Photobleaching Problems Research Articles

Plasmon-enhanced fluorescence for biophotonics and bio-analytical applications.

Fluorescence spectroscopy serves as an ultrasensitive sophisticated tool where background noises which serve as a major impediment to the detection of the desired signals can be safely avoided for detections down to the single-molecule levels. One such way of bypassing background noise is plasmon-enhanced fluorescence (PEF), where the interactions of fluorophores at the surface of metals or plasmonic nanoparticles are probed. The underlying condition is a significant spectral overlap between the localized surface plasmon resonance (LSPR) of the nanoparticle and the absorption or emission spectra of the fluorophore. The rationale being the coupling of the excited state of the fluorophore with the localized surface plasmon leads to an augmented emission, owing to local field enhancement. It is manifested in enhanced quantum yields concurrent with a decrease in fluorescence lifetimes, owing to an increase in radiative rate constants. This improvement in detection provided by PEF allows a significant scope of expansion in the domain of weakly emitting fluorophores which otherwise would have remained unperceivable. The concept of coupling of weak emitters with plasmons can bypass the problems of photobleaching, opening up avenues of imaging with significantly higher sensitivity and improved resolution. Furthermore, amplification of the emission signal by the coupling of free electrons of the metal nanoparticles with the electrons of the fluorophore provides ample opportunities for achieving lower detection limits that are involved in biological imaging and molecular sensing. One avenue that has attracted significant attraction in the last few years is the fast, label-free detection of bio-analytes under physiological conditions using plasmonic nanoparticles for point-of-care analysis. This review focusses on the applications of plasmonic nanomaterials in the field of biosensing, imaging with a brief introduction on the different aspects of LSPR and fabrication techniques.

A target-triggered fluorescence-SERS dual-signal nano-system for real-time imaging of intracellular telomerase activity

Telomerase (TE) is a promising diagnostic and prognostic biomarker for many cancers. Quantification of TE activity in living cells is of great significance in biomedical and clinical research. Conventional fluorescence-based sensors for quantification of intracellular TE may suffer from problems of fast photobleaching and auto-fluorescence of some endogenous molecules, and hence are liable to produce false negative or positive results. To address this issue, a fluorescence-SERS dual-signal nano-system for real-time imaging of intracellular TE was designed by functionalizing a bimetallic Au@Ag nanostructure with 4-p-mercaptobenzoic acid (internal standard SERS tag) and a DNA hybrid complex consisted of a telomerase primer strand and its partially complimentary strand modified with Rhodamine 6G. The bimetallic Au@Ag nanostructure serves as an excellent SERS-enhancing and fluorescence-quenching substrate. Intracellular TE will trigger the extension of the primer strand and cause the shedding of Rhodamine 6G-modified complimentary strand from the nano-system through intramolecular DNA strand displacement, resulting in the recovery of the fluorescence of Rhodamine 6G and decrease in its SERS signal. Both the fluorescence of R6G and the ratio between the SERS signals of 4-p-mercaptobenzoic acid and Rhodamine 6G can be used for in situ imaging of intracellular TE. Experimental results showed that the proposed nano-system was featured with low background, excellent cell internalization efficiency, good biocompatibility, high sensitivity, good selectivity, and robustness to false positive results. It can be used to distinguish cancer cells from normal ones, identify different types of cancer cells, as well as perform absolute quantification of intracellular TE, which endows it with great potential in clinical diagnosis, target therapy and prognosis of cancer patients.

Begonia-Inspired Slow Photon Effect of a Photonic Crystal for Augmenting Algae Photosynthesis.

Plant photosynthesis is considered to be an environmentally friendly and effective measure for reducing carbon dioxide levels to meet the global objective of carbon neutrality. However, the light energy utilization of photosynthetic pigments is insufficient. Begonia pavonine (B. pavonina) with blue leaves exhibits a photosynthetic quantum yield 10% higher than those of other plants by virtue of their photonic crystal (PC) thylakoids. Inspired by this property, we prepared non-angle-dependent PC hydrogels and assembled them with algae Chlorella pyrenoidosa (C. pyre). The band edge of PC hydrogels matched the absorption peaks of C. pyre, and the resulting slow photon effect increased the interaction time between incident light and photosynthetic pigments, which in turn induced the expression of light-harvesting proteins and the synthesis of pigments, thereby improving the light energy utilization. Further, we introduced an artificial antenna into the assembly, which assisted the slow photon effect in increasing the oxygen evolution and carbon sequestration rate by more than 200%. This method avoids the photobleaching problems faced by methods of synthesizing artificial antenna pigments and the biosafety problems faced by genetically engineered methods of editing pigments or proteins.

Deep-tissue two-photon microscopy with a frequency-doubled all-fiber mode-locked laser at 937 nm

In two-photon microscopy, low illumination powers on samples and a high signal-to-noise ratio (SNR) of the excitation laser are highly desired for alleviating the problems of photobleaching and phototoxicity, as well as providing clean backgrounds for images. However, the high-repetition-rate Ti:sapphire laser and the low-SNR Raman-shift lasers fall short of meeting these demands, especially when used for deep penetrations. Here, we demonstrate a 937-nm laser frequency-doubled from an all-fiber mode-locked laser at 1.8 μm with a low repetition rate of ∼9 MHz and a high SNR of 74 dB. We showcase two-photon excitations with low illumination powers on multiple types of biological tissues, including fluorescence imaging of mouse brain neurons labeled with green and yellow fluorescence proteins (GFP and YFP), DiI-stained and GFP-labeled blood vessels, Alexa Fluor 488/568-stained mouse kidney, and second-harmonic-generation imaging of the mouse skull, leg, and tail. We achieve a penetration depth in mouse brain tissues up to 620 μm with an illumination power as low as ∼10 mW, and, even for the DiI dye with an extremely low excitation efficiency of 3.3%, the penetration depth is still up to 530 μm, indicating that the low-repetition-rate source works efficiently for a wide range of dyes with a fixed excitation wavelength. The low-repetition-rate and high-SNR excitation source holds great potential for biological investigations, such as in vivo deep-tissue imaging.

Shadow Electrochemiluminescence Microscopy of Single Mitochondria

AbstractMitochondria are the subcellular bioenergetic organelles. The analysis of their morphology and topology is essential to provide useful information on their activity and metabolism. Herein, we report a label‐free shadow electrochemiluminescence (ECL) microscopy based on the spatial confinement of the ECL‐emitting reactive layer to image single living mitochondria deposited on the electrode surface. The ECL mechanism of the freely‐diffusing [Ru(bpy)3]2+ dye with the sacrificial tri‐n‐propylamine coreactant restrains the light‐emitting region to a micrometric thickness allowing to visualize individual mitochondria with a remarkable sharp negative optical contrast. The imaging approach named “shadow ECL” (SECL) reflects the negative imprint of the local diffusional hindrance of the ECL reagents by each mitochondrion. The statistical analysis of the colocalization of the shadow ECL spots with the functional mitochondria revealed by classical fluorescent biomarkers, MitoTracker Deep Red and the endogenous intramitochondrial NADH, validates the reported methodology. The versatility and extreme sensitivity of the approach are further demonstrated by visualizing single mitochondria, which remain hardly detectable with the usual biomarkers. Finally, by alleviating problems of photobleaching and phototoxicity associated with conventional microscopy methods, SECL microscopy should find promising applications in the imaging of subcellular structures.

Shadow Electrochemiluminescence Microscopy of Single Mitochondria.

Mitochondria are the subcellular bioenergetic organelles. The analysis of their morphology and topology is essential to provide useful information on their activity and metabolism. Herein, we report a label-free shadow electrochemiluminescence (ECL) microscopy based on the spatial confinement of the ECL-emitting reactive layer to image single living mitochondria deposited on the electrode surface. The ECL mechanism of the freely-diffusing [Ru(bpy)3 ]2+ dye with the sacrificial tri-n-propylamine coreactant restrains the light-emitting region to a micrometric thickness allowing to visualize individual mitochondria with a remarkable sharp negative optical contrast. The imaging approach named "shadow ECL" (SECL) reflects the negative imprint of the local diffusional hindrance of the ECL reagents by each mitochondrion. The statistical analysis of the colocalization of the shadow ECL spots with the functional mitochondria revealed by classical fluorescent biomarkers, MitoTracker Deep Red and the endogenous intramitochondrial NADH, validates the reported methodology. The versatility and extreme sensitivity of the approach are further demonstrated by visualizing single mitochondria, which remain hardly detectable with the usual biomarkers. Finally, by alleviating problems of photobleaching and phototoxicity associated with conventional microscopy methods, SECL microscopy should find promising applications in the imaging of subcellular structures.

Dynamic signal of live biological cells under interferometric scattering (iSCAT) microscopy and its impacts on single-particle tracking

Interferometric scattering (iSCAT) microscopy has been demonstrated as a powerful tool for tracking single nanoparticles at ultrahigh spatial and temporal resolutions. The unmatched performance of iSCAT single-particle tracking (SPT) relies on the fact that the scattering signal is steady and can be linearly increased under strong illumination, circumventing the problems of photobleaching and saturation of fluorescence-based approaches. However, the scattering-based imaging is complicated by the presence of nonspecific scattering background, e.g. in the complex samples of biological cells. To distinguish the signal of interest from the nonspecific scattering background, metallic nanoparticles are often used as the efficient scattering probes. In many applications, reduction of the particle size is favorable because the loading of particle may introduce labeling artifacts. To work with smaller particles of weaker signal, suppression of the heterogeneous background is necessary. In this work, we start with the characterization of dynamic iSCAT signal of living cells by calculating its spatial and temporal Fourier spectra. To reduce the influence of cell background to SPT, a common strategy of background estimation and correction by temporal average filtering is considered. The effect of residual cell background to SPT is evaluated systematically with simulated image data of various signal-to-background ratios. This work benchmarks the localization errors caused by the cell background in SPT, providing a guideline for the interpretation of single-particle diffusion data of iSCAT microscopy.

Robotic Label-Free Precise Oocyte Enucleation for Improving Developmental Competence of Cloned Embryos.

The invisibility of domestic oocyte nucleus in bright field currently forces operators to blindly aspirate nucleus out in oocyte enucleation, usually causing large cytoplasm losses and poor developmental competences of cloned embryos. Although fluorescent labeling of nucleus allows for nucleus localization, the involved photobleaching problems and barriers to the execution of enucleation process limit its online-application in oocyte enucleation. This paper reports a novel label-free oocyte enucleation method for precise removal of the nucleus with less cytoplasm loss. The relative positions between the injection pipette and nucleus for complete removal of nucleus with less cytoplasm loss were determined through a finite element modeling of nucleus aspiration. To position injection pipette to the above positions relative to nucleus, the appropriate oocyte orientation and trajectory of injection pipette inside oocyte were derived according to the offline-calibrated 3-D distribution of nucleus and the simulated dynamic drift of nucleus that occurs as injection pipette is maneuvered inside oocyte. Finally, a robotic label-free precise enucleation procedure was established. The experimental results on more than 1000 porcine oocytes proved that this system is capable of reducing cytoplasm loss by 60% at the same level of enucleation success rate and almost doubling the cleavage rate of clone embryos in comparison to blind aspiration method. The results prove that our method significantly improves the developmental competence of cloned embryos in comparison to manual enucleation method. Our method is expected to improve the extremely low success rate of animal cloning in the future.

A U-Shape Fibre-Optic pH Sensor Based on Hydrogen Bonding of Ethyl Cellulose With a Sol-Gel Matrix

Development of a biocompatible pH sensor is of importance in biomedical applications, particularly for in vivo measurement, providing necessary information for clinical diagnosis and treatment such as chronic wounds and foetal acidosis. Traditional pH-indicator based optical sensors have problems of dye-leaching and photobleaching that restrict their uses in long-term monitoring. In this work, a dye-free fibre optic pH sensor is proposed consisting of a U-shape multimode optical fibre coated with a hybrid organic-inorganic composite film. The film is formed by cross-linking ethyl cellulose with a silica matrix at an optimised ethyl cellulose/silica molar ratio of 0.0065 via weakly interacted hydrogen bonding. This bonding is affected by hydrogen concentration (i.e., pH) in a solution resulting in a morphological change of the polymer aggregation presented in the silica matrix leading to refractive index change of the film. The developed sensor shows a reversible response to pH from 4.5 to 12.5 and exhibits linear correlation between transmitted light power and pH with a limit of agreement (LoA) between the sensor and a commercial probe of ±0.2 pH. For a clinically important range of pH values between 6 and 8 the LoA is ±0.1 pH. The sensor has low cross-sensitivity to temperature as the maximum interpreted pH change attributed to the power change is 0.12 pH when the temperature changes from 21 °C to 39 °C. To demonstrate biomedical relevance, the sensor is used to monitor pH of human serum. An in-house cytotoxicity assay is conducted with mouse fibroblast cell revealing that the film is not cytotoxic.

Accelerated FRET-PAINT microscopy

Recent development of FRET-PAINT microscopy significantly improved the imaging speed of DNA-PAINT, the previously reported super-resolution fluorescence microscopy with no photobleaching problem. Here we try to achieve the ultimate speed limit of FRET-PAINT by optimizing the camera speed, dissociation rate of DNA probes, and bleed-through of the donor signal to the acceptor channel, and further increase the imaging speed of FRET-PAINT by 8-fold. Super-resolution imaging of COS-7 microtubules shows that high-quality 40-nm resolution images can be obtained in just tens of seconds.

Superresolution fluorescence microscopy for 3D reconstruction of thick samples

Three-dimensional (3D) reconstruction of thick samples using superresolution fluorescence microscopy remains challenging due to high level of background noise and fast photobleaching of fluorescence probes. We develop superresolution fluorescence microscopy that can reconstruct 3D structures of thick samples with both high localization accuracy and no photobleaching problem. The background noise is reduced by optically sectioning the sample using line-scan confocal microscopy, and the photobleaching problem is overcome by using the DNA-PAINT (Point Accumulation for Imaging in Nanoscale Topography). As demonstrations, we take 3D superresolution images of microtubules of a whole cell, and two-color 3D images of microtubules and mitochondria. We also present superresolution images of chemical synapse of a mouse brain section at different z-positions ranging from 0 μm to 100 μm.

Super-Resolution Imaging of the Extracellular Space in Living Brain Tissue

The extracellular space (ECS) of the brain has an extremely complex spatial organization, which has defied conventional light microscopy. Consequently, despite a marked interest in the physiological roles ofbrain ECS, its structure and dynamics remain largelyinaccessible for experimenters. We combined 3D-STED microscopy and fluorescent labeling of theextracellular fluid to develop super-resolution shadow imaging (SUSHI) of brain ECS in living organotypic brain slices. SUSHI enables quantitative analysis of ECS structure and reveals dynamics on multiple scales in response to a variety of physiological stimuli. Because SUSHI produces sharp negative images of all cellular structures, it also enables unbiased imaging of unlabeled brain cells with respect to their anatomical context. Moreover, the extracellular labeling strategy greatly alleviates problems of photobleaching and phototoxicity associated with traditional imaging approaches. As a straightforward variant of STED microscopy, SUSHI provides unprecedented access to the structure and dynamics of live brain ECS and neuropil.

Accelerated super-resolution imaging with FRET-PAINT

Super-resolution fluorescence microscopy in the current form is hard to be used to image the neural connectivity of thick tissue samples due to problems such as slow imaging speed, severe photobleaching of fluorescent probes, and high background noise. Recently developed DNA-PAINT solved the photobleaching problem, but its imaging speed is extremely low. We report accelerated super-resolution fluorescence microscopy named FRET-PAINT. Compared to conventional DNA-PAINT, the imaging speed of the microscopy increases up to ~30-fold. As demonstrations, we show that 25-50 second imaging time is long enough to provide super-resolution reconstruction of microtubules and mitochondria of COS-7 cells.

Non-bleaching fluorescence emission difference microscopy using single 808-nm laser excited red upconversion emission.

Optical super-resolution microscopy has become a powerful technique to help scientists to monitor the sample of interest at nanoscale. Fluorescence emission difference (FED) microscopy, a very facile super-resolution method, does not require high depleting laser intensity and is independent on the species of agents, which makes FED microscopy possess great potential. However, to date, the biomarkers applied in FED microscopy usually suffer from a photo-bleaching problem. In this work, by introducing Er3+ activated upconverting nanoparticles with red-color emission and non-photobleaching properties, we demonstrate nonbleaching super-resolution imaging with FED microscopy. The dopant neodymium ions (Nd3+) can work as highly efficient sensitizing ions and enable near infrared 808-nm CW laser excitation of relatively low power, which would potentially reduce high intensity/short-wavelength light induced tissue damage. Both simulations and experiments on monodispersed NaYF4:Nd3+/Yb3+/Er3+@NaYF4:Nd3+ UCNPs also indicate that the easy saturation of the multiphoton properties of these UCNPs is beneficial to resolution enhancement in FED microscopy.

Photostable Ratiometric Pdot Probe for in Vitro and in Vivo Imaging of Hypochlorous Acid.

Developing probes for the detection of reactive oxygen species (ROS), a hallmark of many pathophysiological process, is imperative to both understanding the precise roles of ROS in many life-threatening diseases and optimizing therapeutic interventions. We herein report an all-in-one fluorescent semiconducting polymer based far-red to near-infrared (NIR) Pdot nanoprobe for the ratiometric detection of hypochlorous acid (HOCl). The fabrication takes the advantage of flexible polymer design by incorporating target-sensitive and target-inert fluorophores into a single conjugated polymer to avoid leakage or differential photobleaching problems existed in other nanoprobes. The obtained nanoprobe has improved performance in HOCl sensing, such as high brightness, ideal far-red to NIR optical window, excellent photostability, self-referenced ratiometric response, fast response, and high selectivity. The dual-emission property allows the sensitive imaging of HOCl fluctuations produced in living macrophage cells and peritonitis of living mice with high contrast. This study not only provides a powerful and promising nanoprobe to be potentially used in the investigations of in situ HOCl status of diseases in living systems but also puts forward the design strategy of a new category of ratiometric fluorescent probes facilitating precise and reliable measurement in biological systems.

Unprecedented “All‐in‐One” Lanthanide‐Doped Mesoporous Silica Frameworks for Fluorescence/MR Imaging and Combination of NIR Light Triggered Chemo‐Photodynamic Therapy of Tumors

Designing a single multifunctional nanoparticle that can simultaneously impart both diagnostic and therapeutic functions is considered to be a long‐lasting hurdle for biomedical researchers. Conventionally, a multifunctional nanoparticle can be constructed by integrating organic dyes/magnetic nanoparticles to impart diagnostic functions and anticancer drugs/photosensitizers to achieve therapeutic outcomes. These multicomponents systems usually suffer from severe photobleaching problems and cannot be activated by near‐infrared (NIR) light. Here, it is demonstrated that all‐in‐one lanthanide‐doped mesoporous silica frameworks (EuGdOx@MSF) loaded with an anticancer drug, doxorubicin (DOX) can facilitate simultaneous bimodal magnetic resonance (MR) imaging with approximately twofold higher T1‐MR contrast as compared to the commercial Gd(III)‐DTPA complex and fluorescence imaging with excellent photostability. Upon a very low dose (130 mW cm−2) of NIR light (980 nm) irradiation, the EuGdOx@MSF not only can sensitize formation of singlet oxygen (1O2) by itself but also can phototrigger the release of the DOX payload effectively to exert combined chemo‐photodynamic therapeutic (PDT) effects and destroy solid tumors in mice completely. It is also discovered for the first time that the EuGdOx@MSF‐mediated PDT effect can suppress the level of the key drug resistant protein, i.e., p‐glycoprotein (p‐gp) and help alleviate the drug resistant problem commonly associated with many cancers.

Impacts of quantum dots in molecular detection and bioimaging of cancer.

Introduction: A number of assays have so far been exploited for detection of cancer biomarkers in various malignancies. However, the expression of cancer biomarker(s) appears to be extremely low, therefore accurate detection demands sensitive optical imaging probes. While optical detection using conventional fluorophores often fail due to photobleaching problems, quantum dots (QDs) offer stable optical imaging in vitro and in vivo. Methods: In this review, we briefly overview the impacts of QDs in biology and its applications in bioimaging of malignancies. We will also delineate the existing obstacles for early detection of cancer and the intensifying use of QDs in advancement of diagnostic devices. Results: Of the QDs, unlike the II-VI type QDs (e.g., cadmium (Cd), selenium (Se) or tellurium (Te)) that possess inherent cytotoxicity, the I-III-VI 2 type QDs (e.g., AgInS2, CuInS2, ZnS-AgInS2) appear to be less toxic bioimaging agents with better control of band-gap energies. As highly-sensitive bioimaging probes, advanced hybrid QDs (e.g., QD-QD, fluorochrome-QD conjugates used for sensing through fluorescence resonance energy transfer (FRET), quenching, and barcoding techniques) have also been harnessed for the detection of biomarkers and the monitoring of delivery of drugs/genes to the target sites. Antibody-QD (Ab-QD) and aptamer- QD (Ap-QD) bioconjugates, once target the relevant biomarker, can provide highly stable photoluminescence (PL) at the target sites. In addition to their potential as nanobiosensors, the bioconjugates of QDs with homing devices have successfully been used for the development of smart nanosystems (NSs) providing targeted bioimaging and photodynamic therapy (PDT). Conclusion: Having possessed great deal of photonic characteristics, QDs can be used for development of seamless multifunctional nanomedicines, theranostics and nanobiosensors.

A G protein-coupled receptor (GPCR) in red: live cell imaging of the kappa opioid receptor–tdTomato fusion protein (KOPR–tdT) in neuronal cells

IntroductionIn contrast to green fluorescent protein and variants (GFPs), red fluorescent proteins (RFPs) have rarely been employed for the generation of GPCR fusion proteins, likely because of formation of aggregates and cell toxicity of some RFPs. Among all the RFPs, tdTomato (tdT), one of the non-aggregating RFP, has the highest brightness score (about 3 times that of eGFP) and unsurpassed photostability. MethodsWe fused tdT to the KOPR C-terminus. The KOPR–tdT cDNA construct was transfected into a Neuro2A mouse neuroblastoma cell line (Neuro2A cells) and rat cortical primary neurons for characterization of pharmacological properties and imaging studies on KOPR trafficking. ResultsKOPR–tdT retained KOPR properties (cell surface expression, ligand binding, agonist-induced signaling and internalization) when expressed in Neuro2A cells and rat primary cortical neurons. Live cell imaging of KOPR–tdT enables visualization of the time course of agonist-induced internalization of KOPR in real time for 60min, without photobleaching and apparent cell toxicity. U50,488H-induced KOPR internalization occurred as early as 4min and plateaued at about 30min. A unique pattern of internalized KOPR in processes of primary neurons was induced by U50,488H. DiscussiontdT is an alternative to, or even a better tool than, GFPs for fusion to GPCR for trafficking studies, because tdT has higher brightness and thus better resolution and less photobleaching problems due to the reduced laser power used. It also has advantages associated with its longer-wavelength emission including spectral separation from autofluorescence and GFPs, reduced cell toxicity that the laser may impose, and greater tissue penetration. These advantages of tdT over GPFs may be critical for live cell imaging studies of GPCRs in vitro and for studying GPCRs in vivo because of their low abundance.

Sub‐cellular tumor identification and markerless differentiation in the rat brain in vivo by multiphoton microscopy

Aim of the current study was to localize and differentiate between tumor (glioma) and healthy tissue in rat brains on a cellular level. Near-infrared multiphoton microscopy takes advantage of the simultaneous absorption of two or more photons to analyze various materials such as cell and tissue components via the observation of endogenous fluorophores such as NAD(P)H, FAD, porphyrins, melanin, elastin, and collagen, with a very high resolution, without inducing the problems of photo-bleaching on out-of-focus areas. In vitro and in vivo studies on healthy rat brains as well as C6 glioma cell line allografts have been performed. Near-infrared laser pulses (λ = 690-1060 nm, τ ~140 fs) generated by an ultrafast Ti:Sapphire tunable laser system (Chameleon, Coherent GmbH, Santa Clara, CA) were coupled into a laser scanning microscope (LSM 510 META, Carl Zeiss, Germany) to observe high quality images. Several image acquisitions have been performed by varying the zoom scale of the multiphoton microscope, image acquisition time and the wavelength (765, 840 nm) to detect various tissue components. With a penetration depth of ~200 µm in vitro and about 30-60 µm in vivo into the brain tissue it was possible to differentiate between tumor and healthy brain tissue even through thin layers of blood. Near-infrared multiphoton microscopy allows the observation and possibly differentiation between tumor (glioma) and healthy tissue in rat brains on a cellular level. Our findings suggest that a further miniaturization of this technology might be very useful for scientific and clinical applications in neurosurgery.

Beyond Photobleaching, Laser Illumination Unbinds Fluorescent Proteins

Confocal and two-photon fluorescence microscopy techniques using genetically encoded fluorescent probes are widely used in cell biology. Beyond the common problems of photobleaching and phototoxicity, we present evidence that photounbinding also has the potential to compromise such methods, especially in quantitative studies. We show that laser intensities within excitation regimes typical for imaging approaches such as as fluorescence recovery after photobleaching (FRAP), photolysis, or fluorescence correlation spectroscopy (FCS) experiments can cause the dissociation of antibodies from their ligands. Indeed, both one- and two-photon excitation of a fluorescent anti-GFP antibody caused its dissociation from immobilized GFP in vitro. Importantly, with two-photon excitation, the laser intensity threshold for photobleaching was the same as for photounbinding. By contrast, with single-photon excitation, we found a range of laser intensities where photobleaching can be separated from photounbinding. This photounbinding effect was visualized and measured by rebinding a second fluorescent anti-GFP (Green Fluorescent Protein) antibody, indicating that the GFP remained functional for reassociation following the photoinduced dissociation. Finally, we show that this unbinding effect occurs only when at least one binding partner carries a fluorescent label. Our results show that this photounbinding effect can readily remain masked or be misinterpreted as photobleaching, which can compromise the quantitative interpretation of binding studies made using fluorescence microscopy.