Fluorophores For Imaging Research Articles

On fluorophore imaging by nonlinear diffusion model with dynamical iterative scheme

Fluorescence imaging aims at recovering the absorption coefficient of fluorophore in biological tissues. Due to the nonlinear dependence of excitation and emission fields on the unknown coefficient, such an inverse problem with the boundary measurement as inversion input is nonlinear. We reformulate this inverse problem as an optimization problem for a non-convex cost functional consisting of the unknown absorption coefficient depending both on the excitation field and the emission one with a penalty term. The existence of minimizer of the cost functional is proved rigorously, with explicit expression for the Fréchet derivative of the cost functional. For seeking the local minimizer considered as the approximate solution to the inverse problem efficiently, we develop a dynamical process which makes the non-convex cost functional quadratic at each iteration step, by freezing the unknown coefficient for the excitation field. This new dynamical quadratic optimization process is proven convergent and decreases the amount of computations for the fluorescence imaging, while the reconstruction accuracy keeps almost unchanged. Such advantages are verified by several numerical examples for different configurations of the absorption coefficient to be identified, comparing our proposed scheme with the algorithm for the original non-convex cost functional.

Chemical Approaches to Optimize the Properties of Organic Fluorophores for Imaging and Sensing.

Organic fluorophores are indispensable tools in cells, tissue and in vivo imaging, and have enabled much progress in the wide range of biological and biomedical fields. However, many available dyes suffer from insufficient performances, such as short absorption and emission wavelength, low brightness, poor stability, small Stokes shift, and unsuitable permeability, restricting their application in advanced imaging technology and complex imaging. Over the past two decades, many efforts have been made to improve these performances of fluorophores. Starting with the luminescence principle of fluorophores, this review clarifies the mechanisms of the insufficient performance for traditional fluorophores to a certain extent, systematically summarizes the modified approaches of optimizing properties, highlights the typical applications of the improved fluorophores in imaging and sensing, and indicates existing problems and challenges in this area. This progress not only proves the significance of improving fluorophores properties, but also provide a theoretical guidance for the development of high-performance fluorophores.

Chemical Approaches to Optimize the Properties of Organic Fluorophores for Imaging and Sensing

AbstractOrganic fluorophores are indispensable tools in cells, tissue and in vivo imaging, and have enabled much progress in the wide range of biological and biomedical fields. However, many available dyes suffer from insufficient performances, such as short absorption and emission wavelength, low brightness, poor stability, small Stokes shift, and unsuitable permeability, restricting their application in advanced imaging technology and complex imaging. Over the past two decades, many efforts have been made to improve these performances of fluorophores. Starting with the luminescence principle of fluorophores, this review clarifies the mechanisms of the insufficient performance for traditional fluorophores to a certain extent, systematically summarizes the modified approaches of optimizing properties, highlights the typical applications of the improved fluorophores in imaging and sensing, and indicates existing problems and challenges in this area. This progress not only proves the significance of improving fluorophores properties, but also provide a theoretical guidance for the development of high‐performance fluorophores.

A red-emission and viscosity-ultrasensitive probe for identifying non-alcoholic fatty liver via imaging lysosomal viscosity

Elucidating the intrinsic correlation between non-alcoholic fatty liver (NAFL) and lysosomal viscosity is a challenging work owing to the disadvantages of state-of-the-art technologies for NAFL identification. Herein, we constructed a red emission imaging fluorophore L-TPQ based on D-π-A (donor–π–bridge-acceptor) structure for identifying NAFL tissues by tracking the change in lysosomal viscosity. Specifically, L-TPQ is ultrasensitive to viscosity with a remarkable enhancement in fluorescence intensity (about 910 fold) and fluorescence quantum yield (about 221 fold) through a twisted intra-molecular charge transfer (TICT) mechanism. In addition, L-TPQ shows a single selectivity toward viscosity with an excellent pH stability and insignificant interference from environmental polarity or biomolecules. Moreover, probe L-TPQ is lysosome-specific, which is capable of monitoring the lysosome movement, lysosomal viscosity change, and mitophagy. Importantly, by means of this probe, we have successfully observed an increased viscosity in the pathological model of NAFL mice. Given the comprehensive property of probe L-TPQ, it is expected to serve as a convenient and effective tool for better understanding the pathophysiology of NAFL and the early diagnosis of this disease.

Orchestrated Strategies for Developing Fluorophores for NIR-II Imaging.

Fluorescence imaging (FLI), a non-invasive, real-time, and highly sensitive imaging modality, allows for investigating the molecular/cellular level activities to understand physiological functions and diseases. The emergence of the second near-infrared window (NIR-II, 1000-1700 nm) has endowed fluorescence imaging with deeper tissue penetration and unprecedented clarity. Among the various NIR-II imaging fluorophores, the organic fluorescent probes have occupied a pivotal position in bioimaging due to their higher biocompatibility, safety, and potential for clinical applications compared with those of the inorganic probes. To obtain high-quality organic dyes, diverse strategies have been taken. In this review, different strategies for optimizing NIR-II organic fluorophores are summarized, including traditional chemical modifications, and emerging bioengineering operations, which have not previously been elaborated on and summarized. Moreover, the bioengineering strategies are highlighted using endogenous serum proteins and even exogenous gene-editing proteins, which would provide fresh insights to design good-performance dyes and help develop NIR-II probes with clinical translation potential in the future. A critical perspective on the direction of the design strategies of NIR-II dyes for disease imaging is also proposed.

Unmixing Biological Fluorescence Image Data with Sparse and Low-Rank Poisson Regression.

Multispectral biological fluorescence microscopy has enabled the identification of multiple targets in complex samples. The accuracy in the unmixing result degrades (1) as the number of fluorophores used in any experiment increases and (2) as the signal-to-noise ratio in the recorded images decreases. Further, the availability of prior knowledge regarding the expected spatial distributions of fluorophores in images of labeled cells provides an opportunity to improve the accuracy of fluorophore identification and abundance. We propose a regularized sparse and low-rank Poisson unmixing approach (SL-PRU) to deconvolve spectral images labeled with highly overlapping fluorophores which are recorded in low signal-to-noise regimes. Firstly, SL-PRU implements multi-penalty terms when pursuing sparseness and spatial correlation of the resulting abundances in small neighborhoods simultaneously. Secondly, SL-PRU makes use of Poisson regression for unmixing instead of least squares regression to better estimate photon abundance. Thirdly, we propose a method to tune the SL-PRU parameters involved in the unmixing procedure in the absence of knowledge of the ground truth abundance information in a recorded image. By validating on simulated and real-world images, we show that our proposed method leads to improved accuracy in unmixing fluorophores with highly overlapping spectra. The source code used for this paper was written in MATLAB and is available with the test data at https://github.com/WANGRUOGU/SL-PRU. Supplementary data are available at Bioinformatics online.

Engineering and Validation of a Peptide-Stabilized Poly(lactic-co-glycolic) Acid Nanoparticle for Targeted Delivery of a Vascular Disruptive Agent in Cancer Therapy.

Developing a biocompatible and biodegradable nanoparticle (NP) carrier that integrates drug-loading capability, active targeting, and imaging modality is extremely challenging. Herein, we report an NP with a core of poly(lactic-co-glycolic) acid (PLGA) chemically modified with the drug combretastatin A4 (CA4), a vascular disrupting agent (VDA) in clinical development for ovarian cancer (OvCA) therapy. The NP is stabilized with a short arginine-glycine-aspartic acid-phenylalanine x3 (RGDFFF) peptide via self-assembly of the peptide on the PLGA surface. Importantly, the use of our RGDFFF coating replaces the commonly used polyethylene glycol (PEG) polymer that itself often induces an unwanted immunogenic response. In addition, the RGD motif of the peptide is well-known to preferentially bind to αvβ3 integrin that is implicated in tumor angiogenesis and is exploited as the NP's targeting component. The NP is enhanced with an optical imaging fluorophore label via chemical modification of the PLGA. The RGDFFF-CA4 NPs are synthesized using a nanoprecipitation method and are ∼75 ± 3.7 nm in diameter, where a peptide coating comprises a 2-3 nm outer layer. The NPs are serum stable for 72 h. In vitro studies using human umbilical cord vascular endothelial cells (HUVEC) confirmed the high uptake and biological activity of the RGDFFF-CA4 NP. NP uptake and viability reduction were demonstrated in OvCA cells grown in culture, and the NPs efficiently accumulated in tumors in a preclinical OvCA mouse model. The RGDFFF NP did not induce an inflammatory response when cultured with immune cells. Finally, the NP was efficiently taken up by patient-derived OvCA cells, suggesting a potential for future clinical applications.

Molecular structure analysis of 4-fluoro phenyl(4-methyl piperidin-1-yl) methanone and a comparative study of its optical, antimicrobial and antioxidant activities with 4-chloro phenyl (4-methyl piperidin-1-yl) methanone

A new fluoro derivative of 4-methyl piperidine (FPMPM) was synthesized by Scholten-Baumann condensation reaction, and the crystal structure was elucidated using single-crystal X-ray diffraction intensities. The molecules are arranged in monoclinic P21/n symmetry allowing for a dihedral angle of 36.76 (8)° observed between the mean plane of the benzene ring and the mean plane of the piperidine chair. Essentially projected non-bonded interactions were corroborated through Hirshfeld surface analysis. Predominant H…H and long-ranged halogen interactions packed with weak dispersion forces suggest the sizeable hydrophobic surface area. The chloro analogue (CPMPM) of FPMPM was compared for its FT-IR and FT-Raman spectra displaying their characteristic vibration modes of the functional groups. The bandgaps of 2.82 and 3.97 eV in FPMPM and CPMPM divulged by the Tauc's plots of UV–vis spectral data are concomitant with the optical band gaps disclosed by photoluminescence spectra. A longer delayed fluorescence lifetime of 31.5(8) and 33.1(1) ns with relatively high contribution confirmed the intermediate triplet state in the materials. The fast, prompt fluorescence decay of 3.72 (2) and 2.85 (1) ns in FPMPM and CPMPM with 54 (1) and 69 (1) % percent fractional contribution are promising shreds of evidence for the materials to act as an optical imaging fluorophore and an imminent scintillating material at moderate temperatures. Relatively moderate values of nonlinear susceptibility (3.44 and 3.85) × 10−6 (e. s. u.) and positive nonlinear absorption coefficient with z-scan measurements supported thermal concave lensing behavior and reverse saturable absorption in both the materials. Thermogravimetry coupled with Differential Scanning Calorimetry (DSC) declares the moderately low melting point (70.4 and 107 °C) for the material, FPMPM, and CPMPM, respectively. The F substituent in FPMPM possesses high hydrophobicity and lipophilicity, mitigating antibacterial activity, while moderate H…F and C-H…π interactions allow for susceptible antimicrobial activities. CPMPM exhibits robust antifungal activity against Trichoderma with reference to amphotericin-B. The antioxidant scavenging property is reflected by relatively high proton reactivity sites in FPMPM and hence relatively low % DPPH activity in CPMPM.

Highly lipophilic coumarin fluorophore with excimer-monomer transition property for lipid droplet imaging

Lipid droplet (LD) fluorescent imaging plays an important role in the detection of lipid-related diseases. Due to their poor photostability and low hydrophobicity of currently available LD imaging fluorophores, LD imaging is limited by its short imaging period and low imaging contrast. Herein, we reasonably designed a highly lipophilic compound Cou-Flu with excellent photostability and excimer-monomer transition property. It exhibited weak excimer emission in cytoplasm, but strong monomer emission in LDs, enabling high contrast LD imaging and LD movement tracing in cells. Zebrafish imaging study demonstrated that Cou-Flu was also suitable for in vivo LD detection with excellent sensitivity. We anticipate that Cou-Flu could be widely applied to understand LD-related intracellular activities and even LD-related diseases in the future.

Heme-Based Gas Sensors in Nature and Their Chemical and Biotechnological Applications

Sensing is an essential feature of life, where many systems have been developed. Diatomic molecules such as O2, NO and CO exhibit an important role in life, which requires specialized sensors. Among the sensors discovered, heme-based gas sensors compose the largest group with at least eight different families. This large variety of proteins also exhibits many distinct ways of sensing diatomic molecules and promote a response for biological adaptation. Here, we briefly describe a story of two impressive systems of heme-based oxygen sensors, FixL from Rhizobium and DevS(DosS)/DosT from Mycobacterium tuberculosis. Beyond this, we also examined many applications that have emerged. These heme-based gas sensors have been manipulated to function as chemical and biochemical analytical systems to detect small molecules (O2, CO, NO, CN−), fluorophores for imaging and bioanalysis, regulation of processes in synthetic biology and preparation of biocatalysts among others. These exciting features show the robustness of this field and multiple opportunities ahead besides the advances in the fundamental understanding of their molecular functioning.

Graphene Quantum Dots for Fluorescent Labeling of Gelatin‐Based Shear‐Thinning Hydrogels

The efficiency of injectable biomaterials as minimally invasive therapeutics significantly relies on biomaterial's characteristics, such as stability, biodegradation rate, and interaction with the host tissue, which requires real‐time tracking of the biomaterials. Fluorescence imaging is considered as a noninvasive technique for monitoring biomaterials; however, the commonly used fluorescent agents are often accompanied by photobleaching and toxicity. Herein, graphene quantum dots (GQDs) are introduced as a biocompatible and stable fluorophore for imaging and noninvasive monitoring of a physically cross‐linked injectable shear‐thinning biomaterial (STB) of gelatin–silicate nanoplatelets. Silicate nanoplatelets and GQDs serve as the physical cross‐linkers of gelatin making electrostatic interaction with gelatin chains. Different STB‐GQDs formulations are assessed in terms of fluorescence intensity, injectability, thermal stability, and cellular biocompatibility. STB‐GQDs with 0.06% GQDs, 6% solid material, and 50% silicate in the solid material show the strongest in vitro fluorescence and the highest thermal stability. In vivo monitoring of STB‐GQDs is also achieved through fluorescent imaging where incorporated GQDs exhibit a robust and stable signal, suggesting their promising applications in long‐term tracking of gelatin‐based STBs.

A potent peptide-steroid conjugate accumulates in cartilage and reverses arthritis without evidence of systemic corticosteroid exposure.

On-target, off-tissue toxicity limits the systemic use of drugs that would otherwise reduce symptoms or reverse the damage of arthritic diseases, leaving millions of patients in pain and with limited physical mobility. We identified cystine-dense peptides (CDPs) that rapidly accumulate in cartilage of the knees, ankles, hips, shoulders, and intervertebral discs after systemic administration. These CDPs could be used to concentrate arthritis drugs in joints. A cartilage-accumulating peptide, CDP-11R, reached peak concentration in cartilage within 30 min after administration and remained detectable for more than 4 days. Structural analysis of the peptides by crystallography revealed that the distribution of positive charge may be a distinguishing feature of joint-accumulating CDPs. In addition, quantitative whole-body autoradiography showed that the disulfide-bonded tertiary structure is critical for cartilage accumulation and retention. CDP-11R distributed to joints while carrying a fluorophore imaging agent or one of two different steroid payloads, dexamethasone (dex) and triamcinolone acetonide (TAA). Of the two payloads, the dex conjugate did not advance because the free drug released into circulation was sufficient to cause on-target toxicity. In contrast, the CDP-11R-TAA conjugate alleviated joint inflammation in the rat collagen-induced model of rheumatoid arthritis while avoiding toxicities that occurred with nontargeted steroid treatment at the same molar dose. This conjugate shows promise for clinical development and establishes proof of concept for multijoint targeting of disease-modifying therapeutic payloads.

On Fluorophore Imaging by Diffusion Equation Model: Decompositions and Optimizations

Fluorescence imaging is a target-specific molecular imaging technology using absorption coefficient of fluorophore. For this imaging model governed by an inverse problem for the coupled diffusion system, which describes the interaction of the excitation field from several boundary sources and the corresponding emission field, we reformulate it as an optimization problem. For solving this non-quadratic optimizing problem, we propose a decomposition scheme, which extracts the horizontal information of the target from the boundary measurement data directly. The realizability of this hybrid imaging scheme is rigorously proved mathematically for cubic and ellipsoid targets, by constructing an indicator function for the horizontal location of the target explicitly. Then based on this horizonal location as a good initial guess for the iteration process, the cost functional is optimized efficiently using the trust domain scheme. Numerical implementations are provided to show the validity of the proposed scheme.

The effect of pH and ionic strength on the fluorescence properties of a red emissive DNA-stabilized silver nanocluster

DNA-stabilized silver nanoclusters (DNA-AgNCs) are a class of promising fluorophores for imaging and sensing applications. All aspects of their spectroscopic properties are not yet fully characterized, leaving this field still with a number of fundamental studies to be addressed. In this work, we studied the spectroscopic properties of red-emitting DNA-AgNCs at different pH (5 to 9) and ionic strength μ (0.005 to 0.5). The photophysical properties of high performance liquid chromatography (HPLC) purified DNA-AgNCs proved to be constant over a large range of pH and μ, with absorption, emission and fluorescence decay times only being affected at very high pH and μ values. Non-purified DNA-AgNCs were also unaffected by pH and/or μ variations, but significant differences can be observed between the rotational correlation times of purified and non-purified DNA-AgNCs.

Biotechnological Method of Obtaining Nanoparticles of Silver, Cadmium, and Zinc Sulfides. Physico-chemical Characteristics. Creation of Polymeric Nanocomposites

A simple and environmentally safe method for obtaining stable nanoparticles of metal sulfides nanoparticles—NpAg2S, NpCdS, and NpZnS—was developed using different strains of microorganisms in an aqueous solution of metal salts and sulfur sources at the National Research Center Kurchatov Institute—GosNIIgenetika. The concentration of nanoparticles is 1‒4 mg/mL in aqueous suspensions. Using the methods of electron microscopy, spectrofluorimetry, and dynamic light scattering, the main characteristics of biogenic nanoparticles were determined: shape, size distribution, crystal structure, effective diameter, luminescent spectrum, and zeta potential. According to their characteristics, these nanoparticles are referred to quantum dots. It is established that the stability of nanoparticles in aqueous suspensions is due to protein molecules adsorbed on the surface of nanoparticles, which are supplied by cells of microorganisms. Effective immobilization of biogenic nanoparticles on the surface of various polymer supports was carried out. Biogenic nanoparticles along with nanoparticles obtained by physico-chemical methods can be used as fluorophores for imaging of biological processes, also as photocatalysts, solar cells, and for new nanocomposite materials.

Biotechnological method of obtaining nanoparticles of silver, cadmium and zinc sulfides. Physico-chemical characteristics. Creation of polymeric nanocomposites

A simple and environmentally safe method for obtaining stable nanoparticles of metal sulfides nanoparticles – NpAg2S, NpCdS and NpZnS was developed using different strains of microorganisms in an aqueous solution of metal salts and sulfur sources at the Research Center "Kurchatov Institute" – GosNIIgenetika. The concentration of nanoparticles is 1–4 mg/ml in aqueous suspensions. Using of the methods of electron microscopy, spectrofluorimetry, dynamic light scattering determined the main characteristics of biogenic nanoparticles: shape, size distribution, crystal structure, effective diameter, luminescent spectrum, zeta potential. According to its characteristics, these nanoparticles are referred to quantum dots. It is established that the stability of nanoparticles in aqueous suspensions is due to protein molecules adsorbed on the surface of nanoparticles, which are supplied by cells of microorganisms. Effective immobilization of biogenic nanoparticles on the surface of various polymer supports has been carried out. Biogenic nanoparticles along with nanoparticles obtained by physico-chemical methods can be used as fluorophores for imaging of biological processes, also as photocatalysts, solar cells and for new nanocomposite materials.

Simultaneous dual-channel imaging to quantify interdependent protein recruitment to laser-induced DNA damage sites

ABSTRACTFluorescence microscopy in combination with the induction of localized DNA damage using focused light beams has played a major role in the study of protein recruitment kinetics to DNA damage sites in recent years. Currently published methods are dedicated to the study of single fluorophore/single protein kinetics. However, these methods may be limited when studying the relative recruitment dynamics between two or more proteins due to cell-to-cell variability in gene expression and recruitment kinetics, and are not suitable for comparative analysis of fast-recruiting proteins. To tackle these limitations, we have established a time-lapse fluorescence microscopy method based on simultaneous dual-channel acquisition following UV-A-induced local DNA damage coupled with a standardized image and recruitment analysis workflow. Simultaneous acquisition is achieved by spectrally splitting the emitted light into two light paths, which are simultaneously imaged on two halves of the same camera chip. To validate this method, we studied the recruitment of poly(ADP-ribose) polymerase 1 (PARP1), poly (ADP-ribose) glycohydrolase (PARG), proliferating cell nuclear antigen (PCNA) and the chromatin remodeler ALC1. In accordance with the published data based on single fluorophore imaging, simultaneous dual-channel imaging revealed that PARP1 regulates fast recruitment and dissociation of PARG and that in PARP1-depleted cells PARG and PCNA are recruited with comparable kinetics. This approach is particularly advantageous for analyzing the recruitment sequence of fast-recruiting proteins such as PARP1 and ALC1, and revealed that PARP1 is recruited faster than ALC1. Split-view imaging can be incorporated into any laser microirradiation-adapted microscopy setup together with a recruitment-dedicated image analysis package.

Imaging γ-Glutamyltranspeptidase for tumor identification and resection guidance via enzyme-triggered fluorescent probe

Development of high selectivity, accurate targeted and noninvasive fluorescent probe for monitoring specific enzyme activity associated with the tumor is urgent needed for early diagnosis of cancer and clinical fluorescence interventional resection guidance treatment. Owing to the invasion of malignant tumor cells, tumor cells are mixed with normal cells in the actual tumor location, which make it quite difficult for clinician to diagnose early diagnosis of tumor as well as resection of tumor. To overcome aforementioned obstacle, herein, an ingenious enzyme-activated one and two-photon fluorescent probe TCF-GGT was constructed and synthesized including γ-GGT enzyme specific identification site and far-red fluorophore for imaging. Under simulative physiological condition, probe TCF-GGT demonstrated high selectivity, sensitivity (DL 0.014 mU/mL), rapid response (Te 14 min) for the detection of γ-GGT enzyme. By virtue of its biocompatibility, probe was employed for the identification of ovarian cancer cells (A2780 cells) from normal cells (NIH-3T3 cells), particularly in mixed cultivation dish (simulate the actual environment of tumor) through 2D&3D fluorescence imaging with “dual” mark (Nucleic acid labeling used by Hoechst 33342 dye and γ-GGT enzyme labeling used by probe TCF-GGT) for the first time. Probe TCF-GGT could be visualize endogenous γ-GGT activity in HepG-2 cells and zebrafish on the two-photon confocal platform, which is conduce to estimate the inhibitor of γ-GGT enzyme in vivo. Through NaBu (a potential anticancer drug) stimulation, the changes of γ-GGT activity were observed in living MCF-7 cells by using this probe. More importantly, the deep tissue penetration ability of far-red fluorescence allowed the two-photon fluorescent probe TCF-GGT to real-time track γ-GGT activity in tissue slices and tumor xenotransplantation model of mice by the tail vein injection, which showed that this enzyme-triggered fluorogenic probe would be a potential tool for preclinical applications of tumor resection.

Multiple Click-Selective tRNA Synthetases Expand Mammalian Cell-Specific Proteomics.

Bioorthogonal tools enable cell-type-specific proteomics, a prerequisite to understanding biological processes in multicellular organisms. Here we report two engineered aminoacyl-tRNA synthetases for mammalian bioorthogonal labeling: a tyrosyl ( ScTyrY43G) and a phenylalanyl ( MmPheT413G) tRNA synthetase that incorporate azide-bearing noncanonical amino acids specifically into the nascent proteomes of host cells. Azide-labeled proteins are chemoselectively tagged via azide-alkyne cycloadditions with fluorophores for imaging or affinity resins for mass spectrometric characterization. Both mutant synthetases label human, hamster, and mouse cell line proteins and selectively activate their azido-bearing amino acids over 10-fold above the canonical. ScTyrY43G and MmPheT413G label overlapping but distinct proteomes in human cell lines, with broader proteome coverage upon their coexpression. In mice, ScTyrY43G and MmPheT413G label the melanoma tumor proteome and plasma secretome. This work furnishes new tools for mammalian residue-specific bioorthogonal chemistry, and enables more robust and comprehensive cell-type-specific proteomics in live mammals.

Compact 3D printed module for fluorescence and label-free imaging using evanescent excitation

Total internal reflection fluorescence (TIRF) microscopy is widely used for selective excitation and high-resolution imaging of fluorophores, and more recently label-free nanosized objects, with high vertical confinement near a liquid–solid interface. Traditionally, high numerical aperture objectives (>1.4) are used to simultaneously generate evanescent waves and collect fluorescence emission signals which limits their use to small area imaging (<0.1 mm2). Objective-based TIRFs are also expensive as they require dichroic mirrors and efficient notch filters to prevent specular reflection within the objective lenses. We have developed a compact 3D module called cTIRF that can generate evanescent waves in microscope glass slides via a planar waveguide illumination. The module can be attached as a fixture to any existing optical microscope, converting it into a TIRF and enabling high signal-to-noise ratio (SNR) fluorescence imaging using any magnification objective. As the incidence optics is perpendicular to the detector, label-free evanescent scattering-based imaging of submicron objects can also be performed without using emission filters. SNR is significantly enhanced in this case as compared to cTIRF alone, as seen through our model experiments performed on latex beads and mammalian cells. Extreme flexibility and the low cost of our approach makes it scalable for limited resource settings.