High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution

High-resolution four-dimensional (4-D) optical tomography of human skin based on multiphoton autofluorescence imaging and second harmonic generation (SHG) was performed with the compact femtosecond laser imaging system DermaInspect as well as a modified multiphoton microscope. Femtosecond laser pulses of 80 MHz in the spectral range of 750 to 850 nm, fast galvoscan mirrors, and a time-correlated single-photon counting module have been used to image human skin in vitro and in vivo with subcellular spatial and 250-ps temporal resolution. The nonlinear induced autofluorescence originates from naturally endogenous fluorophores and protein structures such as reduced nicotinamide adenine dinucleotide phosphate, flavins, collagen, elastin, porphyrins, and melanin. Second harmonic generation was used to detect collagen structures. Tissues of patients with dermatological disorders such as psoriasis, fungal infections, nevi, and melanomas have been investigated. Individual intratissue cells and skin structures could be clearly visualized. Intracellular components and connective tissue structures could be further characterized by fluorescence excitation spectra, by determination of the fluorescence decay per pixel, and by fluorescence lifetime imaging. The novel noninvasive multiphoton autofluorescence-SHG imaging technique provides 4-D (x,y,z,tau) optical biopsies with subcellular resolution and offers the possibility of introducing a high-resolution optical diagnostic method in dermatology.     

Time-resolved microspectrofluorometry and fluorescence lifetime imaging of photosensitizers using picosecond pulsed diode lasers in laser scanning microscopes

This work describes the time-resolved fluorescence characteristics of two different photosensitizers in single cells, in detail mTHPC and 5-ALA induced PPIX, which are currently clinically used in photodynamic therapy. The fluorescence lifetime of the drugs was determined in the cells from time-gated spectra as well as single photon counting, using a picosecond pulsed diode laser for fluorescence excitation. The diode laser, which emits pulses at 398 nm with 70 ps full width at half maximum duration, was coupled to a confocal laser scanning microscope. For time-resolved spectroscopy a setup consisting of a Czerny Turner spectrometer and a MCP-gated and -intensified CCD camera was used. Time-gated spectra within the cells were acquired by placing the laser beam in "spot scan" mode. In addition, a time-correlated single photon counting module was used to determine the fluorescence lifetime from single spots and to record lifetime images. The fluorescence lifetime of mTHPC decreased from 7.5 to 5.5 ns during incubation from 1 to 6 h. This decrease was probably attributed to enhanced formation of aggregates during incubation. Fluorescence lifetime imaging showed that longer lifetimes were correlated with accumulation in the cytoplasm in the neighborhood of the cell nucleus, whereas shorter lifetimes were found in the outer cytoplasm. For cells that were incubated with 5-ALA, a fluorescence lifetime of 7.4 ns was found for PPIX; a shorter lifetime at 3.6 ns was probably attributed to photoproducts and aggregates of PPIX. In contrast from fluorescence intensity images alone, different fluorescence species could not be distinguished. However, in the lifetime image a structured fluorescence distribution in the cytoplasm was correlated with the longer lifetime and probably coincides with mitochondria. In conclusion, picosecond diode lasers coupled to a laser scanning microscope equipped with appropriate detection units allows time-resolved spectroscopy and lifetime imaging with high spatial resolution and provides numerous possibilities in cellular and pharmaceutical research.     

Red-shifted fluorescence of sound dental hard tissue

Autofluorescence spectra were recorded in vitro from dentin, enamel, and whole teeth. The spectra exhibited a broad peak shifted by about 50 to 75 nm from the excitation wavelength and the shape of the spectra remained similar regardless of the excitation wavelength. The maximum of the autofluorescence spectra also exhibited a red-shift that depended upon the laser excitation wavelength. The amplitude of the red-shifted fluorescence spectra produced by 444 and 532 nm excitation lasers were compared to that produced by a 405 nm excitation laser. It was determined that the autofluorescence amplitude was not proportional to the inverse fourth power of the excitation laser wavelength. Therefore, the red-shifted fluorescence is not compatible with the previously proposed mechanism of Raman scattering. Instead, the mechanism giving rise to the laser-induced dental autofluorescence is explained by the red-edge-excitation effect.     

Fluorescence lifetime imaging of unstained tissues: early results in human breast cancer

Fluorescence lifetime imaging (FLIM) depends on the fluorescence decay differences between tissues to generate image contrast. In the present study FLIM has been applied to fixed (but unstained) breast cancer tissues to demonstrate the feasibility of this approach for histopathological assessment. As the FLIM method relies on natural autofluorescence, it may be possible to circumvent tissue processing altogether and so FLIM has the potential to be a powerful new method of in vivo tissue imaging via an endoscopic or per-operative approach in a variety of organs, as well as a research tool for in vivo animal models of disease. Unstained, alcohol-fixed tissue samples from 13 patients were stimulated by laser pulses at 415 nm. The temporal decay of the autofluorescence was imaged over a period of 2 ns after cessation of the pulse. The decay rate at each image pixel was calculated as the 'lifetime' factor tau. A tissue classification scheme was used to define regions in each image. The average lifetimes of different tissue regions were compared. A total of 167 tissue regions were measured. Within individual fields, stroma had a larger tau (slower decay) than epithelium (p < 0.001). Within individual patients (taking the mean tau of a given tissue type across all fields from each patient), there was a statistically significant difference between benign and malignancy-associated stroma (p < 0.05). Also, benign collagen had a longer tau than benign epithelium (p < 0.05). Multivariate analysis showed a significant difference between benign stroma, malignancy-associated stroma, blood vessels, and malignant epithelium (p < 0.05). Statistically significant differences between benign and malignancy-associated stroma were obtained even with small patient numbers, indicating that lifetime-based instruments can be developed for real-time diagnostic imaging with microscopic resolution.     

Investigation of NAD（P） H Fluorescence Decay in Living Cardiomyocytes with Spectrally-resolved Fluorescence Lifetime Spectroscopy

Objective：To study the mitochondrial redox state in experimental animals to sensitively detect early signs of mitochondrial function in pathophysiologieal conditions, such as isehemia. Methods： Fluorescence of nieotinamide adenine dinucleotide （phosphate） , or NAD（P）H, the principal electron donor in mitochondrial respiration responsible for vital ATP supply of cardiomyocytes, is studied for non-invasive fluorescent probing of the mitochondrial function. Examination of NAD （P）H fluorescence in living cardiomyocytes following excitation by UV-pulsed laser diode and detection by spectrally-resolved time-correlated single photon counting （TCSPC） , is based on the simultaneous measurement of the fluorescence spectra and lifetime. Results ： The dynamic characteristics of NAD （P） H fluorescence decay in living rat cardiomyocytes show that at least a 3-exponential decay model, with 0.4 - 0.7 ns, 1.2 - 1.9 ns and 8.0 - 13.0 ns lifetimes, is necessary to describe cardiomyocyte autofluorescenee （AF）. Decay-associated spectra （DSA） revealed the presence of 4 spectrally-distinct populations of NADH molecules in eardiomyocytes with spectral maximum at 470 nm for short-lifetime pool for the first time, and emission peaks at 450 nm, 470 nm and 490 nm for intermediate and long-lifetime pools. Increased mitochondrial NADH content ratio by ketone bodies enhanced the AF intensity, without the significant change in fluorescent lifetimes. Rotenone, the inhibitor of Complex I of the mitochondrial respiratory chain, increased AF and shortened the average fluorescence lifetime. Dinitrophenol （DNP）, an uncoupling agent of the mitochondrial oxidative phosphorylation, lowered AF,broadened the spectral shoulder at 520 nm and increased the average lifetime. These effects, comparable to the changes in the concentration and in the rate of dehydrogenation of NADH in vitro, were also examined under ischemia-mimetic conditions. Conclusion： Our findings anticipate a contribution of both conformational NADH changes and energy transfer from NADH to lipoamide dehydrogenase （LipDH）-bound flavins, to explain observed fluorescence kinetics. Presented spectrally resolved fluorescence lifetime approach provides promising new tool for analysis of mitochondrial NAD （P） H in living cardiomyocytes, and hence for investigation of energy metabolism and mitoehondrial dysfunction at a cellular level.     

Determination of the modulation transfer function for a time-gated fluorescence imaging system

The use of fluorescence for cancer detection is currently under investigation. Presently, steady-state fluorescence detection methods are in use, but have limitations due to poor contrast between the fluorescence of the tumor and background autofluorescence. Improved contrast can be obtained with time-resolved techniques because of the differing lifetimes between autofluorescence and exogenous photosensitizers that selectively accumulate within tumor tissue. An imaging system is constructed using a fast-gated (200-ps) charge-coupled device (CCD) camera and a pulsed 635-nm laser diode. To characterize the ability of the system to transfer object contrast to an image, the modulation transfer function (MTF) of the system is acquired by employing an extended knife-edge technique. A knife-edge target is assembled by drilling a rectangular well into a block of polymethyl methacrylate (PMMA). The imaging system records images of the photosensitizer, chloroaluminum phthalocyanine tetrasulfonate (AlPcTS), within the well. AlPcTS was chosen to test the system because of its strong absorption of 635-nm, high fluorescence yield, and relatively long fluorescence lifetime (approximately 7.5 ns). The results show that the system is capable of resolving 10(-4) M AlPcTS fluorescence as small as 1 mm. The findings of this study contribute to the development of a time-gated imaging system using fluorescence lifetimes.     

Characterization of powdered epidermal vaccine delivery with multiphoton microscopy

Multiphoton laser scanning microscopy (MPLSM) has been adapted to non-invasively characterize hand-held powdered epidermal vaccine delivery technology. A near infrared femtosecond pulsed laser, wavelength at approximately 920 nm, was used to evoke autofluorescence of endogenous fluorophores within ex vivo porcine and human skin. Consequently, sub cellular resolution three-dimensional images of stratum corneum and viable epidermal cells were acquired and utilized to observe the morphological deformation of these cells as a result of micro-particle penetration. Furthermore, the distributional pattern of micro-particles within the specific skin target volume was quantified by measuring the penetration depth as revealed by serial optical sections in the axial plane obtained with MPLSM. Additionally, endogenous fluorescence contrast images acquired at the supra-basal layer reveal cellular structures that may pertain to dendritic Langerhans cells of the epidermis. These results show that MPLSM has advantages over conventional histological approaches, since three-dimensional functional images with sub-cellular spatial resolution to depths beyond the epidermis can be acquired non-invasively. Accordingly, we propose that MPLSM is ideal for investigations of powdered epidermal vaccine delivery.     

Imaging of cancer cells by multiphoton microscopy using gold nanoparticles and fluorescent dyes

Due to their unique optical properties, optical probes, including metal nanoparticles (NPs) and fluorescent dyes, are increasingly used as labeling tools in biological imaging. Using multiphoton microscopy and fluorescence lifetime imaging (FLIM) at 750-nm excitation, we recorded intensity and FLIM images from gold NPs (30 nm) and the fluorescent dye Alexa 488 (A488) conjugated with monoclonal ACT-1 antibodies as well as Hoechst 33258 (H258) after incubation with the lymphoma cell line (Karpas-299). From the FLIM images, we can easily discriminate the imaging difference between cells and optical probes according to their distinct fluorescence lifetimes (cellular autofluorescence: 1 to 2 ns; gold NPs: <0.02 ns; A488: 3.5 ns; H258: 2.5 ns). The NP-ACT-1 and A488-ACT-1 conjugates were bound homogeneously on the surface of cells, whereas H258 stained the cell nucleus. We demonstrate that the emission intensity of gold NPs is about ten times stronger than that of the autofluorescence of Karpas-299 cells at the same excitation power. Compared with fluorescent dyes, stronger emission is also observed from gold NPs. Together with their high photostability, these observations suggest that gold NPs are a viable alternative to fluorescent dyes for cellular imaging and cancer diagnosis.     

Wavelength-resolved measurements of fluorescence lifetime of indocyanine green

We study fluorescence lifetime of indocyanine green (ICG) using femtosecond laser and sensitive detection based on time-correlated single-photon counting. A time-resolved multichannel spectral system is constructed and applied for determination of the fluorescence lifetime of the ICG in different solvents. Emission properties of ICG in water, milk, and 1% intralipid solution are investigated. Fluorescence of the fluorophore of different concentrations (in a range of 1.7-160 μM) dissolved in different solutions is excited by femtosecond pulses generated with the use of Ti:Sa laser tuned within the range of 740-790 nm. It is observed that fluorescence lifetime of ICG in water is 0.166 ± 0.02 ns and does not depend on excitation and emission wavelengths. We also show that for the diffusely scattering solvents (milk and intralipid), the lifetime may depend on the dye concentration (especially for large concentrations of ICG). This effect should be taken into account when analyzing changes in the mean time of arrival of fluorescence photons excited in ICG dissolved in such optically turbid media.     

Diagnosis of meningioma by time-resolved fluorescence spectroscopy

We investigate the use of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as an adjunctive tool for the intraoperative rapid evaluation of tumor specimens and delineation of tumor from surrounding normal tissue. Tissue autofluorescence is induced with a pulsed nitrogen laser (337 nm, 1.2 ns) and the intensity decay profiles are recorded in the 370 to 500 nm spectral range with a fast digitizer (0.2 ns resolution). Experiments are conducted on excised specimens (meningioma, dura mater, cerebral cortex) from 26 patients (97 sites). Spectral intensities and time-dependent parameters derived from the time-resolved spectra of each site are used for tissue characterization. A linear discriminant analysis algorithm is used for tissue classification. Our results reveal that meningioma is characterized by unique fluorescence characteristics that enable discrimination of tumor from normal tissue with high sensitivity (>89%) and specificity (100%). The accuracy of classification is found to increase (92.8% cases in the training set and 91.8% in the cross-validated set correctly classified) when parameters from both the spectral and the time domain are used for discrimination. Our findings establish the feasibility of using TR-LIFS as a tool for the identification of meningiomas and enables further development of real-time diagnostic tools for analyzing surgical tissue specimens of meningioma or other brain tumors.     

Optical imaging in microfluidic bioreactors enables oxygen monitoring for continuous cell culture

For the first time, a fluorescence lifetime calibration method for an oxygen-sensitive dye ruthenium tris(2,2'-dipyridyl) dichloride hexahydrate (RTDP) is applied to image oxygen levels in poly(dimethyl siloxane) (PDMS) bioreactors containing living C2C12 mouse myoblasts. PDMS microsystems are broadly used in bioengineering applications due to their biocompatibility and ease of handling. For these systems, oxygen concentrations are of significance and are likely to play an important role in cell behavior and gene expression. Fluorescence lifetime imaging microscopy (FLIM) bases image contrast on fluorophore excited state lifetimes, which reflect local biochemistry. Unique attributes of the widefield, time-domain FLIM system include tunable excitation (337.1 to 960 nm), large temporal dynamic range (> or =600 ps), high spatial resolution (1.4 microm), calibrated detection (0 to 300+/-8 microM of oxygen), and rapid data acquisition and processing times (10 s). Oxygen levels decrease with increasing cell densities and are consistent with model outcomes obtained by simulating bioreactor oxygen diffusion and cell proliferation. In single bioreactor loops, FLIM detects spatial heterogeneity in oxygen levels with variations as high as 20%. The fluorescence lifetime-based imaging approach we describe avoids intensity-based artifacts (including photobleaching and concentration variations) and provides a technique with high spatial discrimination for oxygen monitoring in continuous cell culture systems.     

Design of an endoscopic optical reference to be used for autofluorescence bronchoscopy with a commercially available diagnostic autofluorescence endoscopy (DAFE) system

We present the design of a sterilizable optical reference to characterize and quantify the inter-patient variations in tissue autofluorescence during autofluorescence bronchoscopy with Richard Wolf's diagnostic autofluorescence endoscopy (DAFE) system. The reference was designed to have optical and spectral properties similar to those of the human bronchial wall in spectral conditions corresponding to autofluorescence bronchoscopy conducted with the DAFE system (fluorescence excitation at 390-470 nm and red backscattering light at 590-680 nm). The reference's effective attenuation coefficient and reflectance were measured at 675 nm. In addition, its fluorescence emission spectrum was determined under 430 nm wavelength excitation. The reference is photostable, reproducible, biocompatible and small enough to be easily inserted through the working channel of a conventional bronchofibrescope. This cylindrical (length: 2 mm; diameter: 2 mm) optical reference was validated in a clinical environment.     

Choroidal laser Doppler flowmeter with enhanced sensitivity based on a scattering plate

A portable choroidal laser Doppler flowmeter (LDF) with enhanced sensitivity based on a scattering plate is developed. The portable LDF is weighted 2 kg operated at center wavelength of 780 nm, leading to a better penetration into the eye fundus in contrast to the previous LDF operated at center wavelength of 670 nm. Enhancement of number of detected photons that undergo Doppler scattering and improved measured speed of choroidal blood flow are achieved with the use of a scattering plate positioned in front of the eye. The mechanism of detection and sensitivity enhancement is theoretically analyzed. Evaluation of system performance is done by in vivo measurements on ten volunteers. The results demonstrate that an increased percentage of backscattering light at high Doppler shift frequency is collected due to utilization of the scattering plate. However, this kind of light detection influences spatial resolution of the system and decreases the total signal measured. The proposed method for detection and sensitivity enhancement might be useful in a case where the perception of very slight alternation of blood flow is pursued and the spatial resolution is not as critical as that in a choroidal vascular bed.     

Fluorescence resonance energy transfer determinations using multiphoton fluorescence lifetime imaging microscopy to characterize amyloid-beta plaques

We describe the implementation of a commercial fluorescence lifetime imaging microscopy (FLIM) instrument used in conjunction with a commercial laser scanning multiphoton microscope. The femtosecond-pulsed near-infrared laser is an ideal excitation source for time-domain fluorescence lifetime measurements. With synchronization from the x-y scanners, fluorescence lifetimes can be acquired on a pixel-by-pixel basis, with high spatial resolution. Multiexponential curve fits for each pixel result in two-dimensional fluorescence resonance energy transfer (FRET) measurements that allow the determination of both proximity of fluorescent FRET pairs, as well as the fraction of FRET pairs close enough for FRET to occur. Experiments are described that characterize this system, as well as commonly used reagents valuable for FRET determinations in biological systems. Constructs of CFP and YFP were generated to demonstrate FRET between this pair of green fluorescent protein (GFP) color variants. The lifetime characteristics of the FRET pair fluorescein and rhodamine, commonly used for immunohistochemistry, were also examined. Finally, these fluorophores were used to demonstrate spatially resolved FRET with senile plaques obtained from transgenic mouse brain. Together these results demonstrate that FLIM allows sensitive measurements of protein-protein interactions on a spatial scale less than 10 nm using commercially available components.     

Three-dimensional fluorescence lifetime tomography

Near-infrared fluorescence tomography using molecularly targeted lifetime-sensitive, fluorescent contrast agents have applications for early-stage cancer diagnostics. Yet, although the measurement of fluorescent lifetime imaging microscopy (FLIM) is extensively used in microscopy and spectroscopy applications, demonstration of fluorescence lifetime tomography for medical imaging is limited to two-dimensional studies. Herein, the feasibility of three-dimensional fluorescence-lifetime tomography on clinically relevant phantom volumes is established, using (i) a gainmodulated intensified charge coupled device (CCD) and modulated laser diode imaging system, (ii) two fluorescent contrast agents, e.g., Indocyanine green and 3-3'-Diethylthiatricarbocyanine iodide differing in their fluorescence lifetime by 0.62 ns, and (iii) a two stage approximate extended Kalman filter reconstruction algorithm. Fluorescence measurements of phase and amplitude were acquired on the phantom surface under different target to background fluorescence absorption (70:1, 100:1) and fluorescence lifetime (1:1, 2.1:1) contrasts at target depths of 1.4-2 cm. The Bayesian tomography algorithm was employed to obtain three-dimensional images of lifetime and absorption owing to the fluorophores.     

Corneal multiphoton microscopy and intratissue optical nanosurgery by nanojoule femtosecond near-infrared pulsed lasers

Multiphoton microscopy including multiphoton autofluorescence imaging (MAI) and second-harmonic generation (SHG) is being used as a novel diagnostic tool to perform tissue nonlinear optical tomography with submicron resolution. The three-dimensional corneal ultrastructure of whole depth has been viewed without any staining or mechanical slicing. Compared with photodisruptive surgical effects occurring at TW/cm2 light intensity, multiphoton imaging can be induced at MW-GW/ cm2 photon intensity. The intratissue surgical effect including nanojoule (nJ) femtosecond laser ablation and flap generation was induced through multiphoton nonlinear absorption at a wavelength of 800 nm and ascertained by the histological outcomes. More interesting, the multiphoton microscopy based on nonlinear absorption of femtosecond laser pulses at the wavelength of 715-930 nm emitted from solid-state Ti:sapphire system is acting as a precise non-invasive monitoring tool to determine the interest of region, to visualize and verify the outcomes in in vivo intrastromal laser nanosurgery. Overall, these data suggest that multiphoton microscopy is a highly sensitive and promising technique for studying the morphometric and biomechanical properties of biological tissues and that the nJ ultrashort Lasers can be used as a ultra-precise nanoscalpel for performing intratissue surgery.     

Autofluorescence characteristics of immortalized and carcinogen-transformed human bronchial epithelial cells

Tissue autofluorescence has been explored as a potential method of noninvasive pre-neoplasia (pre-malignancy) detection in the lung. Here, we report the first studies of intrinsic cellular autofluorescence from SV40 immortalized and distinct tobacco-carcinogen-transformed (malignant) human bronchial epithelial cells. These cell lines are useful models for studies seeking to distinguish between normal and pre-neoplastic human bronchial epithelial cells. The cells were characterized via spectrofluorimetry and confocal fluorescence microscopy. Spectrofluorimetry revealed that tryptophan was the dominant fluorophore. No change in tryptophan emission intensity was observed between immortalized and carcinogen-transformed cells. Confocal autofluorescence microscopy was performed using a highly sensitive, spectrometer-coupled instrument capable of limiting emission detection to specific wavelength ranges. These studies revealed two additional endogenous fluorophores, whose excitation and emission characteristics were consistent with nicotinamide adenine dinucleotide (NADH) and flavins. In immortalized human bronchial epithelial cells, the fluorescence of these species was localized to cytoplasmic granules. In contrast, the carcinogen-transformed cells showed an appreciable decrease in the fluorescence intensity of both NADH and flavins and the punctate, spatial localization of the autofluorescence was lost. The observed autofluorescence decrease was potentially the result of changes in the redox state of the fluorophores. The random cytoplasmic fluorescence pattern found in carcinogen-transformed cells may be attributed to changes in the mitochondrial morphology. The implications of these results to pre-neoplasia detection in the lung are discussed.     

Novel optical detection system for in vivo identification and localization of cervical intraepithelial neoplasia

A noncontact optical detection system is developed for the in vivo identification and localization of high-grade cervical intraepithelial neoplasia (CIN 2,3). Diagnostic scans of the entire human cervix are performed following acetic acid application employing three integrated optical measurements: laser-induced fluorescence spectroscopy, white light diffuse reflectance spectroscopy, and video imaging. Full cervical scans comprising 499 interrogation locations at 1-mm spatial resolution are completed in 12 s. Diffuse reflectance and fluorescence spectra with signal-to-noise ratios of better than 100-to-1 are collected between 360 and 720 nm in increments of 1 nm, with an inherent spectral resolution of 8 nm. Glare reduction and optical vignetting are handled with a novel illumination scheme and subsequent spectral arbitration algorithms. The system is designed and found to be well below acceptable safe optical exposure levels. Typical reproducibility across multiple systems is approximately 5%, providing reliable and accurate detection of in vivo cervical neoplasia in normal clinical use.     

Two postprocessing techniques for the elimination of background autofluorescence for fluorescence lifetime imaging microscopy

The analysis of fluorescence lifetime imaging microscopy (FLIM) data under complex biological conditions can be challenging. Particularly, the presence of short-lived autofluorescent aggregates can confound lifetime measurements in fluorescence energy transfer (FRET) experiments, where it can become confused with the signal from exogenous fluorophores. Here we report two techniques that can be used to discriminate the contribution of autofluorescence from exogenous fluorphores in FLIM. We apply the techniques to transgenic mice that natively express yellow fluorescence protein (YFP) in a subset of cortical neurons and to histological slices of aged human brain tissue, where we study the misfolding of intracellular tau protein in the form of neurofibrillary tangles.     

Fluorescence lifetime imaging study of a thin protein layer on solid surfaces

Understanding the fundamental interactions between proteins and solid surfaces is essential in the area of implantable medical devices. Fluorescence methods offer the sensitivity required to study the formation of the initial thin protein layers that mediate biocompatibility of materials. Thin protein layers (bovine serum albumin labelled with 1-anilino-8-naphthalenesulfonate, BSA-ANS) deposited on several surfaces (glass, silicon, stainless steel, polystyrene, and silver island film) were studied using confocal frequency domain Fluorescence Lifetime Imaging Microscopy (FLIM) and single-point multifrequency lifetime analysis techniques. FLIM provides spatial information about both fluorophores located on the surface and physicochemical parameters of the surface microenvironment. The average fluorescence lifetimes (tau(av)) of the adsorbed BSA-ANS generated by the contact between a protein solution and the material surface were measured by the multifrequency modulation and phase shift. Results indicate that tau(av) values of the albumin complexes on the surfaces (approximately 12 ns) are, in general, shorter than tau(av) found in the bulk solution (approximately 14 ns). For some surfaces, like polystyrene and silver island film the differences in tau(av) of the adsorbed BSA-ANS were found to be much greater. The differences in fluorescence lifetimes may indicate structural changes in the BSA protein induced by contact with the surface.