Collegio Alessandro Volta | University of Pavia | www.collegiovolta.org

20th March 2018.  
Rainer Heintzmann, University of Jena          

The 5th seminar of the series on Light Microscopy entitled Super resolution microscopy will be given by Rainer Heintzmann of the University of Jena on the 20 March, 4.00 pm in the College lecture theatre. The poster of the series can be downloaded here.

Abstract
In the past decade revolutionary advances have been made in the field of microscopy imaging, some of which have been honoured by the Nobel prize in Chemistry 2014. One high-resolution method is based on transforming conventionally unresolvable details into measurable patterns with the help of an effect most people have already personally experienced: the Moiré effect.  If two fine periodic patterns overlap, coarse patterns emerge. This is typically seen on a finely weaved curtain folding back onto itself. Another example is fast moving coarse patterns on both fences of a bridge above a motorway, when approaching it with the car. The microscopy method of structured illumination utilizes this effect by projecting a fine grating onto the sample and imaging the resulting coarser Moiré patterns containing the information about invisibly fine sample detail. With the help of computer reconstruction based on several such Moiré images, a high-resolution image of the sample can then be assembled. Another way to obtain a high-resolution map of the sample is to utilize the blinking behaviour inherent in most molecules, used to stain the sample. Recent methodological advances (Cox et al., Nature Methods 9, 195-200, 2012) enable us to create pointillist high-resolution maps of molecular locations in a living biological sample, even if in each of the required many individual images, these molecules are not individually discernible. Examples will be shown as a film of a cell at 30 millionths of millimeter resolution and 6 seconds between the individual movie frames. 

Two further recently developed modes of lightsheet imaging will also be presented. Lightwedge microscopy aims at mesoscopic imaging of fixed and optically cleared samples at 1 µm isotropic resolution without the need for sample rotation. The key-idea is to focus a lightsheet at an unusually high NA (thus the name “lightwedge”) and still obtain a large field of view due to refocusing of the lightwedge and stitching the multiple small regions of thin illumination back together. This has been simplified by electrical tunable lens technology, which has become available recently. The second mode is hyperspectral Raman imaging in a lightsheet illumination configuration [1]. To recover the spectral information a full-field Fourier-spectroscopic approach has been chosen. The difficulty here is that in a Michelson approach, it would be technically very hard to maintain the angular stability and common path approaches usually tolerate a relatively low product of étendue and maximal optical path difference. We thus developed an optically stable Mach-Zehnder like scheme based on the use of retro-reflecting corner cubes, which is inherently stable. This enabled us to obtain full spectrally-resolved Raman images consisting of over four million spectra in about 10 minutes. Advantages over the conventional Raman imaging are the reduced maximum power on the sample and out of focus heating, the lightsheet-inherent good suppression of crosstalk from the illumination side and the avoidance of glass close to the sample mounting. Light sheet illumination for Raman imaging at few specific wavelengths was previously reported [2]. With a total laser power of 2W at an illumination wavelength of 577 nm, we obtained images (2048 × 2048 pixels) of polystyrene beads, zebrafish and a root cap of a snowdrop at a spectral resolution of 4.4 cm-1 with only few minutes of exposure. The olefinic and aliphatic C-H stretching modes, as well as the fingerprint region are clearly visible along with the broad water peak of the embedding medium. Spectrally resolved spontaneous Raman microscopy therefore promises high-throughput imaging for biomedical research and on-the-fly clinical diagnostics.

[1] W. Müller, M. Kielhorn, M. Schmitt, J. Popp, R. Heintzmann, Light sheet Raman micro-spectroscopy, Optica 3, 452-457, 2016.
[2] Ishan Barman, Khay Ming Tan, Gajendra Pratap Singh, “Optical sectioning using single-plane-illumination Raman imaging”, J. Raman Spectrosc., 41, 1099–1101 (2010)