FEATURE MICROSCOPY
➤ ratio. ‘In our experiments, we can detect NIR fluorescent proteins at a depth of 18mm,’ said Verkhusha. ‘If you compare this depth, the signal to background ratio is 20-fold higher for NIR fluorescent proteins as compared to the most far-red shifted protein from the GFP family. This means a 20-fold better sensitivity, or 20-fold smaller number of cells we can detect.’ Whereas deep cell imaging of live cells is typically associated with two-photon excitation, the NIR fluorescent proteins can use single-photon excitation techniques because they already absorb in the NIR. Two-photon microscopy is generally chosen for its ability to achieve higher resolutions with less photobleaching than single-photon excitation techniques such as confocal microscopy. Two-photon lasers excite by using near simultaneous absorption of two long wavelength (around 800nm) photons and, because excitation only happens near the focal plane where the laser light is most concentrated, there is little tissue damage to the surrounding areas and a higher signal to background ratio. However, two-photon microscopy has an imaging depth limit of around several hundred micrometres, according to Verkhusha. So, in order to take advantage of the 18mm depths
POCKET MICROSCOPY
Earlier this year, UK start-up company IoLight launched a digital microscope powerful enough to observe the structure of plant and animal cells, but that can fit inside a jacket pocket. Capable of achieving resolutions
of 1µm, it unfolds to record and share 5 megapixel still images and real-time high-definition video at a magnification of x200 on an iPad. ‘There have been digital microscopes available on the market for several years… these use high quality mobile phone lenses and optics to produce a small, low cost microscope that is fine for low magnification applications, such as looking at insects,’ commented IoLight co- founder Andrew Monk. ‘However, these microscopes cannot see smaller subjects, like individual cells, because they are handheld and it is impossible to hold your hand still enough. They can be
mounted on a robust stand, but then they are not really portable anymore.’ To make the microscope truly
portable, IoLight has used these same components, but has added a fixed sample stage, just like a lab microscope would have, which holds the sample securely in front of the head. The optical head folds into the stage so that the package fits into a jacket pocket, and contains the camera and top and bottom illuminators, both of which are adjustable. Although it’s still early days, the microscopy has already been sold into micro-engineering applications such as electronics; microbiology research; universities and science centres, including the Eden Project; animal health; and for home use and education.
‘IoLight sees its role as
developing the portable functionality, giving more people
that these new NIR probes offer, techniques such as structured illumination, single-sheet illumination, adaptive optics microscopy, and photoacoustic imaging can be used. ‘These new imaging techniques will allow users to have high resolution in deep tissue using one- photon NIR excitation. They use lower power than two-photon microscopy, so there is less damage to the tissue. Because of this low level of damage, live tissue imaging is more feasible in one-photon regimes with NIR probes,’ Verkhusha noted. ‘As a result, I think these imaging techniques will become more popular as biologists adopt these new probes, because two-photon microscopy has existed for about 20 years and, during this time, I have not seen much development,’ Verkhusha continued. ‘These days, wavefront engineering imaging techniques are becoming more and more popular. NIR fluorescent proteins, which we develop, fit well with them.’
Label-free imaging
As well as engineering dyes that are more compatible with live tissue, scientists are
The goal is to
image in a similar way to fluorescence, but without having to add any dyes or stains
improving the methods for imaging live cells without the use of labels. Whereas fluorescence microscopy is often the method of choice for observing structures or dynamic processes in biological samples, in some cases, labelling isn’t practical. ‘There are certain situations where you just cannot label – for example, for medical diagnostic applications where the labels could be toxic, or when it is simply too labour-intensive to label and more convenient to image in a label- free fashion,’ explained Dr Chistian Freudiger, vice president of research and development at Invenio Imaging, which develops technology for non-destructive microscopy.
‘If scientists want to image very small molecules – for example,
metabolites such as glucose – the components are a lot smaller than the labels you would need to attach for imaging. If you were to label it, you would really perturb the function of these molecules that you’re trying to study,’ Freudiger said. ‘On occasions where you can’t do it, you need to start looking at label-free methods – the goal is to image in a similar
➤
IoLight’s portable microscope fits in a jacket pocket
access to better microscopy in more places,’ Monk said. ‘We think that existing microscope manufacturers will continue to make beautifully engineered and expensive microscopes, which will have a place in the lab for the foreseeable future. However, high resolution portable microscopes will
add another dimension of flexibility, optimising workflow and image sharing.
‘There will be applications that
go portable very quickly and others that take longer to adapt. We are looking forward to learning what the market wants from a portable microscope.’
32 ELECTRO OPTICS l OCTOBER 2016
@electrooptics |
www.electrooptics.com
IoLight
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