  I realized that the interference pattern could be modulated instead by changing the incidence angle of excitation light, and that this could be done by a straightforward modification to a standard epi-fluorescence microscope. A first-year graduate student (at the time), Prasad Ganesan, and I carried out an experimental test of this new technique - dubbed variable incidence angle fluorescence interference contrast microscopy (VIA-FLIC) - using supported membranes, and found that it could indeed accurately determine the z-position of fluorescent objects. Prasad is now working to apply VIA-FLIC to answer some biologically relevant questions in the area of cell adhesion.