The excited state lifetime of a fluorophore together with its fluorescence emission spectrum provide information that can yield valuable insights into the nature of a fluorophore and its microenvironment. However, it is difficult to obtain both channels of information in a conventional scheme as detectors are typically configured either for spectral or lifetime detection. We present a fiber-based method to obtain spectral information from a multiphoton fluorescence lifetime imaging (FLIM) system. This is made possible using the time delay introduced in the fluorescence emission path by a dispersive optical fiber coupled to a detector operating in time-correlated single-photon counting mode. This add-on spectral implementation requires only a few simple modifications to any existing FLIM system and is considerably more cost-efficient compared to currently available spectral detectors.
Spinal cord injury (SCI) causes permanent paralysis below the damaged area. SCI is linked to neuronal death, demyelination, and limited ability of neuronal fibers to regenerate. Regeneration capacity is limited by the presence of many inhibitory factors in the spinal cord environment. The use of poly(lactide-co-glycolide) (PLG) bridges has demonstrated the ability to sustain long-term regeneration after SCI in a cervical hemisection mouse model. Critically, imaging of regenerating fibers and the myelination status of these neuronal filaments is a severe limitation to progress in SCI research. We used a transgenic mouse model that selectively expresses fluorescent reporters (eGFP) in the neuronal fibers of the spinal cord. We implanted a PLG bridge at C5 vertebra after hemisection and evaluated in live animals’ neuronal fibers at the bridge interface and within the bridge 8 weeks postimplant. These in vivo observations were correlated with in situ evaluation 12 weeks postimplantation. We sectioned the spinal cords and performed fluorescent bioimaging on the sections to observe neuronal fibers going through the bridge. In parallel, to visualize myelination of regenerated axons, we exploited the characteristics of the third-harmonic generation arising from the myelin structure in these fixed sections.
Atomic force microscopes (AFM) provide topographical and mechanical information of the sample with very good axial resolution, but are limited in terms of chemical specificity and operation time-scale. An optical microscope coupled to an AFM can recognize and target an area of interest using specific identification markers like fluorescence tags. A high resolution fluorescence microscope can visualize fluorescence structures or molecules below the classical optical diffraction limit and reach nanometer scale resolution. A stimulated emission depletion (STED) microscopy superresolution (SR) microscope coupled to an AFM is an example in which the AFM tip gains nanoscale manipulation capabilities. The SR targeting and visualization ability help in fast and specific identification of subdiffraction-sized cellular structures and manoeuvring the AFM tip onto the target. We demonstrate how to build a STED AFM and use it for biological nanomanipulation aided with fast visualization. The STED AFM based bionanomanipulation is presented for the first time in this article. This study points to future nanosurgeries performable at single-cell level and a physical targeted manipulation of cellular features as it is currently used in research domains like nanomedicine and nanorobotics.
Instruments with single-molecule level detection capabilities can potentially benefit a wide variety of fields, including medical diagnostics. However, the size, cost, and complexity of such devices have prevented their widespread use outside sophisticated research laboratories. Fiber-only devices have recently been suggested as smaller and simpler alternatives, but thus far, they have lacked the resolution and sensitivity of a full-fledged system, and accurate alignment remains a critical requirement. Here we show that through-space reciprocal optical coupling between a fiber and a microscope objective, combined with wavelength division multiplexing in optical fibers, allows a drastic reduction of the size and complexity of such an instrument while retaining its resolution. We demonstrate a 4×4×18 cm3 sized fluorescence correlation spectrometer, which requires no alignment, can analyze kinetics at the single-molecule level, and has an optical resolution similar to that of much larger microscope based devices. The sensitivity can also be similar in principle, though in practice it is limited by the large background fluorescence of the commonly available optical fibers. We propose this as a portable and field deployable single molecule device with practical diagnostic applications.
In India, the pedagogy of science education is “believe what text book says”. Providing schools with appropriate teaching materials to enhance teaching has always been a challenge in a developing country like India. Generally it is not possible for a normal school in India to afford the expensive teaching materials to teach through demonstrations and experiments. Thus students are forced to believe what text book says rather than learning concepts through experiments. The International School of Photonics SPIE (International Society for Optical Engineering) student chapter came up with ‘Optics kit’ to supplement the teaching of optics in school level. ‘Optics kit’, developed with indigenously procured components, could be sold at an affordable prize for an average Indian School. The chapter is currently selling the kit for less than $20. The content of the kit is at par with many kits already available commercially in developed countries, and the price is just 10% compared to those kits. The kit is aimed to higher secondary level students in India, where students are taught Ray optics and basics of Wave Optics. The content of the kit is developed based on this syllabus. The Optics Kit contains simple optical elements like lens, grating, polarizer, mirror, diode laser etc. The kit can be used to demonstrate optics phenomena like interference, diffraction, polarization etc. The kit was developed based on the feedback gathered by the chapter through its outreach activities. The syllabus for the kit was developed through thorough discussion with educational experts in the field of Physics. The student community welcomed the optics kit with overwhelming enthusiasm and hence the project proved to be successful in giving an opportunity for students to “See and Believe” what they are learning.