Posts with the tag: microscopy

No benefit to structured illumination microscopy for scattering imaging

Comments
Add One

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.
Read more…

Imaging through multi-mode fibres using model-based calibration

Comments
Add One

In the last few years, several papers have looked at how it might be possible to use a multimode fibre as an ultra-narrow endoscope (see this post and this post for a bit of background). The most common approach is to use a spatial light modulator to shape the wavefront entering the fibre. If this is done in precisely the right way, interference between light coupled into the different modes of the fibre will result in a focused spot at the far end. By adjusting the input wavefront it’s then possible to scan the spot in two dimensions, allowing point-by-point imaging. Of course, we need to know what wavefronts to use, making it necessary to perform a calibration which requires access to the far end of the fibre. Unfortunately, this calibration is highly dependent on the configuration of the fibre – if the fibre is bent then the calibration changes. This means the technique is only applicable to rigid probes, greatly limiting the scope of potential applications. Now, in a paper published in Nature Photonics, Tomáš Čižmár and colleagues from the University of Dundee have suggested a possible solution to this problem.

Read more…

Deep Tissue Imaging by Collective Accumulation of Single-Scatterers

Comments
Add One

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”.

Read more…

Fourier Ptychographic Microscopy

Comments
Add One

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.

Read more…

Imaging Through Scattering Media Using the Speckle Memory Effect

Comments
Add One

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.

Read more…

Capsule OCT Endomicroscopy

Comments
Add One

A team from Massachusetts General Hospital (MGH) has developed a tethered endomicroscopy capsule, offering a potential alternative to endoscopic tissue biopsy. It uses optical coherence tomography (OCT) to generate high-resolution cross-sections through the walls of the oesophagus. Capsules are already in use for video endoscopy, and OCT has been shown to have value in the diagnosis of Barrett’s oesophagus, but this is the first time the two technologies have been combined.
Read more…

High Resolution Fluorescence Endomicroscopy Using a Multi-mode Fibre

Comments
Add One

I recently discussed a paper by a group from Korea University,Seoul, who had developed a reflection-mode widefield endomicroscope using a multi-mode fibre. Other people have been thinking along the same lines, including a Swiss group who have recently published details of another multi-mode fibre based device. Unlike the widefield implementation, this method uses digital phase conjugation to scan a point of light at the far-end of the fibre, making fluorescence imaging possible. The basic idea isn’t new, but the group was able to demonstrate better resolution than previous reports through the use of a very high NA, double clad fibre.
Read more…

High Speed Multi-photon PLIM

Comments
Add One

Phosphorescence lifetime imaging microscopy (PLIM) allows substances or tissues with different phosphorescence lifetimes to be identified with high spatial resolution. PLIM hasn’t found many practical applications so far, but it could be useful as a way of measuring oxygen concentration in tissues. Depth resolved images can be obtained using multi-photon excitation, a technique which ensures that all the signal comes from the focal plane. Unfortunately, relatively long phosphorescent lifetimes make the point-by-point scanning used in multi-photon microscopy very time consuming. Attempts to improve the frame rate using parallel excitation can result in cross-talk between pixels and blurring of the image. Now, a group from Cornell University has devised a way to acquire parallel excitation PLIM images which are free from cross-talk.

Read more…

Dual Mode Endomicroscopy for Assessing Gene Transfection

Comments
Add One

Fibre bundle endomicroscopes usually operate in fluorescence mode: the tissue is stained with a fluorescent dye which, when illuminated at a certain wavelength, emits light at a longer wavelength. Collecting this fluorescent emission tends to produce clear, high contrast images, and also allows back-reflections from the fibre bundle to be removed using wavelength selective filters. Reflectance mode endomicroscopes, which create an image from the light back-scattered from the tissue, have been demonstrated several times, but have found little practical application. Now, Cha et al. from John Hopkins University have developed a dual-mode device that simultaneously collects both fluorescence and reflectance images. They have used this device to measure the efficacy of gene transfection – the deliberate insertion of genes into cancerous cells.
Read more…

Mosaicing of Widefield Endomicroscopy Images

Comments
Add One

The Bioengineering Department at Rice University in Texas has been developing fibre-bundle based widefield endomicroscopes for several years. While these devices lack the depth sectioning capabilities of confocal endomicroscopes, they can still produce useful images from certain tissues if a suitable topical fluorophore is applied. A recent paper from Richards-Kortum’s Group at Rice has demonstrated ‘real time’ mosaicing using their endomicroscope, allowing characterisation of much larger areas of tissue than would otherwise be possible.
Read more…