Widefield fluorescence lifetime imaging of protoporphyrin IX for fluorescence‐guided neurosurgery: An ex vivo feasibility study

Achieving a maximal safe extent of resection during brain tumor surgery is the goal for improved patient prognosis. Fluorescence‐guided neurosurgery using 5‐aminolevulinic acid (5‐ALA) induced protoporphyrin IX has thereby become a valuable tool enabling a high frequency of complete resections and a prolonged progression‐free survival in glioblastoma patients. We present a widefield fluorescence lifetime imaging device with 250 mm working distance, working under similar conditions such as surgical microscopes based on a time‐of‐flight dual tap CMOS camera. In contrast to intensity‐based fluorescence imaging, our method is invariant to light scattering and absorption while being sensitive to the molecular composition of the tissue. We evaluate the feasibility of lifetime imaging of protoporphyrin IX using our system to analyze brain tumor phantoms and fresh 5‐ALA‐labeled human tissue samples. The results demonstrate the potential of our lifetime sensing device to go beyond the limitation of current intensity‐based fluorescence‐guided neurosurgery.      

Inside Front Cover: Widefield multifrequency fluorescence lifetime imaging using a two‐tap complementary metal‐oxide semiconductor camera with lateral electric field charge modulators (J. Biophotonics 5/2019)

A novel Widefield frequency‐domain fluorescence lifetime imaging system based on a ultrafast sCMOS sensor is reported. It can do parallel multi‐frequency FLIM up to 620MHz in one measurement with 64 phase images. With this system, measurement of FRET efficiency at multi‐frequencies is demonstrated in living cells. Meanwhile, the temperature change of living cells can be measured at each pixel based on FLIM of Rhodamine B at different frequencies. Further details can be found in the article by Hongtao Chen, Ning Ma, Keiichiro Kagawa, et al. ( e201800223 ).

     

Reference‐independent wide field fluorescence lifetime measurements using Frequency‐Domain (FD) technique based on phase and amplitude crossing point

Fluorescence lifetime imaging microscopy (FLIM) is an essential tool in many scientific fields such as biology and medicine thanks to the known advantages of the fluorescence lifetime (FLT) over the classical fluorescence intensity (FI). However, the frequency domain (FD) FLIM technique suffers from its strong dependence on the reference and its compliance to the sample. In this paper, we suggest a new way to calculate the FLT by using the crossing point (CRPO) between the modulation and phase FLTs measured over several light emitting diode (LED) DC currents values instead of either method alone. This new technique was validated by measuring homogeneous substances with known FLT, where the CRPO appears to be the optimal measuring point. Furthermore, the CRPO method was applied in heterogeneous samples. It was found that the CRPO in known mixed solutions is the weighted average of the used solutions. While measuring B16 and lymphocyte cells, the CRPO of the DAPI compound in single FLT regions was measured at 3.5 ± 0.06 ns and at 2.83 ± 0.07 ns, respectively, both of which match previous reports and multi‐frequency analyses. This paper suggests the CRPO as a new method to extract the FLT in problematic cases such as high MCP gains and heterogeneous environments.

In traditional FD FLIM measurements, the variation in phase angle and modulation are measured. By measuring over varying DC currents, another variation is detected in the FLT determined through the phase and modulation methods, with the CRPO indicating the true FLT.     

Lightsheet fluorescence lifetime imaging microscopy with wide‐field time‐correlated single photon counting

We report on wide‐field time‐correlated single photon counting (TCSPC)‐based fluorescence lifetime imaging microscopy (FLIM) with lightsheet illumination. A pulsed diode laser is used for excitation, and a crossed delay line anode image intensifier, effectively a single‐photon sensitive camera, is used to record the position and arrival time of the photons with picosecond time resolution, combining low illumination intensity of microwatts with wide‐field data collection. We pair this detector with the lightsheet illumination technique, and apply it to 3D FLIM imaging of dye gradients in human cancer cell spheroids, and C. elegans.     

High-precision FLIM-FRET in fixed and living cells reveals heterogeneity in a simple CFP-YFP fusion protein

We have used widefield photon-counting FLIM to study FRET in fixed and living cells using control FRET pairs. We have studied fixed mammalian cells expressing either cyan fluorescent protein (CFP) or a fusion of CFP and yellow fluorescent protein (YFP), and living fungal cells expressing either Cerulean or a Cerulean-Venus fusion protein. We have found the fluorescence behaviour to be essentially identical in the mammalian and fungal cells. Importantly, the high-precision FLIM data is able to reproducibly resolve multiple fluorescence decays, thereby revealing new information about the fraction of the protein population that undergoes FRET and reducing error in the measurement of donor-acceptor distances. Our results for this simple control system indicate that the in vivo FLIM-FRET studies of more complex protein-protein interactions would benefit greatly from such quantitative measurements.     

Fluorescence lifetime imaging.

We describe a new fluorescence imaging methodology in which the image contrast is derived from the fluorescence lifetime at each point in a two-dimensional image and not the local concentration and/or intensity of the fluorophore. In the present apparatus, lifetime images are created from a series of images obtained with a gain-modulated image intensifier. The frequency of gain modulation is at the light-modulation frequency (or a harmonic thereof), resulting in homodyne phase-sensitive images. These stationary phase-sensitive images are collected using a slow-scan CCD camera. A series of such images, obtained with various phase shifts of the gain-modulation signal, is used to determine the phase angle and/or modulation of the emission at each pixel, which is in essence the phase or modulation lifetime image. An advantage of this method is that pixel-to-pixel scanning is not required to obtain the images, as the information from all pixels is obtained at the same time. The method has been experimentally verified by creating lifetime images of standard fluorophores with known lifetimes, ranging from 1 to 10 ns. As an example of biochemical imaging we created life-time images of Yt-base when quenched by acrylamide, as a model for a fluorophore in distinct environments that affect its decay time. Additionally, we describe a faster imaging procedure that allows images in which a specific decay time is suppressed to be calculated, allowing rapid visualization of unique features and/or regions with distinct decay times. The concepts and methodologies of fluorescence lifetime imaging (FLIM) have numerous potential applications in the biosciences. Fluorescence lifetimes are known to be sensitive to numerous chemical and physical factors such as pH, oxygen, temperature, cations, polarity, and binding to macromolecules. Hence the FLIM method allows chemical or physical imaging of macroscopic and microscopic samples.     

Fluorescence lifetime imaging of calcium using Quin-2.

We describe the use of a new imaging technology, fluorescence lifetime imaging (FLIM), for the imaging of the calcium concentrations based on the fluorescence lifetime of a calcium indicator. The fluorescence lifetime of Quin-2 is shown to be highly sensitive to [Ca2+]. We create two-dimensional lifetime images using the phase shift and modulation of the Quin-2 in response to intensity-modulated light. The two-dimensional phase and modulation values are obtained using a gain-modulated image intensifier and a slow-scan CCD camera. The lifetime values in the 2D image were verified using standard frequency-domain measurements. Importantly, the FLIM method does not require the probe to display shifts in the excitation or emission spectra, which may allow Ca2+ imaging using other Ca2+ probes not in current widespread use due to the lack of spectral shifts. Fluorescence lifetime imaging can be superior to stationary (steady-state) imaging because lifetimes are independent of the local probe concentration and/or intensity, and should thus be widely applicable to chemical imaging using fluorescence microscopy.     

Parallelized shifted‐excitation Raman difference spectroscopy for fluorescence rejection in a temporary varying system

A fluorescence background is one of the common interference factors of the Raman spectroscopic analysis in the biology field. Shifted‐excitation Raman difference spectroscopy (SERDS), in which a slow (typically 1 Hz) modulation to excitation wavelength is coupled with a sequential acquisition of alternating shifted‐excitation spectra, has been used to separate Raman scattering from excitation‐shift insensitive background. This sequential method is susceptible to spectral change and thus is limited only to stable samples. We incorporated a fast laser modulation (200 Hz) and a mechanical streak camera into SERDS to effectively parallelize the SERDS measurement in a single exposure. The developed system expands the scope of SERDS to include temporary varying system. The proof of concept is demonstrated using highly fluorescent samples, including living algae. Quantitative performance in fluorescence rejection and the robustness of the method to the dynamic spectral change during the measurement are manifested.      

A readily usable two‐photon fluorescence lifetime microendoscope

A two‐photon fluorescence lifetime (2P‐FLIM) microendoscope, capable of energetic metabolism imaging through the intracellular nicotinamide adenine dinucleotide (NADH) autofluorescence, at sub‐cellular resolution, is demonstrated. It exhibits readily usable characteristics such as convenient endoscope probe diameter (≈2 mm), fiber length (>5 m) and data accumulation rate (16 frames per second (fps)), leading to a FLIM refreshing rate of ≈0.1 to 1 fps depending on the sample. The spiral scanning image formation does not influence the instrument response function (IRF) characteristics of the system. Near table‐top microscope performances are achieved through a comprehensive system including a home‐designed spectro‐temporal pulse shaper and a custom air‐silica double‐clad photonic crystal fiber, which enables to reach up to 40 mW of ≈100 fs pulses @ 760 nm with a 80 MHz repetition rate. A GRadient INdex (GRIN) lens provides a lateral resolution of 0.67 μm at the focus of the fiber probe. Intracellular NADH fluorescence lifetime data are finally acquired on cultured cells at 16 fps.      

Investigating the healing mechanisms of an angiogenesis‐promoting topical treatment for diabetic wounds using multimodal microscopy

Impaired skin wound healing is a significant comorbid condition of diabetes that is caused by poor microcirculation, among other factors. Studies have shown that angiogenesis, a critical step in the wound healing process in diabetic wounds, can be promoted under hypoxia. In this study, an angiogenesis‐promoting topical treatment for diabetic wounds, which promotes angiogenesis by mimicking a hypoxic environment via inhibition of prolyl hydroxylase resulting in elevation or maintenance of hypoxia‐inducible factor, was investigated utilizing a custom‐built multimodal microscopy system equipped with phase‐variance optical coherence tomography (PV‐OCT) and fluorescence lifetime imaging microscopy (FLIM). PV‐OCT was used to track the regeneration of the microvasculature network, and FLIM was used to assess the in vivo metabolic response of mouse epidermal keratinocytes to the treatment during healing. Results show a significant decrease in the fluorescence lifetime of intracellular reduced nicotinamide adenine dinucleotide, suggesting a hypoxic‐like environment in the wounded skin, followed by a quantitative increase in blood vessel density assessed by PV‐OCT. Insights gained in these studies could lead to new endpoints for evaluation of the efficacy and healing mechanisms of wound‐healing drugs in a setting where delayed healing does not permit available methods for evaluation to take place.      

FLIM on a wide field fluorescence microscope

Summary FLIM (Fluorescence Lifetime Imaging Microscopy) is a new tool to detect interaction between proteins. The proteins under investigation are fused with fluorescent donor and acceptor molecules. Interaction between the two proteins is accompanied by direct energy transfer from donor to acceptor (FRET), resulting in a shorter lifetime of the fluorescence emitted by the donor molecule. This change in lifetime is detected by FLIM. Fluorescence lifetime imaging can now be done on a widefield fluorescence microscope by using an attachment that is easy to install and simple to operate. The new LIFA attachment is equipped to use different excitation sources. High brightness modulated LEDs as well as lasers modulated by an Accousto Optical Modulator can be used as excitation light source. A modulated image intensifier with digital camera is used as a detector. Power supplies and signal generator are integrated in one control unit that is connected to the light source, detector and computer. All parameters for image acquisition, processing and viewing are easy accessible in the user interface of the software package that uses a modular structure. Lifetime images showing FRET in MCF7 cells with ErbB1-GFP as donor and Py72/Cy3 as acceptor that were taken at EMBL, Heidelberg are shown.     

Dual light‐emitting diode‐based multichannel microscopy for whole‐slide multiplane,multispectral and phase imaging

We report the development of a multichannel microscopy for whole‐slide multiplane, multispectral and phase imaging. We use trinocular heads to split the beam path into 6 independent channels and employ a camera array for parallel data acquisition, achieving a maximum data throughput of approximately 1 gigapixel per second. To perform single‐frame rapid autofocusing, we place 2 near‐infrared light‐emitting diodes (LEDs) at the back focal plane of the condenser lens to illuminate the sample from 2 different incident angles. A hot mirror is used to direct the near‐infrared light to an autofocusing camera. For multiplane whole‐slide imaging (WSI), we acquire 6 different focal planes of a thick specimen simultaneously. For multispectral WSI, we relay the 6 independent image planes to the same focal position and simultaneously acquire information at 6 spectral bands. For whole‐slide phase imaging, we acquire images at 3 focal positions simultaneously and use the transport‐of‐intensity equation to recover the phase information. We also provide an open‐source design to further increase the number of channels from 6 to 15. The reported platform provides a simple solution for multiplexed fluorescence imaging and multimodal WSI. Acquiring an instant focal stack without z‐scanning may also enable fast 3‐dimensional dynamic tracking of various biological samples.      

Toward the measurement of multiple fluorescence lifetimes in flow cytometry: maximizing multi‐harmonic content from cells and microspheres

Flow cytometry is a powerful means for in vitro cellular analyses where multi‐fluorescence and multi‐angle light scattering can indicate unique biochemical or morphological features of single cells. Yet, to date, flow cytometry systems have lacked the ability to capture complex fluorescence dynamics due to the transient nature of flowing cells. In this contribution we introduce a simple approach for measuring multiple fluorescence lifetimes from a single cytometric event. We leverage square wave modulation, Fourier analysis, and high frequency digitization and show the ability to resolve more than one fluorescence lifetime from fluorescently‐labelled cells and microspheres.

Illustration of a flow cytometer capable of capturing multiple fluorescence lifetime measurements; creating potential for multi‐parametric, time‐resolved signals to be captured for every color channel.     

A flexible wide‐field FLIM endoscope utilising blue excitation light for label‐free contrast of tissue

Fluorescence lifetime imaging (FLIM) has previously been shown to provide contrast between normal and diseased tissue. Here we present progress towards clinical and preclinical FLIM endoscopy of tissue autofluorescence, demonstrating a flexible wide‐field endoscope that utilised a low average power blue picosecond laser diode excitation source and was able to acquire ～mm‐scale spatial maps of autofluorescence lifetimes from fresh ex vivo diseased human larynx biopsies in ～8 seconds using an average excitation power of ～0.5 mW at the specimen. To illustrate its potential for FLIM at higher acquisition rates, a higher power mode‐locked frequency doubled Ti:Sapphire laser was used to demonstrate FLIM of ex vivo mouse bowel at up to 2.5 Hz using 10 mW of average excitation power at the specimen. (© 2014 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Large‐depth‐of‐field full‐field optical angiography

A large‐depth‐of‐field full‐field optical angiography (LD‐FFOA) method is developed to expand the depth‐of‐field (DOF) using a contrast pyramid fusion algorithm (CPFA). The absorption intensity fluctuation modulation effect is utilized to obtain full‐field optical angiography (FFOA) images at different focus positions. The CPFA is used to process these FFOA images with different focuses. By selecting high‐contrast areas, the CPFA can highlight the characteristics and details of blood vessels to obtain LD‐FFOA images. In the optimal case of the proposed method, the DOF for FFOA is more than tripled using 10 differently focused FFOA images. Both the phantom and animal experimental results show that the LD‐FFOA resolves FFOA defocusing issues induced by surface and thickness inhomogeneities in biological samples. The proposed method can be potentially applied to practical biological experiments.      

Fluorescence lifetime imaging in an optically sectioning programmable array microscope (PAM).

BACKGROUND: The programmable array microscopes (PAMs) are a family of instruments incorporating arbitrary control of the patterns of illumination and/or detection. The PAM can be used in sectioning and nonsectioning modes, thereby constituting a useful platform for fluorescence lifetime imaging. METHODS AND RESULTS: We used a PAM for acquisition of optically sectioned and widefield fluorescence lifetime images, in which contrast was increased predominantly by suppressing out-of-focus light contributions. We simulate, display, and discuss the effects of blurring and fluorophore heterogeneity on lifetime imaging in widefield and confocal configurations. CONCLUSION: Sectioning improves the quality of lifetime images of samples with multiple fluorophores or spatially varying F?rster resonance energy transfer.     

In vitro and in vivo phasor analysis of stoichiometry and pharmacokinetics using short‐lifetime near‐infrared dyes and time‐gated imaging

We introduce a simple new approach for time‐resolved multiplexed analysis of complex systems using near‐infrared (NIR) dyes, applicable to in vitro and in vivo studies. We show that fast and precise in vitro quantification of NIR fluorophores' short (subnanosecond) lifetime and stoichiometry can be done using phasor analysis, a computationally efficient and user‐friendly representation of complex fluorescence intensity decays obtained with pulsed laser excitation and time‐gated camera imaging. We apply this approach to the study of binding equilibria by Förster resonant energy transfer using two different model systems: primary/secondary antibody binding in vitro and ligand/receptor binding in cell cultures. We then extend it to dynamic imaging of the pharmacokinetics of transferrin engagement with the transferrin receptor in live mice, elucidating the kinetics of differential transferrin accumulation in specific organs, straightforwardly differentiating specific from nonspecific binding. Our method, implemented in a freely‐available software, has the advantage of time‐resolved NIR imaging, including better tissue penetration and background‐free imaging, but simplifies and considerably speeds up data processing and interpretation, while remaining quantitative. These advances make this method attractive and of broad applicability for in vitro and in vivo molecular imaging and could be extended to applications as diverse as image‐guided surgery or optical tomography.      

The squared distance approach to frequency domain time‐resolved fluorescence analysis

A frequency‐domain (FD) analysis of fluorescence lifetime (FLT) is a unique and rapid method for cellular and intracellular classifications that can serve for medical diagnostics purposes. Nevertheless, its data analysis process demands nonlinear fitting algorithms that may distort the resolution of the FLT data and hence diminish the classification ability of the method. This research suggests a sample classification technique that is unaffected by the analysis process as it is based on the squared distance (D²) between the raw frequency response data (FRD). In addition, it presents the theory behind this technique and its validation in two simulated data sets of six groups with similar widely and closely spaced FLT data as well as in experimental data of 43 samples from bacterial and viral infected and non‐infected patients. In the two simulated tests, the classification accuracy was above 95% for all six groups. In the experimental data, the classification of 41 out of 43 samples matched earlier report and 29 out of 31 agreed with preliminary physician diagnosis. The D² approach has the potential to promote FD‐time resolved fluorescence measurements as a medical diagnostic technique with high specifity and high sensitivity for many of today's conventional diagnostic procedures.      

Quantifying intracellular equilibrium dissociation constants using single‐channel time‐resolved FRET

Quantification of the intracellular equilibrium dissociation constant of the interaction, K_d, is challenging due to the variability of the relative concentrations of the interacting proteins in the cell. Fluorescence lifetime imaging microscopy (FLIM) of the donor provides an accurate measurement of the molecular fraction of donor involved in FRET, but the fraction of bound acceptor is also needed to reliably estimate K_d. We present a method that exploits the spectroscopic properties of the widely used eGFP – mCherry FRET pair to rigorously determine the intracellular K_d based on imaging the fluorescence lifetime of only the donor (single‐channel FLIM). We have assessed the effect of incomplete labelling and determined its range of application for different K_d using Monte Carlo simulations. We have demonstrated this method estimating the intracellular K_d for the homodimerisaton of the oncogenic protein 3‐phosphoinositide‐dependent kinase 1 (PDK1) in different cell lines and conditions, revealing a competitive mechanism for its regulation. The measured intracellular K_d was validated against in‐vitro data. This method provides an accurate and generic tool to quantify protein interactions in situ.

     

Fluorescence life‐time imaging and steady state polarization for examining binding of fluorophores to gold nanoparticles

Nanocomposites as multifunctional agents are capable of combing imaging and cell biology technologies. The conventional methods used for validation of the conjugation process of nanoparticles (NPs) to fluorescent molecules such as spectroscopy analysis and surface potential measurements, are not sufficient. In this paper we present a new and highly sensitive procedure that uses the combination of (1) fluorescence spectrum, (2) fluorescence lifetime, and (3) steady state fluorescence polarization measurements. We characterize and analyze gold NPs with Lucifer yellow (LY) surface coating as a model. We demonstrate the ability to differentiate between LY‐GNP (the conjugated complex) and a mixture of coated NP and free dyes. We suggest the approach for neuroscience applications where LY is used for detecting and labeling cells, studying morphology and intracellular communications.

Histograms of Fluorescence lifetime imaging (FLIM) of free LY dye (Left) in comparison to the conjugated dye to gold nanoparticles, LY‐GNP (Middle) enable the differentiation between LY‐GNP (the conjugated complex) and a mixture of coated NP and free dyes (Right).