Transfected cells expressing fluorescent proteins contain information on the spatial organization of specific target proteins that is accurate at the molecular level. However, conventional optical microscopy is limited by diffraction to imaging on a scale coarser by two orders of magnitude.  Together, my colleague Harald Hess and I invented PALM, a microscope which uses the serial photoconversion and localization of hundreds of thousands of individual molecules, including fluorescent proteins or photoswitchable dyes, to build an image at near-molecular resolution.  At Janelia, we have each pursued various extensions and applications of this technology, including multicolor and live cell imaging in my lab, and three dimensional interferometric PALM in Harald’s.  

 

 

 

 

Although many methods exist to image fixed, frozen, or sectioned cells at high resolution, fluorescence microscopy is unique in its ability to study the dynamics of specific target proteins and organelles within living cells.  However, since cells evolve in three dimensions, a key challenge is how to image fast enough to follow this evolution continuously in time throughout the entire cellular volume.  In my lab, we have developed a variant of plane illumination microscopy that uses scanned Bessel beams to image living cells with 3D isotropic resolution at hundreds of image frames per second.

For high speed 3D imaging in scattering tissues, my colleague Na Ji and I have developed another technology, the passive pulse splitter -- a self-contained unit that, when placed in the path of an ultrafast laser, can increase the pulse repetition rate, and hence the imaging speed, by more than a hundred-fold.  Potential applications include high throughput anatomical mapping of neural circuits or rapid functional imaging of activity in neural populations.  

 

 

Sadly, even optical microscopy is somewhat invasive: extended imaging of living cells or tissues often leads to light-induced damage.  Furthermore, in fluorescence microscopy, every sample contains a finite number of fluorescent molecules, each of which will emit a finite number of photons before irrevocably bleaching.  In other words, every fluorescent sample has a finite photon budget.  As spatiotemporal resolution improves, it becomes imperative to devise methods that use this budget efficiently.  By confining the light excitation to a plane of submicron thickness, our Bessel beam plane illumination microscope substantially reduces photobleaching and phototoxicity compared to popular epi-illumination techniques (e.g., widefield or confocal microscopy), thereby permitting hundreds of 3D image stacks encompassing tens of thousands of image frames to be acquired from a single living cell.


The high peak excitation intensity required in nonlinear optical imaging methods, such as two photon fluorescence microscopy, can also lead to significant photodamage.  Together, Na, I, and our colleague Jeff Magee have shown that our pulse splitter, when used with N-fold splitting and N^1/2 higher average laser power, produces the same signal level as a two photon system without splitting, but with up to nine-fold less photobleaching in vivo and a six- to twenty-fold reduction in the rate of photodamage during calcium recording of neural activity in acute brain slices.  

 

Optical microscopes achieve diffraction-limited performance only when imaging through the immersion medium for which they are designed – usually water in the case of in vivo imaging.  However, nearly all biological specimens larger than a single cell are rife with refractive index inhomogeneities.  As a result, many biologists do not approach the level of spatial resolution in their images that they might assume.  Similar problems, confronted by astronomers when imaging through Earth’s turbulent atmosphere, have long been addressed by using adaptive optics.  Motivated by their success, Na Ji and I have developed an approach for adaptive optics suited to the constraints of biological microscopy.  Using the approach, we can recover near-diffraction-limited performance in a variety of specimens, including those exhibiting large amplitude and/or spatially complex aberrations.  

 

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