We have demonstrated the first application of the stimulated-emission technique to fluorescence microscopy. By measuring the fluorescence signal at the cross-correlation frequency, pump—probe fluorescence microscopy can provide superior spatial resolution and effective off-focal background rejection compared to conventional one-photon microscopy. Due to the wavelengths used in the one-photon pumping and probing processes, this technique has better spatial resolution than two-photon excitation microscopy, and comparable spatial resolution to confocal microscopy. Furthermore, imaging at low-frequency harmonics eliminates the need of using a fast optical detector in time-resolved imaging of biological systems. The technical development of pump—probe microscopy is still in its early stages. Substantial future improvements are expected. The implementation of transient absorption mode in pump—probe microscopy will allow ground-state kinetics to be directly monitored. The addition of two-photon excitation would improve the microscope spatial resolution as well as making wavelength-dependent spectroscopy possible. The implementation of the pump—probe technique in the time domain would make the microscopy alignment and automation more difficult but may take time-resolved microscopy solidly into the femtosecond time scale. The pump—probe microscopy technique has the potential to radically transform the field of time-resolved microscopy.

We present the implementation of intensity-modulated laser diodes for applications in frequency-domain pump-probe fluorescence microscopy. Our technique, which is based on the stimulated-emission approach, uses two sinusoidally modulated laser diodes. One laser (635 nm) excites the chromophores under study, and the other laser (680 nm) is responsible for inducing stimulated emission from excited-state molecules. Both light sources are modulated in the 80-MHz range but with an offset of 5 kHz between them. The result of the interaction of the pump and the probe beams is that a cross-correlation fluorescence signal at 5 kHz is generated primarily at the focal volume. Microscope imaging at the cross-correlation signal results in images with high contrast, and time-resolved high-frequency information can be acquired without high-speed detection. A detailed experimental arrangement of our methodology is presented along with images acquired from a 4.0-mum-diameter fluorescent sphere and TOTO-3-labeled mouse STO cells. (TOTO-3 is a nucleic acid stain.) Our results demonstrate the feasibility of using sinusoidally modulated laser diodes for pump-probe imaging, creating the exciting possibility of high-contrast time-resolved imaging with low-cost laser-diode systems.

We report the application of pump-probe fluorescence microscopy in time-resolved polarization imaging. We derived the equations governing the pump-probe stimulated emission process and characterized the pump and probe laser power levels for signal saturation. Our emphasis is to use this novel methodology to image polarization properties of fluorophores across entire cells. As a feasibility study, we imaged a 15-μm orange latex sphere and found that there is depolarization that is possibly due to energy transfer among fluorescent molecules inside the sphere. We also imaged a mouse fibroblast labeled with CellTracker Orange CMTMR (5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethyl-rhodamine). We observed that Orange CMTMR complexed with gluthathione rotates fast, indicating the relatively low fluid-phase viscosity of the cytoplasmic microenvironment as seen by Orange CMTMR. The measured rotational correlation time ranged from ∼30 to ∼150 ps. This work demonstrates the effectiveness of stimulated emission measurements in acquiring high-resolution, time-resolved polarization information across the entire cell.

Microscopy is traditionally a tool for determining biological structures. Many recent advances in optical microscopy involves the incorporation of spectroscopy techniques to monitor biochemical states of microscopic structures in living cells and tissues. By minimizing tissue photodamage, two-photon excitation microscopy provides a new opportunity to study the dynamics of biological systems on time scales from nanoseconds to hours. This review will focus on a number of these new methods: two-photon time-lapse microscopy, two-photon photoactivation, two-photon correlated spectroscopy, two-photon single particle tracking and two-photon lifetime microscopy.

Microscopy is traditionally a tool for determining biological structures. Many recent advances in optical microscopy involves the incorporation of spectroscopy techniques to monitor biochemical states of microscopic structures in living cells and tissues. By minimizing tissue photodamage, two-photon excitation microscopy provides a new opportunity to study the dynamics of biological systems on time scales from nanoseconds to hours. This review will focus on a number of these new methods: two-photon time-lapse microscopy, two-photon photoactivation, two-photon correlated spectroscopy, two-photon single particle tracking and two-photon lifetime microscopy.

Two‐photon fluorescence lifetime imaging microscopy was used noninvasively to monitor a fluorescent antigen during macrophage‐mediated endocytosis, intracellular vacuolar encapsulation, and protease‐dependent processing. Fluorescein‐conjugated bovine serum albumin (FITC–BSA) served as the soluble exogenous antigen. As a relatively nonfluorescent probe in the native state, the antigen was designed to reflect sequential intracellular antigen processing events through time‐dependent changes in fluorescence properties. Using two‐photon lifetime imaging microscopy, antigen processing events were monitored continuously for several hours. During this time, the initial fluorescein fluorescence lifetime of 0.5 ns increased to α 3.0 ns. Control experiments using fluorescein conjugated poly‐l‐lysine and poly‐d‐lysine demonstrated that the increase in fluorescence parameters observed with FITC–BSA were due to intracellular proteolysis since addition of the inert d‐isomer did not promote an increase in fluorescence lifetime or intensity. Comparisons of intravacuolar and extracellular FITC–dextran concentration suggested active localization of dextran in the vacuoles by the macrophage. In addition, the kinetics of degradation observed using two‐photon microscopy were similar to results obtained on the flow cytometer, thus validating the use of flow cytometry for future studies.

Todd E French, Albert E Cerussi, Sergio Fantini, Maria Angela Franceschini, and Enrico Gratton. Measurement of absolute fluorescence quantum yield in turbid media. 41st Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 1997. Biophys J. 1997; 72(2 Pt 2), Tu-Pos424. Abstract We demonstrate the measurement of the absolute fluorescence quantum yields performed inside a multiple scattering medium. A model for frequency-domain fluorescence spectroscopy in multiple scattering media that allows the accurate recovery of fluorescence parameters such as the quantum yield has recently been verified. Our work uses this model to determine the absolute quantum yield of a fluorophore that is uniformly distributed throughout a multiple scattering medium. Using this technique, it is not necessary to have a reference fluorophore of known quantum yield. The only reference compound that is necessary is one which is used to calibrate the spectral response of the detection system. This technique does not require the calibration of the excitation and emission light paths. Because of the multiply scattering of the medium (titanium dioxide particles suspended in water), the excitation and emission geometries are identical. We present the quantum yield of Rhodamine B in water measured in both infinite and semi-infinite geometries. Supported by NIH RR03155 and CA5702, a joint Whitaker-NIH grant RR10966, and Sandia CRADA SC93/01 177.

We use the lipophilic fluorescence probe Laurdan to study cell membranes. The generalized polarization (GP) of Laurdan-labeled cells contains useful information about membrane fluidity and polarity. A high GP is usually associated with low fluidity, low polarity, or high cholesterol content of the membranes, and a low GP is the opposite. We have combined the GP method and two-photon fluorescence microscopy to provide an alternative approach to study cell membranes. Using two-photon excitation in a conventional microscope offers great advantages for studying biological samples. These advantages include efficient background rejection, low photodamage, and improved depth discrimination. We performed GP measurements on mouse fibroblast cells and observed that both intensity and GP images are not spatially uniform. We tested for possible GP artifacts arising from cellular autofluorescence and lifetime quenching, using a procedure for background fluorescence subtraction and by direct lifetime measurements in the microscope. GP measured in a single cell displays a broad distribution, and the GP of 40 different cells grown on the same cover glass is also statistically distributed. The correlations between intensity and GP images were analyzed, and no monotonic dependence between the two was found. By digitally separating high and low GP values, we found that high GP values often associate with the regions of the plasma membrane and low GP values link with the nuclear membranes. Our results also show local GP variations within the plasma and nuclear membranes.

We have developed a high sensitivity time-resolved two-photon scanning microscope. At an excitation wavelength of 960 nm, a spatial point spread function of 0.3 μm (FWHM) radially and 0.9 μm (FWHM) axially is measured for an 1.25 N.A. objective. The light source is a mode-locked titanium-sapphire laser. The time resolution is 400 ps with common chromophores used in microscopy. Time resolution is obtained using the frequency-domain heterodyning technique in which the laser is synchronized at a very high cross-correlation frequency to the rest of the electronics. We demonstrate spatial and time resolution using well-characterized fluorescent microspheres. We show two applications of two-photon time-resolved fluorescence microscopy: time-resolved imaging of multiple dye labeled cells and quantitative cellular calcium concentration using a lifetime indicator.

We report the development of a scanning lifetime fluorescence microscope using the asynchronous, pump-probe (stimulated emission) approach. There are two significant advantages of this technique. First, the cross-correlation signal produced by overlapping the pump and probe lasers results in i) an axial sectioning effect similar to that in confocal and two-photon excitation microscopy, and ii) improved spatial resolution compared to conventional one-photon fluorescence microscopy. Second, the low-frequency, cross-correlation signal generated allows lifetime-resolved imaging without using fast photodetectors. The data presented here include 1) determination of laser sources' threshold powers for linearity in the pump-probe signal; 2) characterization of the pump-probe intensity profile using 0.28 microns fluorescent latex spheres; 3) high frequency (up to 6.7 GHz) lifetime measurement of rhodamine B in water; and 4) lifetime-resolved images of fluorescent latex spheres, human erythrocytes and a mouse fibroblast cell stained by rhodamine DHPE, and a mouse fibroblast labeled with ethidium bromide and rhodamine DHPE.

Two photon fluorescence microscopy was first proposed by Denk et al. in 1990. There are a number of advantages which this new kind of microscopy can offer, including the high spatial resolution, rejection of out of plane fluorescence and reducing photobleaching. We have constructed a fluorescence microscope similar in concept to that of Denk et al. with the addition of lifetime fluorescence capability. The microscope system is built around a Zeiss Axiovert unit. The light source is a Coherent Mira 900 Ti:Sapphire laser with about 150 fs pulse width at a repetition of 80 MHz. The average laser power in the 720 nm-960 nm range varies between 0.5 W to about 1.5 W. The laser scanning system is a digital scanner from Cambridge Research with a resolution of 16 bits. A digital controller interfaced with the data acquisition unit permits positioning and scanning in an arbitrary area of the field of view. Two channels of light emission are available. Typical frame acquisition times are less than a second for the intensity only acquisition mode and about 2-3 seconds for the intensity and lifetime acquisition mode. In a typical run about 15 to 20 frames at a resolution of 257 x 256 are acquired and stored at different z-positions for off-line 3-D reconstruction. The lifetime mode of operation is based on the frequency-domain technique.

Time-resolved fluorescence imaging can enhance the contrast of microscope images and it can also provide important information about the micro-environment in cellular systems. We have developed a fluorescence microscope which can measure fluorescence lifetimes over an entire image. Fluorescence lifetimes are measured by using heterodyne frequency domain techniques. Heterodyning is accomplished by using an intensity modulated laser light source and a fast scan CCD camera coupled with a gain modulated microchannel plate as the detector. The high duty cycle of this method allows us to generate a phase resolved image with about five seconds integration time. Operating in the fast scan mode, the systematic uncertainties in lifetime determination caused by photobleaching are less severe than those of slow-scan cameras. The microchannel plate can be modulated at frequencies up to 300 MHz, which allows us to measure lifetimes as short as 500 ps with resolution of 50 ps. The modulation of the microchannel plate only slightly degrades the spatial resolution of the image from the diffraction limit; 0.8 micron resolution is maintained with 500 nm laser excitation.

Todd E French, Peter T C So, Keith M Berland, and Enrico Gratton. A time-resolved fluorescence microscope. 37th Annual Meeting of the Biophysical Society, Washington, DC, February 1993. Biophys J. 1993; 64(2 Pt 2): A110, M-Pos507. Abstract We have constructed a time-resolved fluorescence microscope system that can collect images which show differences in fluorescence decay times. The microscope system has the abilities of the more common steady-state fluorescence imaging microscope and, additionally, can determine fluorescence intensity as a function of time. A modulated laser beam is the light source and a modulated microchannel plate coupled to a CCD camera serves as the detector. Data arrim at the detector at a chosen frequency (DC-150 MHz) and is heterodyned by the microchannel plate to a frequency slower than the camera's sampling rate (frame rate). Alternatively, the microchannel plate can be set to homodyne the incoming signal at which point the system becomes capable of real time analysis. The homodyning or heterodyning process maintains the spatial coherence of the images, which may have a resolution of 0.5 microns. The frequency domain images (DC intensity, AC intensity, phase shift and demodulation) can be interpreted directly or new combinations can be created for further analysis (e.g., lifetime images or phase-resolved images). Lifetimes as short as about 2 ns can be resolved with a resolution of about 50 pa. Using phase resolved techniques, fractional components of lifetimes at each pixel can be distinguished, potentially with only a single frequency data run. Supported by NIH RR03155.

Our laboratory has developed a modular laser tomography system, with pulsed or amplitude modulated (MHz to GHz), near infrared lasers that deliver a probing beam to the tissue of interest through a fiber optic. After the incoming light is scattered and attenuated by the tissue, a detector or imaging fiber optic bundle delivers it to a point detector (photomultiplier tube) which is heterodyned with the modulation frequency to yield the phase delay and demodulation resulting from the light-tissue interaction. The CCD electronics are phase-locked with those of the digitizer to minimize pixel jitter and, in addition, an external clock synchronizes the detector units with the modulated laser source. The digitized time slices are integrated into four bins corresponding to four quadrants of the cross correlation period. The final processing step, a fast Fourier transform, generates the phase shift, demodulation, and average intensity data suitable for image reconstruction.

William W Mantulin, Enrico Gratton, Martin J vandeVen, and Todd E French. Imaging of mammalian tissue. 36th Annual Meeting of the Biophysical Society, Houston, Texas, 9-13 February 1992. Biophys J. 1992; 61(2 Pt 2): A447, 2572. Abstract Phase resolved techniques can be used successfully to image the internal structures of mammalian tissue. Our method observes the diffusion of a modulated infrared laser beam through a biological sample. The transmitted wavefront is read by a modulated CCD camera and the DC intensity, AC intensity, phase shift and demodulation are recorded. Images obtained characterize the absorption and scattering of the material. The modulation frequency of the beam controls the spatial resolution of the images whereas the inherent contrast of the images can be changed by using a different illumination wavelength. Hand and teeth images created under various experimental conditions will be presented and compared to x-ray images. The resolution of these images is limited primarily by the camera to about 0.1 mm. Currently, the system is comparable to an x-ray machine but without the ionizing radiation. In the future, it seems feasible that MRI reconstruction techniques or computer aided tomography could be applied to our process to create volumetric images.This work is supported by NIH grant PHS-P41-RR03155.