Rice University

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Seminar

Chemistry

Speaker: Robert Pal
Research Fellow in the Department of Chemistry
Durham University

In the pursuit of higher resolution in live cell imaging … and little perks of microscopy

Tuesday, March 21, 2017
4:00 PM  to 5:00 PM

337  Space Science Building
Rice University
6100 Main St
Houston, Texas, USA

The optical probes and cellular stains commonly used in microscopy are usually fluorescent organic molecules or recombinant proteins which have been used in many areas of cellular biology leading to an enhanced understanding of cellular processes and molecular interactions. However, many of these dyes have inherent drawbacks, such as issues associated with their toxicity, photostability and selectivity. For almost a century, the resolution of optical microscopy was thought to be governed by Abbe’s law where the highest achievable spatial resolution is dictated by the wavelength of excitation light d ~ ?(exc.)/2 [2]. This gives a resolution limit of, in the lateral and in the axial domain (1.4 NA), which until recently left the nanoscopic realm accessible only by electron microscopy; a method that is so far incompatible when natural homeostatic studies are desired. The invention of confocal microscopy, the first technique to truly reach this barrier and remaining to this day the most popular live cell imaging method, paved the way to the development of new optical (hardware) and software based super-resolution methodologies. At the turn of the millennium, aided by new emerging novel optical microscopy techniques [3] and fluorophores, the field of optical microscopy finally surpassed the diffraction barrier, a milestone achievement that has been recognized by the 2014 Nobel Prize in Chemistry. Almost all current super-resolution methods rely heavily on the properties of the applied fluorophores to improve predominantly lateral resolution. Crucially they extract no more ‘true’ spatial information from the studied sample and are, regrettably, also limited by their well-known experimental drawbacks. The attempt to truly visualize live nanostructures in detail has been first satisfied by the use of Moiré-fringes generated in Structured Illumination Microscopy (SIM) [4]. Since its early conception and first use in biological imaging, SIM has proven to be a popular tool in the biologist’s arsenal for probing structures of nanometer scale. SIM has seen, and continues to see, many advancements in design, such as harnessing the non-linear properties of fluorophores to provide a theoretically infinite resolution [5]; the use of spatial light modulators to increase image acquisition speed favorable for live-cell imaging [6]; and most recently Image Scanning Microscopy (ISM) and its many successful variants [7]. Despite all of these developments, SIM, like all other super-resolution techniques currently exploited, still has its limitations and shortfalls. Drawing form this, and incorporating more recent achievements in light-shaping and patterned-scanning [8]. we have set out to break the diffraction barrier and develop a novel super-resolution technique called Phase Modulation Nanoscopy (PhMoNa) [9]. This technique is based on a novel combined structured illumination technique, boosting a custom Electro-optical modulator that allows frequency matched spatiotemporally modulated laser cluster excitation to be achieved with subsequent detection of cellular substructures with an 8 fold reduced voxel size compared to standard LSCM. In essence PhMoNa operates by utilizing an in situ generated optical grid pattern projected by the raster scanned excitation beam that subsequently introduces high spatial frequency mixing of the sub-diffraction size excitation cluster with the observed finite objects spatial frequencies. This approach allows experimental resolution in both lateral and axial domains to be improved by at least a factor of 2. Combining this novel technique with newly synthesized functionalised Lanthanide(III) complexes as organelle probes, remarkable sub-diffraction ‘true experimental’ resolution of ~60 nm (lateral and ~190 nm axial) was achieved in live-cell LSCM experiments, rendering this technique free from any unnecessary time consuming post-image processing deconvolution algorithms. The advantageous properties of the Ln(III) based probes have been further exploited in Durham in recent years, allowing high resolution visualization of selected cellular organelles in long term live-cell experiments, whilst also reporting on the micro-chemical environment [10]. This lecture aims to highlight the evolution of lanthanide based cellular stains and expand the theory of SIM through a chronological overview of the key innovations in the field. We continuously seek to develop this fascinating technique further to allow its use by the broad imaging community, so saturation eliminated sub-diffraction spatio- and high temporal resolution 3D reconstructions can be created by simply incorporating modular SIM/ISM attachments into any existing LSCM or epi-fluorescence setup. The lecture will also include some of the fascinating and entertaining ‘side-tracks’ of our microscopy research, such as what is common in a 65 million years old ancient armor-plated slug and a ‘newly-formed’ Nebula thousands of light years away …

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