Elimination Of Autofluorescence Research Articles

A Highly Bright Near‐Infrared Afterglow Luminophore for Activatable Ultrasensitive In Vivo Imaging

AbstractAfterglow luminescence imaging probes, with long‐lived emission after cessation of light excitation, have drawn increasing attention in biomedical imaging field owing to their elimination of autofluorescence. However, current afterglow agents always suffer from an unsatisfactory signal intensity and complex systems consisting of multiple ingredients. To address these issues, this study reports a near‐infrared (NIR) afterglow luminophore (TPP‐DO) by chemical conjugation of an afterglow substrate and a photosensitizer acting as both an afterglow initiator and an energy relay unit into a single molecule, resulting in an intramolecular energy transfer process to improve the afterglow brightness. The constructed TPP‐DO NPs emit a strong NIR afterglow luminescence with a signal intensity of up to 108 p/s/cm2/sr at a low concentration of 10 μM and a low irradiation power density of 0.05 W/cm2, which is almost two orders of magnitude higher than most existing organic afterglow probes. The highly bright NIR afterglow luminescence with minimized background from TPP‐DO NPs allows a deep tissue penetration depth ability. Moreover, we develop a GSH‐activatable afterglow probe (Q‐TPP‐DO NPs) for ultrasensitive detection of subcutaneous tumor with the smallest tumor volume of 0.048 mm3, demonstrating the high potential for early diagnosis and imaging‐guided surgical resection of tumors.

A Molecular Imaging "Skin A Time-resolving Intraoperative Imager for Microscopic Residual Cancer Detection Using Enhanced Upconverting Nanoparticles.

Optimal cancer therapy requires targeted and individualized treatment of all tumor cells, including both gross and microscopic disease. Intraoperatively hard to visualize and often left behind, microscopic foci of residual cancer cells significantly increase the risk of cancer recurrence and treatment failure rates. Fluorescently-tagged targeted molecular labels are employed to guide surgery, but conventional fluorescent intraoperative imagers suffer from lack of sensitivity and maneuverability, limiting practicality in small tumor cavities owing to their cumbersome sizes driven by optics. This work does away with conventional lenses and filters and introduces an optics-free molecular imaging "skin" consisting of only a $25\mu \mathrm{m}$ thin CMOS contact imager that synergistically integrates the long emission lifetimes of upconverting nanoparticles (UCNP) combined with upconversion to use a time domain approach to acquire the image coupled with infrared illumination allowing deep tissue penetration and elimination of autofluorescence. Using this strategy, we are able to visualize UCNPs at fluences (W/cm2) compatible with intraoperative use, opening the door to visualize targeted areas with microscopic sensitivity and facilitate residual microscopic disease detection during surgery, and laying the groundwork for precision post-operative radiation.

Elimination of autofluorescence in fluorescence correlation spectroscopy using the AzaDiOxaTriAngulenium (ADOTA) fluorophore in combination with time-correlated single-photon counting (TCSPC)

Fluorescence correlation spectroscopy (FCS) is a frequently applied technique that allows for the precise and sensitive analysis of molecular diffusion and interactions. However, the potential of FCS for in vitro or ex vivo studies has not been fully realized due in part to artifacts originating from autofluorescence (fluorescence of inherent components and fixative-induced fluorescence). Here, we propose the azadioxatriangulenium (ADOTA) dye as a solution to this problem. The lifetime of the ADOTA probe, about 19.4 ns, is much longer than most components of autofluorescence. Thus, it can be easily separated by time-correlated single-photon counting methods. Here, we demonstrate the suppression of autofluorescence in FCS using ADOTA-labeled hyaluronan macromolecules (HAs) with Rhodamine 123 added to simulate diffusing fluorescent background components. The emission spectrum and decay rate of Rhodamine 123 overlap with the usual sources of autofluorescence, and its diffusion behavior is well known. We show that the contributions from Rhodamine 123 can be eliminated by time gating or by fluorescence lifetime correlation spectroscopy (FLCS). While the pairing of ADOTA and time gating is an effective strategy for the removal of autofluorescence from fluorescence imaging, the loss of photons leads to erroneous concentration values with FCS. On the other hand, FLCS eliminates autofluorescence without such errors. We then show that both time gating and FLCS may be used successfully with ADOTA-labeled HA to detect the presence of hyaluronidase, the overexpression of which has been observed in many types of cancer.

Autofluorescence generation and elimination: a lesson from glutaraldehyde

Glutaraldehyde causes especially high autofluorescence. It reacted with proteins and peptides to generate visible to near-IR emitters. A model indicated that ethylenediamine and a secondary amine in the molecule were key components for the formation of emissive species. The mechanism enables us to control the generation and elimination of autofluorescence.

Abstract 3236: High-sensitivity, high-resolution multispectral fluorescence imaging of small (and very small) animals

Abstract in vivo multispectral imaging (MSI) increases the sensitivity and usefulness of small animal models of development and disease, including the most popular and promising experimental systems. In mice, MSI can essentially eliminate autofluorescence that otherwise limits sensitivity, reducing detection limits by as much as 100-fold. Fluorescence imaging is a key technology for determining the where, the when and the how much of gene expression. However, the ability to image and quantitate fluorescently labeled markers in vivo has typically been limited by the bright autofluorescence of animal tissues. Small-animal imaging, which often means the imaging of mouse models, encounters autofluorescence primarily from components in skin (collagen, which fluoresces green) and food (chlorophyll, which fluoresces red). However, other small animal model systems, such as nematodes (C. elegans), zebrafish (D. rerio) embryos or adult fish, and fruit flies (D. melanogaster) pose similar problems due to the presence of autofluorescent structures that may render common labeling strategies, including the use of green fluorescent protein (GFP) labels, problematic. Various solutions have been proposed for the reduction or elimination of autofluorescence. The most prevalent is simply to use narrow bandpass emission filters in an effort to isolate the desired fluorescence signal [1]. More recently, there has been a move to use infrared-emitting labels that are excited at wavelengths that are much less likely to induce autofluorescence signals. However, autofluorescence still limits sensitivity. Fortunately, the low signal-to-background contrast problem created by autofluorescence can be substantially eliminated using MSI techniques. Moreover, MSI permits the simultaneous use of multiple labels, such as GFP and RFP, even when they spectrally overlap with themselves or autofluorescence [2]. Presented here are the results of applying an MSI strategy to the whole-animal in vivo imaging of mice, C. elegans, D. rerio and D. melanogaster at a variety of magnifications, ranging from the imaging of three whole mice simultaneously down to microscopic images of whole animals and embryos. This is accomplished using a liquid crystal tunable filter (LCTF)-based imaging system that has been adapted to either a C-mount on a fluorescence microscope or macroscope, or incorporated into a whole-animal imaging system. In each case, the sensitivity of the fluorescence measurement was greatly improved by using a MSI approach. Rapid, quantitative, imaging of GFP signals of up to a few hundred flies simultaneously in a few seconds can be easily achieved, as can high-sensitivity microscopic imaging of GFP in nematode worms. These types of imaging experiments are of value to anyone with whole-animal fluorescence-based models. [1] Niswender KD et al, J Microsc 180, 109-116 (1995) [2] Levenson, et al, Cytometry A. 2006 Aug; 69(8):748-58 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3236.

Spectral Imaging for Separation of Fluorescent Signals from Autofluorescence

Autofluorescence, also known as adventitious fluorescence or background fluorescence, often poses a significant problem in many applications of fluorescence microscopy. It contributes to unwanted noise and can overwhelm the desired signal. Particularly difficult samples to image include many pathology specimens that have been processed using crosslinking fixatives (typically formaldehyde). This procedure dramatically increase the autofluorescence level, leading to bright, broad spectrum emissions, particularly from connective tissue components. Unprocessed plant tissue and neuronal tissue also have extremely high levels of endogenous autofluorescence that can make many convenient labeling strategies, including most green fluorescent protein (GFP) labels, extremely problematic. Various solutions have been proposed for the reduction or elimination of autofluorescence. These include using narrow bandpass emission filters to try to isolate the desired fluorescence signal, the use of labels that can be excited at wavelengths that are much less likely to induce autofluorescence (moving the excitation towards the NIR is effective), and post-processing aldehyde-fixed samples with such reagents as sodium borohydride or toluidine blue to chemically suppress the autofluorescence signal. However, in many cases, these approaches are either infeasible or ineffective. Spectral imaging, that is, the acquisition of a high-resolution optical spectrum at every pixel of an image, offers another approach to elimination of the contribution of autofluorescence. This has recently become possible due to the development of a number of technologies that allow the collection of spectral datasets from fluorescently labeled samples using fluorescence microscopy. Such technologies include liquid crystal tunable filters (LCTFs) and Fourier transform imaging spectrometers, both of which are now commercially available.

Spectral Imaging for Separation of Fluorescent Signals from Autofluorescence

Abstract Autofluorescence, also known as adventitious fluorescence or background fluorescence, ofter poses a significant problem in many applications of fluorescence microscopy. It contributes to unwanted noise and can swamp the desired signal. Particularly difficult samples to image include many pathology specimen that have been processed using crosslinking fixatives (typically formaldehyde). This procedure dramatically increase the autofluorescence level, leading to bright, broad spectrum emissions, particularly from connective tissue components. Unprocessed plant tissue and neuronal tissue also have extremely high levels of endogenous autofluorescence that can make many convenient labeling strategies, including most (green fluorescent protein (GFP) labels, extremely problematic. Various solutions have been proposed for the reduction or elimination of autofluorescence. These include using narrow bandpass emission filters to try to isolate the desired fluorescence signal, the use of labels which can be excited at wavelengths that are much less likely to induce autofluorescence (moving the excitation towards the NIR is effective), and post-processing aldehyde-fixed samples with such reagents as sodium borohydride or toluidine blue to chemically suppress the autofluorescence signal.However, in many cases, these approaches are either infeasible or ineffective.

Elimination of autofluorescence in immunofluorescence microscopy with digital image processing.

Autofluorescence can be a very disturbing factor in immunofluorescence microscopy. We present here a method to eliminate autofluorescence. The method is based on the fact that most autofluorescent compounds have broad-banded excitation and emission spectra, whereas specific fluorescent probes have narrow spectra. Two images are recorded and digitized, one at a wavelength exciting both the fluorescent probe and the autofluorescent molecules, and one at a wavelength exciting only the latter. Subtraction of the autofluorescence signal from the total fluorescence signal, using a self-developed computer program, results in an autofluorescence-free image. The procedure is demonstrated for elimination of elastin-derived autofluorescence in human lung alveoli and for elimination of lipofuscin-derived autofluorescence in human heart muscle. The autofluorescence signal is positively correlated with tissue section thickness (r = 0.93; p < 0.0001), and can be used to correct the specific fluorescence signals for section thickness.

Time-resolved fluroescence microscopy: Elimination of autofluorescence in tissue specimens for image cytometry with fluorescent labelled probes

Time-resolved fluroescence microscopy: Elimination of autofluorescence in tissue specimens for image cytometry with fluorescent labelled probes