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The dynamics of molecular systems can be studied with time-resolved spectroscopy combined with model-based analysis. A Python framework for global and target analysis of time-resolved spectra is introduced with the help of three case studies. The first study, concerning broadband absorption of intersystem crossing in 4-thiothymidine, demonstrates the framework's ability to resolve vibrational wavepackets with a time resolution of ≈10 fs using damped oscillations and their associated spectra and phases. Thereby, a parametric description of the "coherent artifact" is crucial. The second study addresses multichromophoric systems composed of two perylene bisimide chromophores. Here, pyglotaran's guidance spectra and lego-like model composition enable the integration of spectral and kinetic properties of the parent chromophores, revealing a loss process, the undesired production of a radical pair, that reduces the light harvesting efficiency. In the third, time-resolved emission case study of whole photosynthetic cells, a megacomplex containing ≈500 chromophores of five different types is described by a combination of the kinetic models for its elements. As direct fitting of the data by theoretical simulation is unfeasible, our global and target analysis methodology provides a useful 'middle ground' where the theoretical description and the fit of the experimental data can meet. The pyglotaran framework enables the lego-like creation of kinetic models through its modular design and seamless integration with the rich Python ecosystem, particularly Jupyter notebooks. With extensive documentation and a robust validation framework, pyglotaran ensures accessibility and reliability for researchers, serving as an invaluable tool for understanding complex molecular systems.
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Low-temperature fluorescence measurements are frequently used in photosynthesis research to assess photosynthetic processes. Upon illumination of photosystem II (PSII) frozen to 77 K, fluorescence quenching is observed. In this work, we studied the light-induced quenching in intact cells of Chlamydomonas reinhardtii at 77 K using time-resolved fluorescence spectroscopy with a streak camera setup. In agreement with previous studies, global analysis of the data shows that prolonged illumination of the sample affects the nanosecond decay component of the PSII emission. Using target analysis, we resolved the quenching on the PSII-684 compartment which describes bulk chlorophyll molecules of the PSII core antenna. Further, we quantified the quenching rate constant and observed that as the illumination proceeds the accumulation of the quencher leads to a speed up of the fluorescence decay of the PSII-684 compartment as the decay rate constant increases from about 3 to 4 ns- 1. The quenching on PSII-684 leads to indirect quenching of the compartments PSII-690 and PSII-695 which represent the red chlorophyll of the PSII core. These results explain past and current observations of light-induced quenching in 77 K steady-state and time-resolved fluorescence spectra.
Assuntos
Chlamydomonas reinhardtii/fisiologia , Fotossíntese , Complexo de Proteína do Fotossistema II/metabolismo , Chlamydomonas reinhardtii/metabolismo , Chlamydomonas reinhardtii/efeitos da radiação , Clorofila/metabolismo , Temperatura Baixa , Fluorescência , Complexo de Proteína do Fotossistema II/genética , Espectrometria de FluorescênciaRESUMO
In the light-harvesting antenna of the Synechocystis PCC 6803 phycobilisome (PB), the core consists of three cylinders, each composed of four disks, whereas each of the six rods consists of up to three hexamers (Arteni et al., Biochim Biophys Acta 1787(4):272-279, 2009). The rods and core contain phycocyanin and allophycocyanin pigments, respectively. Together these pigments absorb light between 400 and 650 nm. Time-resolved difference absorption spectra from wild-type PB and rod mutants have been measured in different quenching and annihilation conditions. Based upon a global analysis of these data and of published time-resolved emission spectra, a functional compartmental model of the phycobilisome is proposed. The model describes all experiments with a common set of parameters. Three annihilation time constants are estimated, 3, 25, and 147 ps, which represent, respectively, intradisk, interdisk/intracylinder, and intercylinder annihilation. The species-associated difference absorption and emission spectra of two phycocyanin and two allophycocyanin pigments are consistently estimated, as well as all the excitation energy transfer rates. Thus, the wild-type PB containing 396 pigments can be described by a functional compartmental model of 22 compartments. When the interhexamer equilibration within a rod is not taken into account, this can be further simplified to ten compartments, which is the minimal model. In this model, the slowest excitation energy transfer rates are between the core cylinders (time constants 115-145 ps), and between the rods and the core (time constants 68-115 ps).
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Modelos Biológicos , Ficobilissomas/metabolismo , Synechocystis/metabolismo , Simulação por Computador , Transferência de Energia , Ficobilissomas/química , Espectrometria de Fluorescência , Fatores de TempoRESUMO
The phenomenon of non-photochemical quenching (NPQ) was studied in spinach chloroplasts using pulse amplitude modulated (PAM) fluorometry. We present a new analysis method which describes the observed fluorescence quantum yield as the sum of the product of four different states of PSII and their corresponding quantum yields. These four distinct states are PSII in the quenched or unquenched state, and with its reaction center either open or closed depending upon the reduction of the QA site. With this method we can describe the dynamics of the NPQ induction and recovery as well as quantify the percentage of photoinactivated RC throughout the measurement. We show that after one cycle of quenching followed by a period of recovery, approximately 8-9% of the RC are photoinactivated, after two cycles of illumination this number becomes 15-17%. The recovery from the quenching appeared with rates of (50s)-1 and (1h)-1. The new analysis method presented here is flexible, allowing it to be applied to any type of PAM fluorometry protocol. The method allows to quantitatively compare qualitatively different PAM curves on the basis of statistically relevant fitting parameters and to quantify quenching dynamics and photoinactivation. Moreover, the results presented here demonstrate that the analysis of a single PAM fluorometry quenching experiment can already provide information on the relative quantum yield of the four different states of PSII for the intact chloroplasts - something no other form of spectroscopy could provide in a single measurement.
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Complexo de Proteína do Fotossistema II/metabolismo , Percepção de Quorum/fisiologia , Cloroplastos/metabolismo , Fluorescência , Fluorometria/métodos , Cinética , Luz , Spinacia oleracea/metabolismoRESUMO
The regulatory mechanism of state transitions was studied in Chlamydomonas reinhardtii (C.r.) wild type (WT) as well as mutant strains deficient in the photosystem I (PSI) or the photosystem II (PSII) core. Time-resolved fluorescence measurements were obtained on instantly frozen cells incubated beforehand in the dark in aerobic or anaerobic conditions which leads to state 1 (S1) or state 2 (S2). WT data contains information on the light-harvesting complex (LHC) connected to PSI and PSII. The mutants' data contain information on either LHCII-LHCI-PSI or LHCII-PSII, plus information on LHC antennas devoid of a PS core. In a simultaneous analysis of the data from all strains under S1 or S2 conditions a unified model for the excited state dynamics at 77K was created. This yielded the completely resolved LHCII-LHCI-PSI and LHCII-PSII dynamics and quantified the state transitions. In WT cells the fraction of light absorbed by LHCII connected to PSII decreases from 45% in S1 to 29% in S2, while it increases from 0% to 16% for LHCII connected to PSI. Thus (16/45=) 36% of all LHCII is involved in the state transition. In the mutant strains deficient in the PSI core, the red most species peaking at 716nm disappears completely, indicating that this far red Chl pigment is located in the PSI core. In the mutant strain deficient in the PSII core, red shifted species with maxima at 684 and 686nm appear in the LHCII antenna. LHCII-684 is quenched and decays with a rate of (310ps)-1.
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Chlamydomonas reinhardtii/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Clorofila/metabolismo , Luz , Complexos de Proteínas Captadores de Luz/metabolismo , Fosforilação/fisiologia , Espectrometria de Fluorescência/métodos , Tilacoides/metabolismoRESUMO
The color variations of light emitted by some natural and mutant luciferases are normally attributed to collective factors referred to as microenvironment effects; however, the exact nature of these interactions between the emitting molecule (oxyluciferin) and the active site remains elusive. Although model studies of noncomplexed oxyluciferin and its variants have greatly advanced the understanding of its photochemistry, extrapolation of the conclusions to the real system requires assumptions about the polarity and proticity of the active site. To decipher the intricate excited-state dynamics, global and target analysis is performed here for the first time on the steady-state and time-resolved spectra of firefly oxyluciferin complexed with luciferase from the Japanese firefly (Luciola cruciata). The experimental steady-state and time-resolved luminescence spectra of the oxyluciferin/luciferase complex in solution are compared with the broadband time-resolved firefly bioluminescence recorded in vivo. The results demonstrate that de-excitation of the luminophore results in a complex cascade of photoinduced proton transfer processes and can be interpreted by the pH dependence of the emitted light. It is confirmed that proton transfer is the central event in the spectrochemistry of this system for which any assignment of the pH-dependent emission to a single chemical species would be an oversimplification.
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Indóis/química , Indóis/metabolismo , Luciferases de Vaga-Lume/metabolismo , Pirazinas/química , Pirazinas/metabolismo , Domínio Catalítico , Modelos MolecularesRESUMO
Pulse-amplitude modulated (PAM) fluorometry is extensively used to characterize photosynthetic organisms on the slow time-scale (1-1000 s). The saturation pulse method allows determination of the quantum yields of maximal (F(M)) and minimal fluorescence (F(0)), parameters related to the activity of the photosynthetic apparatus. Also, when the sample undergoes a certain light treatment during the measurement, the fluorescence quantum yields of the unquenched and the quenched states can be determined. In the case of cyanobacteria, however, the recorded fluorescence does not exclusively stem from the chlorophyll a in photosystem II (PSII). The phycobilins, the pigments of the cyanobacterial light-harvesting complexes, the phycobilisomes (PB), also contribute to the PAM signal, and therefore, F(0) and F(M) are no longer related to PSII only. We present a functional model that takes into account the presence of several fluorescent species whose concentrations can be resolved provided their fluorescence quantum yields are known. Data analysis of PAM measurements on in vivo cells of our model organism Synechocystis PCC6803 is discussed. Three different components are found necessary to fit the data: uncoupled PB (PB(free)), PB-PSII complexes, and free PSI. The free PSII contribution was negligible. The PB(free) contribution substantially increased in the mutants that lack the core terminal emitter subunits allophycocyanin D or allophycocyanin F. A positive correlation was found between the amount of PB(free) and the rate constants describing the binding of the activated orange carotenoid protein to PB, responsible for non-photochemical quenching.
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Fluorometria/métodos , Modelos Biológicos , Ficobilissomas/química , Synechocystis/química , Simulação por Computador , Fluorescência , Mutação , Complexo de Proteína do Fotossistema I/química , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/metabolismo , Ficobilissomas/metabolismo , Ficocianina/genética , Ficocianina/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Synechocystis/genética , Synechocystis/metabolismo , Fatores de TempoRESUMO
Cyanobacteria have developed responses to maintain the balance between the energy absorbed and the energy used in different pigment-protein complexes. One of the relatively rapid (a few minutes) responses is activated when the cells are exposed to high light intensities. This mechanism thermally dissipates excitation energy at the level of the phycobilisome (PB) antenna before it reaches the reaction center. When exposed to low intensities of light that modify the redox state of the plastoquinone pool, the so-called state transitions redistribute energy between photosystem I and II. Experimental techniques to investigate the underlying mechanisms of these responses, such as pulse-amplitude modulated fluorometry, are based on spectrally integrated signals. Previously, a spectrally resolved fluorometry method has been introduced to preserve spectral information. The analysis method introduced in this work allows to interpret SRF data in terms of species-associated spectra of open/closed reaction centers (RCs), (un)quenched PB and state 1 versus state 2. Thus, spectral differences in the time-dependent fluorescence signature of photosynthetic organisms under varying light conditions can be traced and assigned to functional emitting species leading to a number of interpretations of their molecular origins. In particular, we present evidence that state 1 and state 2 correspond to different states of the PB-PSII-PSI megacomplex.
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Synechocystis/efeitos da radiação , Fluorescência , Luz , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema I/efeitos da radiação , Complexo de Proteína do Fotossistema II/metabolismo , Complexo de Proteína do Fotossistema II/efeitos da radiação , Espectrometria de FluorescênciaRESUMO
When exciting a complex molecular system with a short optical pulse, all chromophores present in the system can be excited. The resulting superposition of electronically and vibrationally excited states evolves in time, which is monitored with transient absorption spectroscopy. We present a methodology to resolve simultaneously the contributions of the different electronically and vibrationally excited states from the complete data. The evolution of the excited states is described with a superposition of damped oscillations. The amplitude of a damped oscillation cos(ωnt)exp(-γnt) as a function of the detection wavelength constitutes a damped oscillation associated spectrum DOASn(λ) with an accompanying phase characteristic φn(λ). In a case study, the cryptophyte photosynthetic antenna complex PC612 which contains eight bilin chromophores was excited by a broadband optical pulse. Difference absorption spectra from 525 to 715 nm were measured until 1 ns. The population dynamics is described by four lifetimes, with interchromophore equilibration in 0.8 and 7.5 ps. We have resolved 24 DOAS with frequencies between 130 and 1649 cm-1 and with damping rates between 0.9 and 12 ps-1. In addition, 11 more DOAS with faster damping rates were necessary to describe the "coherent artefact." The DOAS contains both ground and excited state features. Their interpretation is aided by DOAS analysis of simulated transient absorption signals resulting from stimulated emission and ground state bleach.
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State transitions in the green alga Chlamydomonas reinhardtii serve to balance excitation energy transfer to photosystem I (PSI) and to photosystem II (PSII) and possibly play a role as a photoprotective mechanism. Thus, light-harvesting complex II (LHCII) can switch between the photosystems consequently transferring more excitation energy to PSII (state 1) or to PSI (state 2) or can end up in LHCII-only domains. In this study, low-temperature (77 K) steady-state and time-resolved fluorescence measured on intact cells of Chlamydomonas reinhardtii shows that independently of the state excitation energy transfer from LHCII to PSI or to PSII occurs on two main timescales of <15 ps and â¼ 100 ps. Moreover, in state 1 almost all LHCIIs are functionally connected to PSII, whereas the transition from state 1 to a state 2 chemically locked by 0.1 M sodium fluoride leads to an almost complete functional release of LHCIIs from PSII. About 2/3 of the released LHCIIs transfer energy to PSI and â¼ 1/3 of the released LHCIIs form a component designated X-685 peaking at 685 nm that decays with time constants of 0.28 and 5.8 ns and does not transfer energy to PSI or to PSII. A less complete state 2 was obtained in cells incubated under anaerobic conditions without chemical locking. In this state about half of all LHCIIs remained functionally connected to PSII, whereas the remaining half became functionally connected to PSI or formed X-685 in similar amounts as with chemical locking. We demonstrate that X-685 originates from LHCII domains not connected to a photosystem and that its presence introduces a change in the interpretation of 77 K steady-state fluorescence emission measured upon state transitions in Chalamydomonas reinhardtii.
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Chlamydomonas reinhardtii/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/metabolismoRESUMO
We establish a general kinetic scheme for energy transfer and trapping in the photosystem I (PSI) of cyanobacteria grown under white light (WL) or far-red light (FRL) conditions. With the help of simultaneous target analysis of all emission and transient absorption datasets measured in five cyanobacterial strains, we resolved the spectral and kinetic properties of the different species present in PSI. WL-PSI can be described by Bulk Chl a, two Red Chl a, and a reaction center compartment (WL-RC). The FRL-PSI contains two additional Chl f compartments. The lowest excited state of the FRL-RC is downshifted by ≈ 29 nm. The rate of charge separation drops from ≈900 ns-1 in WL-RC to ≈300 ns-1 in FRL-RC. The delayed trapping in the FRL-PSI (≈130 ps) is explained by uphill energy transfer from the Chl f compartments with Gibbs free energies of ≈kBT below that of the FRL-RC.
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PURPOSE: To validate the clinical performance of point-source corneal topography (PCT) in postpenetrating keratoplasty (PKP) eyes and to compare it with conventional Placido-based topography. METHODS: Corneal elevation maps of the anterior corneal surface were obtained from 20 post-PKP corneas using PCT (VU topographer, prototype; VU University Medical Center, Amsterdam, The Netherlands) and Placido-based topography (Keratron, Optikon 2000, Rome, Italy). Corneal surface parameters are calculated in terms of radius and asphericity. Corneal aberrations were characterized using standard Zernike convention. An artificial surface with quadrafoil feature (SUMIPRO, Almelo, The Netherlands) was measured and used as a reference to assess instrument performance compared with the gold standard. RESULTS: The differences (mean ± std of PCT - Placido) found between the two types of topographers in measurements of post-PKP eyes are 0.02 ± 0.21 mm (p=0.64) for radius of curvature, 0.14 ± 0.49 (p=0.23) for asphericity, -0.19 ± 1.67 µm (p=0.61) for corneal astigmatism, -0.25 ± 1.34 µm (p=0.41) for corneal coma, 0.23 ± 0.82 µm (p=0.23) for corneal trefoil, and 0.15 ± 0.28 µm (p=0.02) for corneal quadrafoil. The PCT measured the artificial surface more accurate (rms error 0.16 µm; 0.12 eq. Dpt.) than the Placido-based topographer (rms error 1.50 µm; 1.15 eq. Dpt.). CONCLUSIONS: PCT is more accurate than Placido-based topography in measuring quadrafoil aberration.
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Topografia da Córnea/métodos , Topografia da Córnea/normas , Transplante de Córnea , Aberrações de Frente de Onda da Córnea/diagnóstico , Cuidados Pós-Operatórios , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
A forward ray tracing (FRT) model is presented to determine the exact image projection in a general corneal topography system. Consequently, the skew ray error in Placido-based topography is demonstrated. A quantitative analysis comparing FRT-based algorithms and Placido-based algorithms in reconstructing the front surface of the cornea shows that arc step algorithms are more sensitive to noise (imprecise). Furthermore, they are less accurate in determining corneal aberrations particularly the quadrafoil aberration. On the other hand, FRT-based algorithms are more accurate and more precise showing that point to point corneal topography is superior compared to its Placido-based counterpart.
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Córnea/anatomia & histologia , Técnicas de Diagnóstico Oftalmológico , Óptica e Fotônica , Algoritmos , Simulação por Computador , Computadores , Córnea/fisiologia , Desenho de Equipamento , Humanos , Processamento de Imagem Assistida por Computador , Modelos AnatômicosRESUMO
This chapter describes the procedure for globally analyzing fluorescence lifetime imaging (FLIM) data for the observation and quantification of Förster resonance energy transfer (FRET) in live plant cells. The procedure is illustrated by means of a case study, for which plant protoplasts were transfected with different visible fluorescent proteins and subsequently imaged using two-photon excitation FLIM. Spatially resolved fluorescence lifetime images were obtained by application of global analysis using the program Glotaran, which is open-source and freely available software. Using this procedure it is possible to extract the fraction and distance of interacting species between, or conformational changes within proteins, from complex experimental FRET-FLIM datasets, even at low signal-to-noise ratios. In addition, the software allows excluding inherently present autofluorescence from the plant cells, which improves the accuracy of the FRET analysis. The results from the case study are presented and interpreted in the context of the current scientific understanding of these biological systems.
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Transferência Ressonante de Energia de Fluorescência/métodos , Microscopia de Fluorescência/métodos , Células Vegetais/ultraestrutura , Proteínas de Fluorescência Verde/química , Imagem Óptica , Protoplastos/ultraestrutura , Razão Sinal-Ruído , SoftwareRESUMO
Time-resolved fluorescence spectroscopy measurements at 77 K on thylakoid membrane preparations and isolated photosynthetic complexes thereof were investigated using target analysis with the aim of building functional compartmental models for the photosystems in the thylakoid membrane. Combining kinetic schemes with different spectral constraints enabled us to resolve the energy transfer pathways and decay characteristics of the different emissive species. We determined the spectral and energetic properties of the red Chl pools in both photosystems and quantified the formation of LHCII-LHCI-PSI supercomplexes in the transition from native to unstacked thylakoid membranes.
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Proteínas de Bactérias/química , Modelos Moleculares , Complexo de Proteínas do Centro de Reação Fotossintética/química , Tilacoides/metabolismo , Proteínas de Bactérias/metabolismo , Clorofila/química , Cianobactérias/metabolismo , Transferência de Energia , Cinética , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Complexo de Proteína do Fotossistema I/química , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/metabolismo , Espectrometria de Fluorescência , Spinacia oleracea/metabolismo , TemperaturaRESUMO
We report for the first time steady-state and time-resolved emission properties of photosystem I (PSI) complexes isolated from the cyanobacterial strain Synechococcus WH 7803. The PSI complexes from this strain display an extremely small fluorescence emission yield at 77 K, which we attribute to the absence of so-called red antenna chlorophylls, chlorophylls with absorption maxima at wavelengths longer than those of the primary electron donor P700. Emission measurements at room temperature with picosecond time resolution resulted in two main decay components with lifetimes of about 7.5 and 18 ps and spectra peaking at about 685 nm. Especially in the red flanks, these spectra show consistent differences, which means that earlier proposed models for the primary charge separation reactions based on ultrafast (â¼1 ps) excitation equilibration processes cannot describe the data. We show target analyses of a number of alternative models and conclude that a simple model (Ant2)* â (Ant1/RC)* â RP2 can explain the time-resolved emission data very well. In this model, (Ant2)* represents chlorophylls that spectrally equilibrate in about 7.5 ps and in which RP2 represents the "final" radical pair P700(+)A0(-). Adding an equilibrium (Ant1/RC)* â RP1, in which RP1 represents an "intermediate" radical pair A(+)A0(-), resulted in the same fit quality. We show that the simple model without RP1 can easily be extended to PSI complexes from cyanobacteria with one or more pools of red antenna chlorophylls and also that the model provides a straightforward explanation of steady-state emission properties observed at cryogenic temperatures.
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Proteínas de Bactérias/química , Complexo de Proteína do Fotossistema I/química , Synechococcus/metabolismo , Proteínas de Bactérias/metabolismo , Clorofila/química , Elétrons , Transferência de Energia , Complexo de Proteína do Fotossistema I/metabolismo , Espectrometria de Fluorescência , Temperatura , Tilacoides/metabolismo , Fatores de TempoRESUMO
PURPOSE: A pseudo forward ray-tracing (PFRT) algorithm is developed to evaluate surface reconstruction in corneal topography. The method can be applied to topographers where one-to-one correspondence between mire and image points can be established. METHODS: The PFRT algorithm was applied on a corneal topographer designed and constructed at the VU University Medical Center, Amsterdam, The Netherlands. Performance of the algorithm was evaluated using artificial test surfaces and two sample eyes. The residual output of the PFRT algorithm is displayed as pixel displacements of actual feature points on the corneal image. Displacement of 1 pixel indicates submicrometer corneal height accuracy. RESULTS: PFRT residual increases with complexity of the measured surface. Using Zernike radial order 6, the mean residual for the artificial surfaces is subpixel. The mean residual for the regular cornea and the irregular cornea is 1.16 and 2.94 respectively. To some extent, increasing the Zernike radial order improves the accuracy. The improvement from order 6 to 20 is factor 2.3 for the irregular cornea. Using the residuals to further improve the accuracy brought local changes as high as 0.28 D in some areas of the reconstructed corneal power map. CONCLUSION: PFRT can be used to evaluate how close a reconstructed corneal surface is to the actual one. The residue information obtained from this algorithm can be displayed simultaneously with the corneal image. This provides accurate information about the corneal shape that is useful for application in laser refractive surgery.