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Company > 30 - September - 2016

TIRF Microscopy Advances in Cellular Biology

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Feature: TIRF Microscopy Advances in Cellular Biology

It is difficult to overestimate the impact fluorescence imaging has had on life sciences. Fluorescence has advanced the field dramatically, and has become the basis for numerous bio-imaging methods and applications. In cellular and molecular biology a large number of molecular-level events surrounding cellular surfaces, such as adhesion, binding of cells by way of hormones, secretion of neurotransmitters, and membrane dynamics have been studied with conventional fluorescence microscopes. Fluorophores that are bound to a specimen's surface provide us a great deal of information about it. The resulting fluorescence from these fluorophores is very low, compared to that of non-bound particles. Total Internal Reflection Fluorescence Microscopy (TIRFM) allows for selective excitation of the surface-bound fluorophores, while non-bound molecules are not excited and do not fluoresce. Due to the sub-micron surface selectivity of surface-bound fluorophores TIRFM has become the method of choice for in vitro single molecule detection and imaging.

TIRF was developed within microscopy circles to restrict the background fluorescence and increase the signal-to-noise ratio (s/n) in the resultant image. This is accomplished by using the ability of induced light to create an evanescent wave at a very limited range within the sample, beyond the interface of two differing refractive index substrates. If the angle of the incident light is greater than the critical angle, the refractive index mismatch will create a wave that is identical to the incident light. Since this wave will experience exponential intensity decay with distance, the resultant fluorescence signal will occur within less than 100 nanometers of the surface. This effectively restricts the excitation of the sample being studied.

TIRFM has potential benefits in any application requiring imaging of minute structures or single molecules in specimens having large numbers of fluorophores located outside of the optical plane of interest, such as molecules in solution in Brownian motion, vesicles undergoing endocytosis or exocytosis, or single protein trafficking within cells.

Much of the trend toward greater utilization of TIRFM is due to the development of versatile biological tools like green fluorescent protein (GFP) and its cyan, blue, yellow, and red derivatives. TIRFM is an ideal tool for investigation of both the mechanisms and dynamics of many of the proteins involved in cell-cell interactions. Live-cell imaging represents one of the most promising applications of the TIRFM technique. Protein interactions at the cell membrane surface, such as those involved in focal adhesions, have tremendous importance to cell biology. An understanding of the signals involved in normal cell growth and the attenuation resulting from cell-cell contact (contact inhibition) can provide insight into abnormal cell growth that occurs in diseases such as cancer.

At the biomolecular level, TIRFM techniques have been utilized to image single molecules of the mutant protein GFP-Rac, which is the protein involved in cell motility. TIRFM is ideal for investigations involved at the cell membrane level, for example, the study of neurotransmitter release and uptake during a synapse. TIRFM also provides the ability to study the vast number of proteins involved in neurobiological processes at a refined level and in a manner never before possible.

The acquisition of image data at high temporal resolution in living cells, at multiple wavelengths, is an area of great promise for TIRFM, and the expanded utilization of various dye combinations is certain to reveal new cellular dynamics in more detail than possible previously. Recently, investigators have reported dual emission-wavelength detection utilizing fluorophores that are be excited by a single wavelength, and the possibility exists to expand TIRFM capabilities further by configuring systems for multiple-wavelength excitation. Single molecule studies will be greatly enhanced with further development in dye characteristics and with continued improvement of various detectors.

Expansion of the TIRFM approach in cellular studies is likely to continue through the refinement of genetic and molecular manipulation techniques, combined with optical detection at the high temporal and spatial resolution afforded by evanescent wave excitation.


1. "Application Notes- TIRFM" - C. Michael Stanley, PhD. Nature Methods

2. "Microscopy U. Nikon TIRFM Overview" - Ross et al Microscopy U.

3. "Multiwavelength TIRF Microscopy Enables Insight into Actin Filaments" - Dan Callen BioPhotonics Sep 2015

Laserglow in Research: Dynamic photothermal interferometric phase microscopy

Laserglow's LRS-532 LabSpec laser was recently used as a light source by a team of researchers at Tel Aviv University, in a study that demonstrates highly dynamic photothermal interferometric phase microscopy for quantitative, selective contrast imaging. The method enables fast analysis of photothermal signals from nanoparticles in live, highly dynamic cells. For the first time, video-rate photothermal signals were obtained. This forms the basis for real-time interferometric phase microscopy with molecular specificity. The team believes that this technique holds great potential for using photothermal imaging in flow cytometry.

In this method, gold nanoparticles were used as biomarkers to bind to specific cells. When stimulating gold nanoparticles at their plasmon-peak wavelength, a localized increase in temperature occurs due to plasmon resonance. This thermal response causes a rapid change of the optical phase of the incident light beam interacting with the sample. These phase changes can be recorded by interferometric phase microscopy and analyzed to form a photothermal image of the nanoparticle binding site in the cells. The analysis is usually done by computational Fourier transforms - which are time consuming, and hence not suitable for applications requiring dynamic imaging or real-time quantitative analysis, such as for analyzing and sorting cells during fast flow cytometry. In order to achieve the temporally improved goal, the researchers have developed new algorithms based on discrete Fourier transform variants. When compared with other methods, the researchers also claim that their setup is more cost effective while still providing a full field of view and photothermal signal.

The team observed accurate capture of dynamic imaging where they needed to compute the data at very few frames per second. The group are confident that this method will be useful for future phase imaging systems, as well as will assist in providing specificity to phase imaging for rapidly changing samples. For a detailed discussion of the results and the experimental setup, access the full research paper at: SPIE.org Full Paper

News: Window to the Brain Could Enable Laser Surgery

Use of a novel material, nanocrystalline yttria-stabilized zirconia (nc-YSZ), to make cranial implants may allow the safe and efficacious use of laser-based therapies to treat brain disorders while combating the bacterial infections that are a leading cause of cranial implant failure. The nc-YSZ is a transparent version of the same ceramic based material used to make dental crowns and hip implants.

The implant under development, which literally provides a 'window to the brain', will allow doctors to deliver minimally invasive, laser-based treatments to patients that are inflicted with life-threatening neurological disorders, such as brain cancers, traumatic brain injuries, neurodegenerative diseases and stroke. Recent studies highlight both the biocompatibility of the implant material and its ability to endure bacterial infections. The internal toughness of YSZ, which is more impact resistant than glass-based materials developed by other researchers, also makes it the only transparent skull implant that may conceivably be used in humans. The two recent studies further support YSZ as a promising alternative to currently available cranial implants. Published July 8 in Lasers in Surgery and Medicine, the most recent study also supports the use of transparent YSZ to allow doctors to combat bacterial infections.

In lab studies, the researchers treated E-Coli infections by aiming laser light through the implant without having to remove it, and without damaging the surrounding tissues. Another recent study published in the journal Nanomedicine: Nanotechnology, Biology and Medicine, explored the biocompatibility of YSZ in an animal model, where it was integrated into the host tissue without causing an immune response or other adverse effects.

The Window to the Brain team is comprised of faculty at UCR's Bourns College of Engineering and School of Medicine together with researchers at the University of California, San Diego and three universities in Mexico: Centro de Investigacion Cientifica y de Educacion Superior de Ensenada (CICESE); Universidad Nacional Autonoma de Mexico (UNAM); and Ruben Ramos-Garcia, Instituto Nacional de Astrofisica, Optica y Electronica (INAOE) in Puebla. Yasaman Damestani, a graduate student in Aguilar's lab was the lead author of these recent research studies. Read the full article at: UCR full article