Imaging of Protein Crystals with Two-Photon Microscopy

Padayatti, Pius S.; Palczewska, Grażyna; Sun, Wenyu; Palczewski, Krzysztof; Salom, David

doi:10.1021/bi201682q

Cited by 27 publications

(19 citation statements)

References 50 publications

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“…S4). The resulting spectra did not contain any SHG signature and were found to be consistent with spectra from photo-activated rhodopsin previously published for 1PO and 2PO excitation (15,33). Thus, these biochemical experiments rule out SHG from visual pigments either in a crystal or solution as the cause of visual pigment activation by IR light.…”

Section: Resultssupporting

confidence: 88%

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Human infrared vision is triggered by two-photon chromophore isomerization

Palczewska

Vinberg

Stremplewski

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Vision relies on photoactivation of visual pigments in rod and cone photoreceptor cells of the retina. The human eye structure and the absorption spectra of pigments limit our visual perception of light. Our visual perception is most responsive to stimulating light in the 400-to 720-nm (visible) range. First, we demonstrate by psychophysical experiments that humans can perceive infrared laser emission as visible light. Moreover, we show that mammalian photoreceptors can be directly activated by near infrared light with a sensitivity that paradoxically increases at wavelengths above 900 nm, and display quadratic dependence on laser power, indicating a nonlinear optical process. Biochemical experiments with rhodopsin, cone visual pigments, and a chromophore model compound 11-cis-retinyl-propylamine Schiff base demonstrate the direct isomerization of visual chromophore by a two-photon chromophore isomerization. Indeed, quantum mechanics modeling indicates the feasibility of this mechanism. Together, these findings clearly show that human visual perception of near infrared light occurs by twophoton isomerization of visual pigments.visual pigment | two-photon absorption | rhodopsin | transretinal electrophysiology | multiscale modeling H uman vision is generally believed to be restricted to a visible light range, although >50% of the sun's radiation energy that reaches earth is in the infrared (IR) range (1). Human rod and cone visual pigments with the 11-cis-retinylidene chromophore absorb in the visible range, with absorption monotonically declining from their maxima of 430-560 nm toward longer wavelengths. The spectral sensitivity of human dim light perception matches well with the absorption spectrum of the rod visual pigment, rhodopsin (2, 3). Activation of visual pigments is temperature independent around their absorption peaks (λ max ), but at longer wavelengths, the lower energy photons must be supplemented by heat to achieve chromophore photoisomerization (4). Long wavelength-sensitive visual pigments of vertebrates exhibit maximal absorption at the ∼500-to ∼625-nm range. Pigments with λ max > 700 nm are theoretically possible, but the high noise due to spontaneous thermal activation would render them impractical (5). At human body temperature and with 1,050-nm stimulation, the sensitivity of the peripheral retina to one-photon (1PO) stimulation is less than 10 −12 of its maximum value at 505 nm (4, 6). Indeed, reports about human IR vision can be found in the literature, although they are fragmentary and do not describe the mechanism of this phenomenon.With the invention of radar during World War II, it was immediately questioned if pilots could detect high intensity radiation in the IR range of the spectrum. Wald and colleagues reported that at wavelengths above 800 nm, rod photoreceptors become more sensitive than cones, resulting in perception of IR signals as white light selectively in the peripheral retina (6). They proposed that relative spectral sensitivity declines monotonically toward longer wavele...

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Section: Resultssupporting

confidence: 88%

“…Additionally, emission of photons caused by 2PO absorption in rhodopsin is possible (Fig. S1) (33). Excitation of rod cells with a 1,000-nm light caused emission with maximum at ∼560 nm.…”

Section: Discussionmentioning

confidence: 99%

Human infrared vision is triggered by two-photon chromophore isomerization

Palczewska

Vinberg

Stremplewski

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…The pure CLLCs, without any Au‐inclusions, show a bright white emission which is spectrally peaked around 570 nm. Similar emission spectra have been reported in several recent studies and have been attributed to the emission from delocalized electrons engaged in hydrogen bonding 30–33. The emission is generic for different types of proteins and occurs both in solutions and single‐crystals of proteins.…”

Section: Resultssupporting

confidence: 86%

Plasmonic Response of Ag‐ and Au‐Infiltrated Cross‐Linked Lysozyme Crystals

Muskens

England

Danos

et al. 2012

Adv Funct Materials

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Metal‐infiltrated protein crystals form a novel class of bio‐nanomaterials of great interest for applications in biomedicine, chemistry, and optoelectronics. As yet, very little is known about the internal structure of these materials and the interconnectivity of the metallic network. Here, the optical response of individual Au‐ and Ag‐infiltrated cross‐linked lysozyme crystals is investigated using angle‐ and polarization‐dependent spectroscopy. The measurements unequivocally show that metallic inclusions formed inside the nanoporous solvent channels do not connect into continuous nanowires, but rather consist of ensembles of isolated spheroidal nanoclusters with aspect ratios as high as a value of four, and which exhibit a pronounced plasmonic response that is isotropic on a macroscopic length scale. Fluorescence measurement in the visible range show a strong contribution from the protein host, which is quenched by the Au inclusions, and a weaker contribution attributed to the molecule‐like emission from small Au‐clusters.

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“…This imaging technique requires a femtosecond pulsed laser to exploit the frequency-doubling effect. Second-harmonic generation can also be complemented by two-photon excited fluorescence (TPEF) caused by the oxidation of tryptophan residues for even better contrast [33]. These techniques have been evaluated for crystals grown in lipidic mesophases, such as LCP [34] where the opacity of the crystallization media prevents visual analysis of crystallization assays.…”

Section: Robots For Protein Crystallizationmentioning

confidence: 99%

Recent progress in robot-based systems for crystallography and their contribution to drug discovery

Ferrer¹,

Larive²,

Bowler

et al. 2013

Expert Opinion on Drug Discovery

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The evolution in robot technology, together with progress in X-ray beam performance and software developments, contributes to a new era in macromolecular X-ray crystallography. Highly integrated experimental environments open new possibilities for crystallography experiments. It is likely that it will also change the way this technique will be used in the future, opening the field to a larger community.

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Imaging of Protein Crystals with Two-Photon Microscopy

Cited by 27 publications

References 50 publications

Human infrared vision is triggered by two-photon chromophore isomerization

Human infrared vision is triggered by two-photon chromophore isomerization

Plasmonic Response of Ag‐ and Au‐Infiltrated Cross‐Linked Lysozyme Crystals

Recent progress in robot-based systems for crystallography and their contribution to drug discovery

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