Posts with the tag: fluorescence
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.
When biopsies are reviewed by pathologists in histology, they are typically viewed in ‘cross-section’ – a slice is cut perpendicular to the surface of the tissue. In contrast, microscopy performed directly on the patient (in vivo or endomicroscopy) usually generates en face images – slices in the plane of the tissue surface. Some endomicroscopes designs allow vertical cross-sections to be generated from stacks of en face images at varying depths, but this is nowhere near to being a real time approach. A group from the University of Michigan has now developed a handheld endomicroscope which can generate vertical cross-sections with a frame rate of 10 Hz.
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.
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.
If we wanted to build a fluorescence imaging system for minimally invasive surgery then there are a few things we would need to consider. We would want it to be simple to implement, reasonably lightweight and, most importantly, compatible with existing laparoscopes. We’d also like to be able to obtain a conventional white light view at the same time as the fluorescence. Researchers at GE have developed a device which meets all of these requirements, and recently published the details in the open access journal Biomedical Optics Express. Their suggested application is to help identify nerves during surgery, but the technique could easily be used for a range of purposes.
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.