Neuromag July 2018 - Page 6

The Path to All Optical Neurophysiology Written by Joe Sheppard

The microscope is one of the most iconic symbols of science , at least equal to the white coat or bubbling neon liquids in its ability to endow scientific credibility in the eyes of TV viewers of shows like The Big Bang Theory or CSI : Miami . Yet the kinds of microscopes that most people imagine , despite being a mainstay of many labs , represent the old-school of microscopy in terms of both their technology and their impact in modern research . The new-school of microscopy involves liquid cooled lasers , glowing cells and the seemingly paradoxical ability to resolve objects smaller than the wavelength of light itself ! How did all of this happen without you knowing ? That I don ’ t know , but I can tell you how we got here , and how it ’ s going to change neuroscience .

The fluorescent revolution

Over the last 30 years , light microscopy has been the subject of continual innovation that has dramatically changed microscope design and capability , making them much more prominent in biological research . This is particularly true in the field of neuroscience where visualizing the intricate and diverse morphologies of neurons ( brain cells ) demands both specific labelling and high-resolution imaging . In fact , it was the desire to view neuronal “ wiring ” in three dimensions that motivated Marvin Minsky to theorize and build the first confocal microscope in the sixties . Already a paradigm shift away from the old-school , confocal microscopes of the time used narrow light beams instead of full-field illumination to repeatedly scan over biological samples . This focused illumination , combined with a pinhole aperture placed in front of the photon detector enabled early confocal microscopy to exclude background light originating outside the plane of interest , thus granting a large increase in contrast which allows for better visualization of neurons . It was not until the eighties that confocal microscopy saw widespread adoption by the scientific community .

The benefit of all types of fluorescence microscopy is that , unlike conventional light microscopy , the fluorescent molecules which compose the image can be targeted to specific features of a cell to highlight it , such as the plasma membrane or nucleus . Since these are the only things

Fluorescence Microscope with sample image on screen . ( Source : Zeiss )

6 | NEUROMAG | July 2018

The Path to All Optical Neurophysiology Written by Joe Sheppard The microscope is one of the most iconic symbols of science, at least equal to the white coat or bub- bling neon liquids in its ability to endow scientific credibility in the eyes of TV viewers of shows like The Big Bang Theory or CSI: Miami. Yet the kinds of microscopes that most people imagine, despite being a mainstay of many labs, represent the old-school of microscopy in terms of both their tech- nology and their impact in modern research. The new-school of microscopy involves liquid cooled lasers, glowing cells and the seemingly paradoxical ability to resolve objects smaller than the wave- length of light itself! How did all of this happen without you knowing? That I don’t know, but I can tell you how we got here, and how it’s going to change neuroscience. The fluorescent revolution Over the last 30 years, light micros- copy has been the subject of contin- ual innovation that has dramatically changed microscope design and capa- bility, making them much more prom- inent in biological research. This is particularly true in the field of neuro- science where visualizing the intricate and diverse morphologies of neurons (brain cells) demands both specific la- belling and high-resolution imaging. In fact, it was the desire to view neuronal “wiring” in three dimensions that mo- tivated Marvin Minsky to theorize and build the first confocal microscope in the sixties. Already a paradigm shift away from the old-school, confocal microscopes of the time used nar- row light beams instead of full-field illumination to repeatedly scan over biological samples. This focused il- lumination, combined with a pinhole aperture placed in front of the photon detector enabled early confocal mi- 6 | NEUROMAG |July 2018 croscopy to exclude background light originating outside the plane of inter- est, thus granting a large increase in contrast which allows for better visu- alization of neurons. It was not unti ��F�RV�v�F�W2F�B6��f�6�֖7&�66���6rv�FW7&VBF�F���'�F�R66�ЦV�F�f�26���V�G��F�R&V�Vf�B�b��G�W2�bf�V�&W2Ц6V�6R֖7&�66���2F�B�V�Ɩ�R6��ЧfV�F����Ɩv�B֖7&�66���F�Rf�V�Ч&W66V�B���V7V�W2v��6�6���6P�F�R��vR6�&RF&vWFVBF�7V6�f�0�fVGW&W2�b6V��F���v�Ɩv�B�B�7V6��2F�R�6��V�'&�R�"�V6�RЧW2�6��6RF�W6R&RF�R��ǒF���w0�f�V�&W66V�6R֖7&�66�Rv�F�6��R��vR��67&VV���6�W&6S��V�72�