Two-photon excited fluorescence enhancement with broadband versus tunable femtosecond laser pulse excitation

The inverse relationship between two-photon excited fluorescence (TPEF) and laser pulse duration suggests that two-photon microscopy (TPM) performance may be improved by decreasing pulse duration. However, for ultrashort pulses of sub-10 femtosecond (fs) in duration, its spectrum contains the effective gain bandwidth of Ti:Sapphire and its central wavelength is no longer tunable. An experimental study was performed to explore this apparent tradeoff between untuned sub-10 fs transform-limited pulse (TLP) and tunable 140 fs pulse for TPEF. Enhancement factors of 1.6, 6.7, and 5.2 are measured for Indo-1, FITC, and TRITC excited by sub-10 fs TLP compared with 140 fs pulse tuned to the two-photon excitation (TPE) maxima at 730 nm, 800 nm, and 840 nm, respectively. Both degenerate (v(1) = v(2)) and nondegenerate (v(1) ≠ v(2)) mixing of sub-10 fs TLP spectral components result in its broad second-harmonic (SH) power spectrum and high spectral density, which can effectively compensate for the lack of central wavelength tuning and lead to large overlap with dye TPE spectra for TPEF enhancements. These pulse properties were also exploited for demonstrating its potential applications in multicolor imaging with TPM.     

Imaging of Caenorhabditis elegans neurons by second-harmonic generation and two-photon excitation fluorescence

Second-harmonic generation (SHG) and two-photon excitation fluorescence (TPEF) are relatively new and promising tools for the detailed imaging of biological samples and processes at the microscopic level. By exploiting these nonlinear phenomena phototoxicity and photobleaching effects on the specimens are reduced dramatically. The main target of this work was the development of a compact inexpensive and reliable experimental apparatus for nonlinear microscopy measurements. Femtosecond laser pulses were utilized for excitation. We achieved high-resolution imaging and mapping of Caenorhabditis elegans (C. elegans) neurons and muscular structures of the pharynx, at the microscopic level by performing SHG and TPEF measurements. By detecting nonlinear phenomena such as SHG and TPEF it is feasible to extract valuable information concerning the structure and the function of nematode neurons.     

Combined multiphoton microscopy and optical coherence tomography using a 12-fs broadband source

A 12-fs broadband (100-nm) source is used to combine multiphoton microscopy (MPM) and optical coherence tomography (OCT) in a single platform. An ultrafast Ti:sapphire laser simultaneously provides short pulses necessary for efficient MPM excitation and the broad bandwidth required for high-resolution OCT. Using 0.3-microm microspheres and a 63x, 0.95 numerical aperture objective, we demonstrate that MPM and OCT channels are coregistered with lateral resolution of approximately 0.5 microm and axial resolution of approximately 1.5 microm. Preliminary studies of a 3-D organotypic epithelial tissue model show that multiphoton images of fluorescence and second harmonic signals are derived from cellular and extracellular matrix structures, respectively, while OCT images are generated from scattering interfaces due to tissue variations in refractive index. The combined MPM/OCT microscope is capable of providing simultaneous functional and structural information from cells and extracellular matrix and is potentially a powerful tool for studying biological processes in thick tissues.     

Biosonar behavior of mustached bats swung on a pendulum prior to cortical ablation

1. The biosonar signal (pulse) of the mustached bat, Pteronotus parnellii parnellii, has four harmonics (H1-4), each consisting of a long constant-frequency component (CF1-4) followed by a short frequency-modulated component (FM1-4). As the bat approaches a target, it systematically modifies its pulses to optimize the extraction of information from the echoes. These behavioral responses include 1) Doppler-shift (DS) compensation in which the bat adjusts the frequency of its pulses to correct for the DS in the echoes. This maintains the echo CF2 at a frequency to which the bat's cochlea is very sharply tuned, slightly above the CF2 frequency of the bat's pulses when it is at rest (Frest, approximately 61 kHz); 2) echo intensity compensation, in which the bat lowers its pulse intensity as it approaches a large target, thus maintaining the echo intensity within a suitable range for auditory processing; and 3) and 4) duration and rate adjustments, in which the bat first increases its pulse duration to facilitate target identification, then shortens its pulse duration while increasing its pulse rate to facilitate target analysis. 2. We examined these responses, especially DS compensation, by swinging bats on a pendulum toward a large target over a distance of 3.6 m. Eight bats were given 15-30 swings per day for 6-25 days. 1) On 97% of all swings the bats showed strong DS compensation as the pendulum approached the target. They did not show DS compensation on the backswing. 2) On 40-50% of all swings, the bats clearly displayed the other responses. The bats typically increased their pulse intensity a small amount early in the pendulum swing, then decreased pulse intensity by as much as 18 dB as the target was more closely approached. They increased their pulse intensity during the backswing. 3) Pulse duration increased from approximately 20 to 23 ms early in the forward swing, decreased to approximately 18 ms as the target was more closely approached, and then increased to 20 ms by the end of the backswing. 4) The instantaneous repetition rate increased from approximately 17 pulses/s at the start of the forward swing to approximately 28 pulses/s near the target, then decreased to approximately 10 pulses/s by the end of the backswing. Pulses usually occurred in trains of 1-2 pulses, with longer trains occasionally occurring near the target. 3. The maximum DS on the pendulum was 1.34 kHz, and the maximum DS compensation was 146 +/- 98 (SD) Hz less than this value.(ABSTRACT TRUNCATED AT 400 WORDS)     

Second-harmonic imaging microscopy of living cells

Second harmonic generation (SHG) has been developed in our laboratories as a high-resolution nonlinear optical imaging microscopy for cellular membranes and intact tissues. SHG shares many of the advantageous features for microscopy of another more established nonlinear optical technique: two-photon excited fluorescence (TPEF). Both are capable of optical sectioning to produce three-dimensional images of thick specimens and both result in less photodamage to living tissue than confocal microscopy. SHG is complementary to TPEF in that it uses a different contrast mechanism and is most easily detected in the transmitted light optical path. It can be used to image membrane probes with high membrane specificity and displays extraordinary sensitivity in reporting membrane potential; it also has the ability to image highly ordered structural proteins without any exogenous labels.     

Recognition of serous ovarian tumors in human samples by multimodal nonlinear optical microscopy

We used a multimodal nonlinear optics microscopy, specifically two-photon excited fluorescence (TPEF), second and third harmonic generation (SHG∕THG) microscopies, to observe pathological conditions of ovarian tissues obtained from human samples. We show that strong TPEF + SHG + THG signals can be obtained in fixed samples stained with hematoxylin and eosin (H&E) stored for a very long time, and that H&E staining enhanced the THG signal. We then used the multimodal TPEF-SHG-THG microscopies in a stored file of H&E stained samples of human ovarian cancer to obtain complementary information about the epithelium∕stromal interface, such as the transformation of epithelium surface (THG) and the overall fibrillary tissue architecture (SHG). This multicontrast nonlinear optics microscopy is able to not only differentiate between cancerous and healthy tissue, but can also distinguish between normal, benign, borderline, and malignant specimens according to their collagen disposition and compression levels within the extracellular matrix. The dimensions of the layers of epithelia can also be measured precisely and automatically. Our data demonstrate that optical techniques can detect pathological changes associated with ovarian cancer.     

Optical detection of intracellular cavitation during selective laser targeting of the retinal pigment epithelium: dependence of cell death mechanism on pulse duration

Selective laser targeting of the retinal pigment epithelium (RPE) is an attractive method for treating RPE-associated disorders. We are developing a method for optically detecting intracellular microcavitation that can potentially serve as an immediate feedback of the treatment outcome. Thermal denaturation or intracellular cavitation can kill RPE cells during selective targeting. We examined the cell damage mechanism for laser pulse durations from 1 to 40 micros ex vivo. Intracellular cavitation was detected as a transient increase in the backscattered treatment beam. Cavitation and cell death were correlated for individual cells after single-pulse irradiation. The threshold radiant exposures for cell death (ED(50,d)) and cavitation (ED(50,c)) increased with pulse duration and were approximately equal for pulses of up to 10 micros. For 20 micros, the ED(50,d) was about 10% lower than the ED(50,c); the difference increased with 40-micros pulses. Cells were killed predominantly by cavitation (up to 10-micros pulses); probability of thermally induced cell death without cavitation gradually increases with pulse duration. Threshold measurements are discussed by modeling the temperature distribution around laser-heated melanosomes and the scattering function from the resulting cavitation. Detection of intracellular cavitation is a highly sensitive method that can potentially provide real-time assessment of RPE damage during selective laser targeting.     

Inactivation of the asymmetrical displacement current in giant axons of Loligo forbesi.

1. Asymmetrical displacement currents ('gating currents') have been recorded in intracellularly perfused squid giant axons by averaging the currents associated with depolarizing and hyperpolarizing pulses. The relation between 'gating current' and Na inactivation was studied by investigating the effect of pulse duration and conditioning pulses. 2. Increasing the pulse duration from 0-3-1 msec to 10-20 msec reduced the off-response of the 'gating current' to 50-70% of its normal size; the time constant was 5 msec at +20 mV and 8 degrees C. The decrease of the Na current during a 10-20 msec pulse was stronger and faster; it decayed to 10-26% with a time constant of 1-35 msec. 3. The effect of pulse duration could also be demonstrated by using only depolarizing pulses. The charge displacement at the end of single or averaged depolarizing pulses was smaller for long pulse durations than for short. A long depolarizing pulse was followed by a small long-lasting tail of inward current. 4. A conditioning depolarizing pulse of 10-20 msec duration to a potential of -30 or +10 mV, followed by a short recovery period at -70 mV, decreased the on-response of the 'gating current'. Its size was reduced to 46-71% and 61-94%, respectively, for a recovery interval of 1-75 and 5 msec at 2-3 degrees C. The reduction of the Na current, measured under similar conditions, was more pronounced; the Na current was decreased to less than 50% of its normal value. 5. The observations about the effect of pulse duration and conditioning pulses on the 'gating current' are qualitatively consistent with those of Bezanilla & Armstrong (1974, 1975) and support the view that part of the asymmetrical charge displacement is inactivated during a 10-20 msec depolarization.     

Second harmonic imaging of membrane potential of neurons with retinal

We present a method to optically measure and image the membrane potential of neurons, using the nonlinear optical phenomenon of second harmonic generation (SHG) with a photopigment retinal as the chromophore [second harmonic retinal imaging of membrane potential (SHRIMP)]. We show that all-trans retinal, when adsorbed to the plasma membrane of living cells, can report on the local electric field via its change in SHG. Using a scanning mode-locked Ti-sapphire laser, we collect simultaneous two-photon excited fluorescence (TPEF) and SHG images of retinal-stained kidney cells and cultured pyramidal neurons. Patch clamp experiments on neurons stained with retinal show an increase of 25% in SHG intensity per 100-mV depolarization. Our data are the first demonstration of optical measurements of membrane potential of mammalian neurons with SHG. SHRIMP could have wide applicability in neuroscience and, by modifying rhodopsin, could in principle be subject for developing genetically engineered voltage sensors.     

Thresholds of intradental A- and C-nerve fibres in the cat to electrical current pulses of different duration

Electrical current pulses of quite variable duration have been used in activation of intradental nerves both in human subjects and experimental animals. It seems, however, that little information is available about the effect of pulse duration on the responses of single pulp nerve units. The aim of the present study was to investigate the effect of pulse duration on excitation thresholds of intradental A- and C-fibres in the cat. In 12 anesthetized cats 61 C- and 53 A-nerve units were identified and recorded. Electrical thresholds were determined with current pulses of different duration from 0.2 to 50.0 ms. The maximal stimulus intensity was 200 microA. Conduction velocities of all recorded units and absolute refractory periods of 20 A- and 20 C-units were determined. Intradental A- and C-fibres had different strength-duration properties. with all pulse durations A-fibres had the lowest thresholds. Part of the C-fibres did not respond to the shortest current pulses even with the maximum stimulus intensity (200 microA). with 0.2 ms pulses only 31.1% of the recorded C-fibres could be activated. In some A-fibres a single current pulse of long duration was capable of inducing several action potentials, when the stimulus intensity was increased suprathreshold. Refractory periods of A-units were less than 2.0 ms and those of C-units 5.0-9.0 ms. It is concluded that in electrical stimulation of teeth duration of current pulses strongly affects responses of single intradental fibre units.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two-photon laser scanning fluorescence microscopy using photonic crystal fiber

We report the application of a simple yet powerful modular pulse compression system based on photonic crystal fibers that improves on incumbent two-photon laser scanning fluorescence microscopy techniques. This system provides more than a sevenfold increase in fluorescence yield when compared with a commercial two-photon microscopy system. From this, we infer pulses of IR radiation of less than 35 fs duration reaching the sample.     

Thresholds of intradental A-and C-nerve fibres in the cat to electrical current pulses of different duration

Electrical current pulses of quite variable duration have been used in activation of intradental nerves both in human subjects and experimental animals. It seems, however, that little information is available about the effect of pulse duration on the responses of single pulp nerve units. The aim of the present study was to investigate the effect of pulse duration on excitation thresholds of intradental A-and C-fibres in the cat. In 12 anesthetized cats 61 C-and 53 A-nerve units were identified and recorded. Electrical thresholds were determined with current pulses of different duration from 0.2 to 50.0 ms. The maximal stimulus intensity was 200 μA.Conduction velocities of all recorded units and absolute refractory periods of 20 A-and 20 C-units were determined. Intradental A-and C-fibres had different strength-duration properties. With all pulse durations A-fibres had the lowest thresholds. Part of the C-fibres did not respond to the shortest current pulses even with the maximum stimulus intensity (200 μA).With 0.2 ms pulses only 31.1% of the recorded C-fibres could be activated. In some A-fibres a single current pulse of long duration was capable of inducing several action potentials, when the stimulus intensity was increased suprathreshold. Refractory periods of A-units were <2.0 ms and those of C-units 5.0–9.0 ms. It is concluded that in electrical stimulation of teeth duration of current pulses strongly affects responses of single intradental fibre units. With long pulses function of A-fibres is pronounced because of repetitive firing in some units. These properties of pulp nerves should also be considered, when electrical stimulation is applied to human teeth.     

Volumetric Reconstruction of the Mouse Meibomian Gland Using High‐Resolution Nonlinear Optical Imaging

Recent studies suggest that mouse meibomian glands (MG) undergo age‐related atrophy that mimics changes seen in age‐related human MG dysfunction (MGD). To better understand the structural/functional changes that occur during aging, this study developed an imaging approach to generate quantifiable volumetric reconstructions of the mouse MG and measure total gland, cell, and lipid volume. Mouse eyelids were fixed in 4% paraformaldehyde, embedded in LR White resin and serially sectioned. Sections were then scanned using a 20× objective and a series of tiled images (1.35 × 1.35 × 0.5 mm) with a pixel size of 0.44 μm lateral and 2 μm axial were collected using a Zeiss 510 Meta LSM and a femtosecond laser to simultaneously detect second harmonic generated (SHG) and two‐photon excited fluorescence (TPEF) signals from the tissue sections. The SHG signal from collagen was used to outline and generate an MG mask to create surface renderings of the total gland and extract relevant MG TPEF signals that were later separated into the cellular and lipid compartments. Using this technique, three‐dimensional reconstructions of the mouse MG were obtained and the total, cell, and lipid volume of the MG measured. Volumetric reconstructions of mouse MG showed loss of acini in old mice that were not detected by routine histology. Furthermore, older mouse MG had reduced total gland volume that is primarily associated with loss of the lipid volume. These findings suggest that mice MG undergo “dropout” of acini, similar to that which occurs in human age‐related MGD. Anat Rec, 2011. © 2010 Wiley‐Liss, Inc.     

Assessment of liver steatosis and fibrosis in rats using integrated coherent anti-Stokes Raman scattering and multiphoton imaging technique

We report the implementation of a unique integrated coherent anti-Stokes Raman scattering (CARS), second-harmonic generation (SHG), and two-photon excitation fluorescence (TPEF) microscopy imaging technique developed for label-free monitoring of the progression of liver steatosis and fibrosis generated in a bile duct ligation (BDL) rat model. Among the 21 adult rats used in this study, 18 rats were performed with BDL surgery and sacrificed each week from weeks 1 to 6 (n = 3 per week), respectively; whereas 3 rats as control were sacrificed at week 0. Colocalized imaging of the aggregated hepatic fats, collagen fibrils, and hepatocyte morphologies in liver tissue is realized by using the integrated CARS, SHG, and TPEF technique. The results show that there are significant accumulations of hepatic lipid droplets and collagen fibrils associated with severe hepatocyte necrosis in BDL rat liver as compared to a normal liver tissue. The volume of normal hepatocytes keeps decreasing and the fiber collagen content in BDL rat liver follows a growing trend until week 6; whereas the hepatic fat content reaches a maximum in week 4 and then appears to stop growing in week 6, indicating that liver steatosis and fibrosis induced in a BDL rat liver model may develop at different rates. This work demonstrates that the integrated CARS and multiphoton microscopy imaging technique has the potential to provide an effective means for early diagnosis and detection of liver steatosis and fibrosis without labeling.     

Asymmetrical displacement currents in the membrane of frog myelinated nerve: Early time course and effects of membrane potential

1. Asymmetrical displacement currents were studied in myelinated nerve fibres from Rana esculenta with a voltage clamp technique. 2. For brief pulses symmetrical with respect to a holding potential of--97mV, the asymmetry current flowing during pulses (on-response) exhibited a rising phase to a peak followed by an approximately exponential decline. After the pulses the rising phase in the off-response could not be resolved; the time constant varied about 2-fold with either size or duration of the pulse. 3. For longer pulses a second slower component could be detected both in on- and off-responses. 4. The rapidly declining on- and off-responses associated with brief pulses carried about the same charges Qon and Qoff. Increasing the duration of the pulse reduced Qoff. For all pulses tested Qoff approached about one fifth of Qmax. The reduction of Qoff was roughly characterised by time constants ranging between 1.5 and 0.5 ms for potentials between--25 and + 23 mV. Analysis of individual membrane currents confirmed that the capacity current after depolarizing pulses decreased with pulse length. 5. The effects of membrane potential on asymmetry current were studied by varying the level from which pulses were applied during 46.9ms prepulses in the range from--97 to--29mV. The fast and slow components of asymmetry current were affected differently. For potentials more positive than--90mV the fast on-response was reduced and reversed its sign at a potential 25mV more negative than the potential estimated from the steady-state charge distribution measured from--97mV.     

Response of cutaneous hair and field mechanoreceptors in cat to paired mechanical stimuli

1. While studying hair- and field-receptor responses to paired mechanical pulses, I observed that in approximately half the receptors the recovery of excitability following generation of an impulse took the form of a damped oscillation. 2. Oscillations could be modified or eliminated by changing the conditions of stimulation: 1) there were no oscillations with an electrical conditioning stimulus; 2) oscillations occurred only when both mechanical pulses were delivered to the surface of the skin, not when the conditioning pulse was given to a hair shaft, and only when both pulses affected the same part of the receptive field; 3) oscillation was reduced by increasing the duration of the pulses; 4) oscillation was enhanced by increasing the distance between the stimulator and the nerve terminal; and 5) maxima on the recovery curve became minima and vice versa when the direction of the conditioning pulse was changed. 3. It was concluded that recovery curve oscillations were due not to the type of hair or field receptor being stimulated, but to the conditions of stimulation. The timing of the oscillations was a function of the duration of the mechanical pulse. Furthermore, interaction was additive when the interval between stimuli was an even multiple of the pulse width. This resulted in a minimal point on the recovery curve. At odd multiples of the pulse width, the converse resulted. The evidence suggests that waves, set up in the skin by the paired mechanical pulses, interact and cause the recovery curve oscillations. Such waves could be set up by a variety of stimuli that contact the skin naturally.     

The statistical nature of the acetylcholine potential and its molecular components

1. When a steady dose of acetylcholine (ACh) is applied to an end-plate, the resulting depolarization is accompanied by a significant increase in voltage noise.2. The characteristic properties of this ACh noise (amplitude and time course) are examined under various experimental conditions. The voltage noise is analysed on the assumption that it arises from statistical fluctuations in reaction rate, and in the frequency of the elementary current pulses (;shot effects') produced by the action of ACh molecules.3. The elementary ACh current pulse (amplitude approximately 10(-11) A), arises from a conductance change of the order of 10(-10) Omega(-1) which lasts for approximately 1 ms (at 20 degrees C), and produces a minute depolarization, of the order of 0.3 muV. It is associated with a net charge transfer of nearly 10(-14) C, equivalent to approximately 5 x 10(4) univalent ions.4. At low temperature, and during chronic denervation, the duration of the elementary current pulse increases, and the elementary voltage change becomes correspondingly larger.5. Curare has little or no effect on the characteristics of the elementary event.6. A comparative study of ACh and carbachol actions shows that carbachol produces considerably briefer, and therefore less effective, current pulses than ACh.     

Improving the therapeutic window of retinal photocoagulation by spatial and temporal modulation of the laser beam

Decreasing the pulse duration helps confine damage, shorten treatment time, and minimize pain during retinal photocoagulation. However, the safe therapeutic window (TW), the ratio of threshold powers for thermomechanical rupture of Bruch's membrane and mild coagulation, also decreases with shorter exposures. Two potential approaches toward increasing TW are investigated: (a) decreasing the central irradiance of the laser beam and (b) temporally modulating the pulse. An annular beam with adjustable central irradiance was created by coupling a 532-nm laser into a 200-μm core multimode optical fiber at a 4-7 deg angle to normal incidence. Pulse shapes were optimized using a computational model, and a waveform generator was used to drive a PASCAL photocoagulator (532 nm), producing modulated laser pulses. Acute thresholds for mild coagulation and rupture were measured in Dutch-Belted rabbit in vivo with an annular beam (154-163 μm retinal diameter) and modulated pulse (132 μm, uniform irradiance "flat-top" beam) with 2-50 ms pulse durations. Thresholds with conventional constant-power pulse and a flat-top beam were also determined. Both annular beam and modulated pulse provided a 28% increase in TW at 10-ms duration, affording the same TW as 20-ms pulses with conventional parameters.     

An aequorin study of a facilitating calcium current in bursting pacemaker neurons of Helix

Bursting pacemaker cells of Helix were injected with aequorin, a protein that reacts with Ca ions to emit light. Depolarizing voltage clamp pulses evoked an increase in light emission with little delay and a return to baseline within seconds after the pulse. The maximum light emissions with single voltage pulses occurred at potentials around +75mV but considerable light responses were observed even between +100 and +200mV; a zero or clear minimum at higher voltages was not obvious. Repetitive depolarizing pulses of 20–100 ms duration produced strongly increasing light emissions suggesting a facilitated Ca entry. Maximal increases in light were attained with about four to ten repetitions of pulses to about +20 mV from holding potentials of ?50 mV. Although overall light output could rise with higher voltages, a decreasing ratio of light emissions for late pulses of a series compared with that of the first pulse became obvious. A transitory increase in emitted light was finally replaced by reduced light output during serial pulses. Pulse intervals between 200 and 500 ms were optimal to produce the facilitated responses. The light output during pulse series was considerably greater than that of a continuous pulse of the same overall duration.Time relations of the light output of pulses are consistent with its being produced by a persistent Ca entry which dominates an early Ca entry during the facilitated light responses. The results extend and corroborate previous findings made with a different method which also suggested a facilitating Ca inward current under voltage clamp.     

Construction of a two-photon microscope and optimisation of illumination pulse duration

 The construction of a two-photon/confocal microscope system is described in detail. For two-photon illumination, a Ti:sapphire modelocked laser generating 62-fs pulses at 715 nm was used. The effect of the optical train on illumination pulse width was examined and the observed increase in pulse duration was almost completely removed by the addition/adjustment of a prism compressor system. The imaging capabilities of the two-photon microscope are demonstrated and it is shown that the imaging performance of the two-photon microscope is similar to that of a conventional confocal microscope. With two-photon illumination, the resolution (full width at half-maximum intensity) was 0.42 μm (x–y) and 0.81 μm axially, while with single-photon illumination (at 488 nm in the same instrument with a confocal pinhole detector) the resolution was 0.3 μm (x–y) and 0.75 μm axially. The results are discussed with regard to the general problem of femtosecond pulse distortion in an optical system and a simple procedure for optimal pulse restoration is described. Received: 18 January 1996 / Received after revision and accepted: 3 April 1996