Low-cost, frequency-domain, fluorescence lifetime confocal microscopy

We describe the theory and implementation of a frequency‐domain fluorescence lifetime confocal microscope using switched diode laser illumination. Standard, communications‐type, radio‐frequency electronics are used to provide inexpensive modulation references and to perform phase‐sensitive detection. This allows the rapid acquisition of fluorescence intensity and lifetime images and their display in real time. We show fluorescence lifetime images of bead objects and fluorescence lifetime images of biological specimens from a single confocal scan.     

A series of flexible design adaptations to the Nikon E‐C1 and E‐C2 confocal microscope systems for UV,multiphoton and FLIM imaging

Multiphoton microscopy is widely employed in the life sciences using extrinsic fluorescence of low‐ and high‐molecular weight labels with excitation and emission spectra in the visible and near infrared regions. For imaging of intrinsic and extrinsic fluorophores with excitation spectra in the ultraviolet region, multiphoton excitation with one‐ or two‐colour lasers avoids the need for ultraviolet‐transmitting excitation optics and has advantages in terms of optical penetration in the sample and reduced phototoxicity. Excitation and detection of ultraviolet emission around 300 nm and below in a typical inverted confocal microscope is more difficult and requires the use of expensive quartz optics including the objective. In this technical note we describe the adaptation of a commercial confocal microscope (Nikon, Japan E‐C1 or E‐C2) for versatile use with Ti‐sapphire and OPO laser sources and the addition of a second detection channel that enables detection of ultraviolet fluorescence and increases detection sensitivity in a typical fluorescence lifetime imaging microscopy experiment. Results from some experiments with this setup illustrate the resulting capabilities.     

Compact non‐contact total emission detection for in vivo multiphoton excitation microscopy

We describe a compact, non‐contact design for a total emission detection (c‐TED) system for intra‐vital multiphoton imaging. To conform to a standard upright two‐photon microscope design, this system uses a parabolic mirror surrounding a standard microscope objective in concert with an optical path that does not interfere with normal microscope operation. The non‐contact design of this device allows for maximal light collection without disrupting the physiology of the specimen being examined. Tests were conducted on exposed tissues in live animals to examine the emission collection enhancement of the c‐TED device compared to heavily optimized objective‐based emission collection. The best light collection enhancement was seen from murine fat (5×–2× gains as a function of depth), whereas murine skeletal muscle and rat kidney showed gains of over two and just under twofold near the surface, respectively. Gains decreased with imaging depth (particularly in the kidney). Zebrafish imaging on a reflective substrate showed close to a twofold gain throughout the entire volume of an intact embryo (approximately 150 μm deep). Direct measurement of bleaching rates confirmed that the lower laser powers, enabled by greater light collection efficiency, yielded reduced photobleaching in vivo. The potential benefits of increased light collection in terms of speed of imaging and reduced photo‐damage, as well as the applicability of this device to other multiphoton imaging methods is discussed.     

Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution

In this paper a detailed discussion is presented of the factors that affect the fluorescence lifetime imaging performance of a scanning microscope equipped with a single photon counting based, two‐ to eight‐channel, time‐gated detection system. In particular we discuss the sensitivity, lifetime resolution, acquisition speed, and the shortest lifetimes that can be measured. Detection systems equipped with four to eight time‐gates are significantly more sensitive than the two time‐gate system. Only minor sensitivity differences were found between systems with four or more time‐gates. Experiments confirm that the lifetime resolution is dominated by photon statistics. The time response of the detector determines the shortest lifetimes that can be resolved; about 25 ps for fast MCP‐PMTs and 300–400 ps for other detectors. The maximum count rate of fast MCP‐PMTs, however, is 10–100 times lower than that of fast PMTs. Therefore, the acquisition speed with MCP‐PMT based systems is limited. With a fast PMT operated close to its maximum count rate we were able to record a fluorescence lifetime image of a beating myocyte in less than one second.     

Simultaneous confocal lifetime imaging of multiple fluorophores using the intensity-modulated multiple-wavelength scanning (IMS) technique

We demonstrate the simultaneous recording of confocal lifetime images of multiple fluorophores. The confocal microscope used in the study combines intensity-modulated laser illumination, lock-in detection and spectral separation of the fluorescent light. A theoretical investigation is presented that describes how the signal-to-noise ratio (SNR) depends on various factors such as modulation frequency, degree of modulation and number of detected photons. Theory predicts that, compared with ordinary intensity images, lifetime images will have a SNR that is, at best, approximately four times lower. Experimental results are presented that confirm this prediction.     

Optimized temporal response in multichannel two-photon fluorescence lifetime microscopy using a photonic crystal fibre

The integration of fibre optics into an imaging system for the convenient delivery and collection of light has resulted in many hybrid forms of novel biomedical optical instrumentation. Although it is extremely robust and cost effective, fibre integration requires special consideration in a time‐domain fluorescence lifetime imaging schema where multipath propagation in the fibre causes significant spread in photon transit times. In this study, we investigated the effect of the length of a multimode collection fibre on the temporal performance of a multichannel fluorescence lifetime microscope and demonstrated the effectiveness of a photonic crystal fibre as a means of optimizing the collection and delivery of emitted fluorescence in terms of temporal resolution. The findings are pertinent to all studies that employ a multimode optical fibre to collect and deliver an emitted fluorescence signal from a sample to a remote detector for measurement of the characteristic fluorescence lifetime.     

Compact multiphoton/single photon laser scanning microscope for spectral imaging and fluorescence lifetime imaging

An inverted fluorescence microscope was upgraded into a compact three-dimensional laser scanning microscope (LSM) of 65 x 62 x 48 cm dimensions by means of a fast kHz galvoscanner unit, a piezodriven z-stage, and a picosecond (ps) 50 MHz laser diode at 405 nm. In addition, compact turn-key near infrared femtosecond lasers have been employed to perform multiphoton fluorescence and second harmonic generation (SHG) microscopy. To expand the features of the compact LSM, a time-correlated single photon counting unit as well as a Sagnac interferometer have been added to realize fluorescence lifetime imaging (FLIM) and spectral imaging. Using this unique five-dimensional microscope, TauMap, single-photon excited (SPE), and two-photon excited (TPE) cellular fluorescence as well as intratissue autofluorescence of water plant leaves have been investigated with submicron spatial resolution, <270 ps temporal resolution, and 10 nm spectral resolution.     

Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation

A scanning microscope utilizing two-photon excitation in combination with fluorescence lifetime contrast is presented. The microscope makes use of a tunable femtosecond titanium:sapphire laser enabling the two-photon excitation of a broad range of fluorescent molecules, including UV probes. Importantly, the penetration depth of the two-photon exciting (infra)red light is substantially greater than for the corresponding single-photon wavelength while photobleaching is significantly reduced. The time structure of the Ti:Sa laser can be employed in a straightforward way for the realization of fluorescence lifetime imaging. The fluorescence lifetime is sensitive to the local environment of the fluorescent molecule. This behaviour can be used for example to quantify concentrations of ions, such as pH and Ca²⁺, or pO₂ and pCO₂. In the set-up presented here the fluorescence lifetime imaging is accomplished by time-gated single photon counting. The performance and optical properties of the microscope are investigated by a number of test measurements on fluorescent test beads. Point-spread functions calculated from measurements on 230-nm beads using an iterative restoration procedure compare well with theoretical expectations. Lifetime imaging experiments on a test target containing two different types of test bead in a fluorescent buffer all with different lifetimes (2.15 ns, 2.56 ns and 3.34 ns) show excellent quantitative agreement with reference values obtained from time correlated single photon counting measurements. Moreover, the standard deviation in the results can be wholly ascribed to the photon statistics. Measurements of acridine orange stained biofilms are presented as an example of the potential of two-photon excitation combined with fluorescence lifetime contrast. Fluorescence lifetime and intensity images were recorded over the whole sample depth of 100 μm. Fluorescence intensity imaging is seriously hampered by the rapid decrease of the fluorescence signal as a function of the depth into the sample. Fluorescence lifetime imaging on the other hand is not affected by the decrease of the fluorescence intensity.     

Generalization of the polar representation in time domain fluorescence lifetime imaging microscopy for biological applications: practical implementation

The polar representation or phasor, which provides a fast and visual indication on the number of exponentials present in the intensity decay of the fluorescence lifetime images is increasingly used in time domain fluorescence lifetime imaging microscopy experiments. The calculations of the polar coordinates in time domain fluorescence lifetime imaging microscopy experiments involve several experimental parameters (e.g. instrumental response function, background, angular frequency, number of temporal channels) whose role has not been exhaustively investigated. Here, we study theoretically, computationally and experimentally the influence of each parameter on the polar calculations and suggest parameter optimization for minimizing errors. We identify several sources of mistakes that may occur in the calculations of the polar coordinates and propose adapted corrections to compensate for them. For instance, we demonstrate that the numerical integration method employed for integrals calculations may induce errors when the number of temporal channels is low. We report theoretical generalized expressions to compensate for these deviations and conserve the semicircle integrity, facilitating the comparison between fluorescence lifetime imaging microscopy images acquired with distinct channels number. These theoretical generalized expressions were finally corroborated with both Monte Carlo simulations and experiments.     

Dynamic behavior of amplitude detection Kelvin force microscopy in ultrahigh vacuum

The acquisition rate of all scanning probe imaging techniques with feedback control is limited by the dynamic response of the control loops. Performance criteria are the control loop bandwidth and the output signal noise power spectral density. Depending on the acceptable noise level, it may be necessary to reduce the sampling frequency below the bandwidth of the control loop. In this work, the frequency response of a vacuum Kelvin force microscope with amplitude detection (AM-KFM) using a digital signal processing (DSP) controller is characterized and optimized. Then, the main noise source and its impact on the output signal is identified. A discussion follows on how the system design can be optimized with respect to output noise. Furthermore, the interaction between Kelvin and distance control loop is studied, confirming the beneficial effect of KFM on topography artefact reduction in the frequency domain. The experimental procedure described here can be generalized to other systems and allows to locate the performance limitations.     

A scanning near-field optical microscope having scanning electron tunnelling microscope capability using a single metallic probe tip

A new microscope system that has the combined capabilities of a scanning near-field optical microscope (SNOM) and a scanning tunnelling microscope (STM) is described. This is achieved with the use of a single metallic probe tip. The distance between the probe tip and the sample surface is regulated by keeping the tunnelling current constant. In this mode of operation, information about the optical properties of the sample, such as its refractive index distribution and absorption characteristics, can be disassociated from the information describing its surface structure. Details of the surface structure can be studied at resolutions smaller than the illumination wavelength. The performance of the microscope is evaluated by analysing a grating sample that was made by coating a glass substrate with gold. The results are then compared with the corresponding SNOM and STM images of the grating.     

A versatile multipurpose scanning probe microscope

A combined scanning probe microscope has been developed that allows simultaneous operation as a non‐contact/tapping mode atomic force microscope, a scattering near‐field optical microscope, and a scanning tunnelling microscope on conductive samples. The instrument is based on a commercial optical microscope. It operates with etched tungsten tips and exploits a tuning fork detection system for tip/sample distance control. The system has been tested on a p‐doped silicon substrate with aluminium depositions, being able to discriminate the two materials by the electrical and optical images with a lateral resolution of 130 nm.     

Three dimensional image restoration in fluorescence lifetime imaging microscopy

A microscope set-up and numerical methods are described which enable the measurement and reconstruction of three-dimensional nanosecond fluorescence lifetime images in every voxel. The frequency domain fluorescence lifetime imaging microscope (FLIM) utilizes phase detection of high-frequency modulated light by homodyne mixing on a microchannel plate image intensifier. The output signal at the image intensifier's phosphor screen is integrated on a charge coupled device camera. A scanning stage is employed to obtain a series of phase-dependent intensity images at equally separated depths in a specimen. The Fourier transform of phase-dependent data gives three-dimensional (3D) images of the Fourier coefficients. These images are deblurred using an Iterative Constrained Tikhonov–Miller (ICTM) algorithm in conjunction with a measured point spread function. The 3D reconstruction of fluorescence lifetimes are calculated from the deblurred images of the Fourier coefficients. An improved spatial and temporal resolution of fluorescence lifetimes was obtained using this approach to the reconstruction of simulated 3D FLIM data. The technique was applied to restore 3D FLIM data of a live cell specimen expressing two green fluorescent protein fusion constructs having distinct fluorescence lifetimes which localized to separate cellular compartments.     

A white light confocal microscope for spectrally resolved multidimensional imaging

Spectrofluorometric imaging microscopy is demonstrated in a confocal microscope using a supercontinuum laser as an excitation source and a custom‐built prism spectrometer for detection. This microscope system provides confocal imaging with spectrally resolved fluorescence excitation and detection from 450 to 700 nm. The supercontinuum laser provides a broad spectrum light source and is coupled with an acousto‐optic tunable filter to provide continuously tunable fluorescence excitation with a 1‐nm bandwidth. Eight different excitation wavelengths can be simultaneously selected. The prism spectrometer provides spectrally resolved detection with sensitivity comparable to a standard confocal system. This new microscope system enables optimal access to a multitude of fluorophores and provides fluorescence excitation and emission spectra for each location in a 3D confocal image. The speed of the spectral scans is suitable for spectrofluorometric imaging of live cells. Effects of chromatic aberration are modest and do not significantly limit the spatial resolution of the confocal measurements.     

Integration of a high‐NA light microscope in a scanning electron microscope

We present an integrated light‐electron microscope in which an inverted high‐NA objective lens is positioned inside a scanning electron microscope (SEM). The SEM objective lens and the light objective lens have a common axis and focal plane, allowing high‐resolution optical microscopy and scanning electron microscopy on the same area of a sample simultaneously. Components for light illumination and detection can be mounted outside the vacuum, enabling flexibility in the construction of the light microscope. The light objective lens can be positioned underneath the SEM objective lens during operation for sub‐10 μm alignment of the fields of view of the light and electron microscopes. We demonstrate in situ epifluorescence microscopy in the SEM with a numerical aperture of 1.4 using vacuum‐compatible immersion oil. For a 40‐nm‐diameter fluorescent polymer nanoparticle, an intensity profile with a FWHM of 380 nm is measured whereas the SEM performance is uncompromised. The integrated instrument may offer new possibilities for correlative light and electron microscopy in the life sciences as well as in physics and chemistry.     

Investigation of Bloch wall fine structures by magnetic force microscopy

Using a magnetic force microscope (MFM), measurements have been performed on single crystal iron whiskers. These samples exhibit a comparatively simple magnetic domain structure and a high degree of crystallographic perfection. STM measurements yield an average surface corrugation of about 3 nm. These nearly ideal boundary conditions permit an investigation of the undistorted surface configuration of Bloch walls in a cubic bulk crystal. Interactions between the stray field configuration of the sample and a ferromagnetic microscope tip have been measured with a new MFM device which is based on direct compliance detection. Measurements have been performed in the constant-current mode, as well as in the constant-compliance, mode. Control observation by means of the Kerr magneto-optic effect permit an interpretation of the obtained MFM data in terms of isolated 180° Bloch walls. The fine structure of these walls has been analysed and compared with results obtained from model calculations.     

Frequency-domain fluorescence microscopy with the LED as a light source

We describe a frequency-domain lifetime fluorometer based on a microscope and a modulated light-emitting diode (LED) excitation source (370/460 nm), which operates in the frequency range 120 Hz–250 MHz. We collected multifrequency phase and modulation fluorescence responses from cellular areas as small as 10–15 µm in diameter. We also collected fluorescence lifetime data from cells stained by a lipophilic coumarin sensitized europium fluorophore, Coum-Eu, with a millisecond lifetime, and Ru(bpy)₂phe-C₁₂, with microsecond lifetime. Nanosecond lifetimes from native nuclei stained with SYTO 14 and SYTO 16 probes were measured as well. We demonstrate that a simple LED excitation source can, for many applications, successfully replace complex and expensive laser systems, which have been used for cellular frequency-domain lifetime measurements. As the LEDs are very stable with low noise, it will be possible to image even smaller sample areas using brighter LEDs. With availability of modulated LEDs emitting at several wavelengths covering almost the entire visible spectrum it is easy to assemble a system for the fluorophore of choice. The ability to select an excitation source for a given fluorophore and low price make such an excitation source even more practical.     

Multifunctional imaging of endogenous contrast by simultaneous nonlinear and optical coherence microscopy of thick tissues

A variety of high resolution optical microscopy techniques have been developed in recent years for basic and clinical studies of biological systems. We demonstrate a trimodal microscope combining optical coherence microscopy (OCM) with two forms of nonlinear microscopy, namely two-photon excited fluorescence (2PF) and second harmonic generation (SHG), for imaging turbid media. OCM combines the advantages of confocal detection and coherence gating for structural imaging in highly scattering tissues. Nonlinear microscopy enables the detection of biochemical species, such as elastin, NAD(P)H, and collagen. While 2PF arises from nonlinear excitation of fluorescent species, SHG is a form of nonlinear scattering observed in materials that lack a center of inversion symmetry, such as type I collagen. Characterization of the microscope showed nearly diffraction-limited spatial resolution in all modalities. Images were obtained in fish scales and excised human skin samples. The primary endogenous sources of contrast in the dermis were due to elastin autofluorescence and collagen SHG. Multimodal microscopy allows the simultaneous visualization of structural and functional information of biological systems.     

Consequences of the background in piezoresponse force microscopy on the imaging of ferroelectric domain structures

The interpretation of ferroelectric domain images obtained with a piezoresponse force microscope (PFM) is discussed. The influence of an inherent experimental background on the domain contrast in PFM images (enhancement, nulling, inversion) as well as on the shape and the location of the domain boundaries are described. We present experimental results to evidence our analysis of the influence of the background on the domain contrast in PFM images.     

A novel fluorescence lifetime imaging system that optimizes photon efficiency

Fluorescence lifetime imaging (FLIM) is a powerful microscopy technique for providing contrast of biological and other systems by differences in molecular species or their environments. However, the cost of equipment and the complexity of data analysis have limited the application of FLIM. We present a mathematical model and physical implementation for a low cost digital frequency domain FLIM (DFD-FLIM) system, which can provide lifetime resolution with quality comparable to time-correlated single photon counting methods. Our implementation provides data natively in the form of phasors. On the basis of the mathematical model, we present an error analysis that shows the precise parameters for maximizing the quality of lifetime acquisition, as well as data to support this conclusion. The hardware and software of the proposed DFD-FLIM method simplifies the process of data acquisition for FLIM, presents a new interface for data display and interpretation, and optimizes the accuracy of lifetime determination.