Two-photon excited UV fluorescence for protein crystal detection

Abstract

Two-photon excited ultraviolet fluorescence (TPE-UVF) microscopy is explored for sensitive protein-crystal detection as a complement to second-order nonlinear optical imaging of chiral crystals (SONICC). Like conventional ultraviolet fluorescence (UVF), TPE-UVF generates image contrast based on the intrinsic fluorescence of aromatic residues, generally producing higher fluorescence emission within crystals than the mother liquor by nature of the higher local protein concentration. However, TPE-UVF has several advantages over conventional UVF, including (i) insensitivity to optical scattering, allowing imaging in turbid matrices, (ii) direct compatibility with conventional optical plates and windows by using visible light for excitation, (iii) elimination of potentially damaging out-of-plane UV excitation, (iv) improved signal to noise through background reduction from out-of-plane excitation and (v) relatively simple integration into instrumentation developed for SONICC.

Figure 1

Instrument schematics for (a) SONICC and TPE-UVF measurements, with insertion of doubling crystal allowing two-photon excitation, and (b) conventional one-photon (UV) measurements.

Figure 2

Comparison of different imaging methods for a lysozyme crystal: (a) bright-field and corresponding TPE-UVF measurements at 25 mW incident power and (b) 18 s and (c) 3 s acquisition. (d) Complementary SONICC measurements gave a weak signal, peaking at 10 000 counts s⁻¹ for ∼230 mW incident power. (e) Analysis of the TPE-UVF measurements, as indicated by the red line, shows a bright signal, producing >500 000 counts s⁻¹ for 25 mW incident power.

Figure 3

Inpact of fluorescence quenching: (a) bright-field, (b) SONICC and (c) TPE-UVF measurements of catalase crystals, with bright SHG images being generated at low power but minimal TPE-UVF signal being generated even at incident powers of >40 mW and an acquisition time of 2 min. This trend is attributed to quenching of the TPE-UVF signal by the heme chromophore in catalase.

Figure 4

Crystal detection of an ABC maltose transporter protein: (a) bright field, (b) SONICC and TPE-UVF at both (c) 3 min and (d) 3 s acquisition time. Complementary SONICC and TPE-UVF measurements demonstrate that the sample is likely to be both crystalline and comprised of protein.

Figure 5

Comparison of one- and two-photon excited UVF of an ABC maltose transporter membrane-protein crystal: (a) bright-field and (b) TPE-UVF measurements completed in situ through the crystallization-plate cover slip; (c) shows a corresponding line scan analysis showing high signal to background for moderate laser power. (d) Bright-field and (e) conventional UVF measurements on the same crystals performed after breaking the well seal and extracting a crystal to allow UVF. (f) Corresponding line scan analysis indicates weaker signal to background compared with TPE-UVF measurements.