Posts related to: Microscopy
Super-resolution microscopy has received a lot of interest in the past few years, culminating in the 2014 Nobel Prize in Chemistry for the development of the STED and STORM/PALM family of techniques. Around the same time, an interesting (and mischievously titled) commentary appeared in Nature Photonics, claiming to resolve (ho ho) a misconception about a third approach to super-resolution – SIM or ‘structured illumination microscopy’. This is a technique which can be used to improve the resolution by a factor of two. The paper argues that structured illumination microscopy only provides true resolution enhancement for fluorescence imaging and none at all for scattering imaging. This is despite recent papers making claims – and apparently providing experimental evidence – to the contrary.
Optical microscopy can only penetrate a few hundred microns into thick tissue, a limit imposed by scattering. High resolution imaging requires single-scattering events, so when we have multiple-scattering from particles above and below the focal plane, the resolution and signal to noise ratio quickly degrade. The thicker the tissue (i.e. the deeper the plane of interest) the more the multiple-scattering events dominate over single scattering. Techniques such as optical coherence tomography (OCT) enhance the penetration depth by rejecting multiple-scattered light using what is effectively a time-of-flight measurement. This works because light that has been scattered multiple times will tend to have travelled further than light that has been scattered only once. However, even with this technique, the penetration depth seldom exceeds 1-2 mm, as some multiple-scattered photons will (by chance) have a time of flight close to that of the single scattered photons. As we try to go deeper, these events will begin to dominate again. Now, a group mainly from Korea University in Seoul have suggested an additional method of discriminating between single and multiple-scattered photons, using a technique they call “collective accumulation of single-scattered waves”.
Two of the key parameters that describe the performance of the optical microscope are its resolution and its field-of-view. In fact, these two parameters are coupled: switching to a higher magnification objective will improve the resolution, but also tend to reduce the field-of-view. This trade-off is encapsulated in the idea of the space-bandwidth product, which is (conceptually at least) a measure of how many useful pixels of information an imaging system can transmit. Typical microscopes and microscope objectives are limited to around 10 Megapixels; if we make these pixels smaller by increasing the resolution then the area covered must be reduced as well. So if we want to image large areas at high resolution, as we might want to do in histology for example, then we have to mechanically scan the slide underneath the microscope and stitch multiple images together.
A research team from the University of Twente has found a way to obtain high resolution images of a fluorescent object through a strongly scattering medium. This has been a goal of bioimaging scientists for some time, as it would allow us to use visible light to image much deeper into tissue. Various methods have been suggested, but they generally need some kind of ‘guide-star’ behind the scattering layer. The new approach uses the ‘memory effect’ of speckle to avoid the need for any calibration or guide-star, potentially making it much more applicable to real situations.
There’s a stark contrast between the elegant simplicity of a conventional widefield microscope and the much more complex apparatus need for point-by-point scanning in confocal microscopy. It’s this point-by-point scanning, together with a pinhole, which gives the confocal microscope its optical sectioning ability. By removing the out of focus blur which would degrade a conventional microscope image, confocal microscopes obtain crisp, clean images of thick samples, or even of in vivo tissue. The additional complexity involved with confocal operation, which includes the requirement to use a laser rather than a thermal light source, is accepted as the price that has to be paid if we want to obtain these kinds of images. But now, HiLo microscopy is offering an alternative approach which could have a number of niche applications, particularly where space or cost preclude the use of a full confocal microscopy setup.
Optical Coherence Tomography (OCT) has a lot of advantages over confocal microscopy, especially for applications where it’s useful to have a large working distance between the probe and the tissue. But a big limitation is that it can only detect reflected light, and so can’t be used with fluorescent stains. Fluorescence is often preferred in conventional microscopy because it allows us to visualise structures that we can’t easily identify in reflectance images. So the race is on to find a way to make OCT work with fluorescent emission as well as reflected light.