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An Evanescent Perspective on Cells

We have optically sectioned living cells to a maximum depth of ~250 nm using a Variable Angle-Total Internal Reflection Fluorescence Microscope (VA-TIRFM). This yields 3D images of cell membranes and nearby organelles similar to that gained by confocal microscopes but with at least an order-of-magnitude greater depth resolution. It also enables cellular membranes to be imaged in near isolation from cell organelles. Key to achieving this resolution was integration of a controllable excitation laser micropositioner into a standard through-the-lens TIRF illuminator and development of a custom culture dish for re-use of expensive high index of refraction cover slips. Images are acquired at several penetration depths by varying the excitation laser illumination angles. At the shallowest penetration depth (~46 nm) just the membrane and a few internal puncta are imaged. As the penetration depth is increased up to 250 nm organelles near the membrane, such as the ER, are imaged as well. The sequence of images from shallow deep is processed to yield a z-stack of images of approximately constant thickness at increasing distance from the coverslip. We employ this method to distinguish membrane-localized fluorophores (α4 GFP β2 nicotinic acetylcholine receptors and pCS2:lyn-mCherry) at the plasma membrane (PM) from those in near-PM endoplasmic reticulum (ERTracker green, α4 GFP β2 nicotinic acetylcholine receptors), on a z-axis distance scale of ~45 to ~250 nm in N2a cells. In doing so we observe occasional smooth ER structures that cannot be resolved as being distinct from the membrane. In a second project substantial progress has been made towards developing a Tip Enhanced Fluorescence Microscope (TEFM) capable of imaging wet biological samples with ~10 nm resolution. A TEFM combines a TIRFM with an Atomic Force Microscope (AFM) to modulate sample fluorescence through near-field dipole-dipole coupling. In the third project the capability to consistently produce high quality nanotube AFM probes was developed and a technique for chemically functionalizing the tip of a nanotube AFM probe was invented.