深紫外荧光寿命成像显微镜技术及其应用

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“Deep-UV荧光寿命成像显微镜是一种使用深紫外线（240-280纳米波长）激发的新型荧光寿命成像显微技术。这项技术被应用于测量熔融石英中由飞秒激光诱导的缺陷相关荧光及其退火过程中的变化。UV-FLIM（紫外线荧光寿命成像）可以与微流控和生物样本一起使用，以在紫外线激发下表征荧光的时间特性，这项功能可以轻松添加到标准显微镜基的FLIM系统中。” 这篇研究论文介绍了深紫外荧光寿命成像显微镜（Deep-UV FLIM）的应用和发展，这是一种利用深紫外光（240-280纳米）进行荧光激发的新型显微技术。该技术的关键在于它能够在非常短的波长下工作，这使得它能够探测到常规可见光无法检测到的特定缺陷或分子结构。 FLIM（荧光寿命成像显微镜）本身是一种非侵入性的成像技术，它通过测量荧光分子从激发态返回基态所需的时间（即荧光寿命）来获取信息。在本文中，研究人员特别关注了在熔融石英中由飞秒激光诱导的缺陷，并通过UV-FLIM观察了这些缺陷的荧光变化以及退火过程。飞秒激光诱导的缺陷可能会改变材料的光学性质，而通过FLIM技术可以精确地了解这些变化。此外，UV-FLIM技术的另一个优点是其适应性，它可以与微流控技术和生物样本结合，用于分析在紫外线激发下荧光的动态特性。这在生物医学领域特别有用，因为许多生物分子具有对紫外线响应的荧光特性。例如，它可以用来研究细胞内部的生物化学反应、标记特定的生物分子或检测细胞内环境的变化。论文的作者来自多个国际知名的研究机构，包括澳大利亚斯温伯恩科技大学的微光子学中心、荷兰埃因霍温科技大学的机械工程系、英国南安普敦大学的光电子研究中心等。他们的研究工作展示了多学科交叉合作的可能性，将光学、材料科学和生物技术结合在一起，推动了显微成像技术的前沿。 Deep-UV荧光寿命成像显微镜是一种先进的成像工具，能够提供有关材料缺陷和生物过程的详细信息，尤其在紫外线激发下的荧光动力学研究中表现出强大的潜力。这项技术的发展对于材料科学、微流控和生物医学研究具有重要意义，有可能开启新的实验方法和应用。

Deep-UV fluorescence lifetime imaging microscopy

Christiaan J. de Jong,

1,2,†

Alireza Lajevardipour,

1,†

Mindaugas Gecevičius,

3,†

Martynas Beresna,

3,†

Gediminas Gervinskas,

1,4,†

Peter G. Kazansky,

3,†

Yves Bellouard,

5,†

Andrew H. A. Clayton,

1,†

and Saulius Juodkazis

1,4,6,

,†

Centre for Micro-Photonics, Faculty of Science, Engineering and Technology, Swinburne University of Technology,

Hawthorn, VIC 3122, Australia

Department of Mechanical Engineering, Eindhoven University of Technology,

Postbus 513, 5600MB Eindhoven, The Netherlands

Optoelectronics Research Centre, University of Southampton, Southampton, SO17 1BJ, UK

Melbourne Centre for Nanofabrication, 151 Wellington Road, Clayton, VIC 3168, Australia

Ecole Polytechnique Fédérale de Lausanne, Rue de la Maladiére 71b, CH—2002 Neuchâtel, Switzerland

Center for Nanotechnology, King Abdulaziz University, Jeddah 21589, Saudi Arabia

*Corresponding author: sjuodkazis@swin.edu.au

Received July 2, 2015; revised August 21, 2015; accepted August 22, 2015;

posted August 24, 2015 (Doc. ID 244203); published September 28, 2015

A novel fluorescence lifetime imaging microscopy (FLIM) working with deep UV 240–280 nm wavelength excita-

tions has been developed. UV-FLIM is used for measurement of defect-related fluorescence and its changes upon

annealing from femtosecond laser-induced modifications in fused silica. This FLIM technique can be used with

microfluidic and biosamples to characterize temporal characteristics of fluorescence upon UV excitation, a capabil-

ity easily added to a standard microscope-based FLIM. UV-FLIM was tested to show annealing of the defects induced

by silica structuring with ultrashort laser pulses. Frequency-domain fluorescence measurements were converted into

OCIS codes: (110.0180) Microscopy; (260.7190) Ultraviolet; (350.3390) Laser materials processing.

http://dx.doi.org/10.1364/PRJ.3.000283

1. INTRODUCTION

Femtosecond laser direct writing is a powerful technique

with applications ranging from optofluidics and optome-

chanics [1–3] to birefringent optical elements [4] and optical

data storage [5]. Under certain experimental conditions,

femtosecond laser irradiation can induce self-assembled

nanostructure in the bulk of transparent material [6–8] or im-

print complex polarization fields onto a surface [9–11]. The

subwavelength arrangement of this modification results in

optical anisotropy (the form birefringence), which provides

control over polarization of traversing light; hence, polariza-

tion controlling optical elements can be made using nanograt-

ings. The nanogratings are observed only in a handful of

materials such as sapphire, tellurium oxide, germanium oxide,

and silica glasses. The mechanisms of nanograting and

surface ripple formation are closely related and are actively

researched (for a recent review [11]). The bulk ripples are

formed at subcritical free carrier plasma densities; the break-

down occurs at the permittivity ε

 0 (real part) when the

critical plasma density is reached. At a higher excitation

and over-critical plasma densities when ε

≤ −1 a surface

wave (the surface plasmon polariton) can be formed, which

is imprinted onto the surface by ablation [11]. In both cases of

the bulk and surface ripples, the period is closely following

Λ  λ

∕nI∕2, where the refractive index n is intensity, I,

dependent, and λ

is the light wavelength in vacuum [11].

Formation of the nanostructure in dielectrics is accompa-

nied by the rearrangement of chemical bonds and formation

of optically, magnetically, and electrically active defects.

Some of them, under UV irradiation, emit strong fluorescence,

which is detectable from the laser-structured areas that can

strongly impede imaging and fluorescence lifetime measure-

ments popular in bio and medical research. The fluorescence

lifetime imaging microscopy (FLIM) has become a popular

technique in cell imaging and is used for investigation of en-

ergy transfer mechanisms and can deliver a super-resolution

capability [12–14]. Addition of UV functionality to FLIM is an

appealing direction due to a possibility of direct excitation of

proteins [15] without their labeling. Further insight into ger-

micidal properties of deep-UV (240–280 nm) light [16–18]

could be achieved with UV-FLIM. LEDs are used in FLIM,

however, not in deep UV range [19].

Here, we show the UV capability, which can be added to

standard microscope-based FLIM setups. This UV-FLIM func-

tionality is demonstrated by fluorescence analysis of laser-

induced defects in silica where polarization converters were

formed by fs-laser writing. It is shown that annealing at

300°C for 2 h fully removes the fluorescence at 650 nm

and longer wavelengths without suppressing the form

birefringence required for polarization conversion. The UV-

fluorescence-lifetime-imaging method revealed the removal

of long-time relaxation components from the fluorescence

time decay in the defect-fluorescence band in silica. This ex-

pands use of fluorescence under UV excitation demonstrated

for recognition of protein crystals [20].

2. METHOD

The frequency domain realization of FLIM is based on the tem-

poral modulation of the excitation and on the synchronized

detection of the phase-shifted signal [12–14]. To characterize

de Jong et al. Vol. 3, No. 5 / October 2015 / Photon. Res. 283

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