Elsevier

Cell Calcium

Volume 37, Issue 6, June 2005, Pages 565-572
Cell Calcium

Flash photolysis using a light emitting diode: An efficient, compact, and affordable solution

https://doi.org/10.1016/j.ceca.2005.03.001Get rights and content

Abstract

Flash photolysis has become an essential technique for dynamic investigations of living cells and tissues. This approach offers several advantages for instantly changing the concentration of bioactive compounds outside and inside living cells with high spatial resolution. Light sources for photolysis need to deliver pulses of high intensity light in the near UV range (300–380 nm), to photoactivate a sufficient amount of molecules in a short time. UV lasers are often required as the light source, making flash photolysis a costly approach. Here we describe the use of a high power 365 nm light emitting diode (UV LED) coupled to an optical fiber to precisely deliver the light to the sample. The ability of the UV LED light source to photoactivate several caged compounds (CMNB-fluorescein, MNI-glutamate, NP-EGTA, DMNPE-ATP) as well as to evoke the associated cellular Ca2+ responses is demonstrated in both neurons and astrocytes. This report shows that UV LEDs are an efficient light source for flash photolysis and represent an alternative to UV lasers for many applications. A compact, powerful, and low-cost system is described in detail.

Introduction

The flash photolysis technique allows the fast and spatially defined application of bioactive molecules and is therefore a powerful tool for the dynamic study of the molecular mechanisms underlying physiological processes at the cellular level. It can be combined to electrophysiological techniques (e.g. patch-clamp) or cellular imaging techniques (e.g. fluorescence microscopy) to monitor cellular responses to photoactivation of the caged molecules. Caged compounds are composed of active molecules that are covalently bound to a photoabsorbing group resulting in a photolabile, biologically inert molecule. Upon UV illumination, the photolabile caged compound releases the free, biologically active molecule along with the free caging group [1]. Several classes of compounds such as neurotransmitters, nucleotides, Ca2+ chelators, fluorescent dyes, or second messengers are commercially available as caged compounds and can be exploited for a wide panel of biological applications [2]. Flash photolysis of caged compounds has many advantages for the study of living cells in real time. For example, caged versions of compounds such as IP3, cyclic AMP or caged Ca2+, can be loaded into the cell and released inside the cell. The photochemical reaction is very fast, usually ranging from submicroseconds to milliseconds, and can be triggered at any moment during the course of the experiment in spatially defined regions of the specimen [3].

Caged compounds are photolyzed in the 300–380 nm range of the UV spectrum [3], and require the use of powerful light sources either pulsed or continuous. Xenon [4], [5], [6] or mercury [7], [8], [9], [10] arc lamps coupled to a electrical or mechanical shutter, as well as flash lamps [11], [12], [13] that provide short pulses of UV light, are often used coupled to a microscope via the epifluorescent port, producing uncaging in defined areas [3]. Because high power flash lamps produce large electromagnetic artifacts and need up to several seconds to re-charge, they are incompatible with certain experimental approaches as discussed by others [14]. The main advantage of flash lamps or arc lamps is the lower cost compared to UV lasers [3].

Lasers of various types (pulsed frequency tripled Nd:YAG lasers [15], frequency doubled ruby laser [16], nitrogen laser [17], [18]), or continuous wave argon laser [17], [19]), have been the source of choice for flash photolysis, since their higher luminous density enables them to release caged compounds in small spatial domains using brief pulses of light. Two-photon excitation using femtosecond infrared lasers is also used for flash photolysis [20], [21] because it allows releasing caged compound in a diffraction-limited volume at the focal point of objectives [22]. Despite the growing interest for flash photolysis, the high cost of UV lasers and the difficulty of implementing the technique have hindered the widespread use of this technique.

Light emitting diode (LED) could be an alternative UV light source for flash photolysis as recent innovations in the semiconductor industry has seen the production of a device that can emit UV light of wavelengths and powers potentially compatible with photolysis applications. The cost of these devices is considerably lower than UV lasers and flash lamps. In the present report, we demonstrate the feasibility of using a high power UV LED as a light source for flash photolysis with several applications for intracellular calcium (Cai2+) homeostasis.

Section snippets

Cell culture

Cortical astrocytes in primary culture were obtained from 1- to 3-day-old OF1 mice as described previously [23]. Briefly, after microdissection and dissociation of the cortex, cells were grown for 2–5 weeks on 12 mm glass coverslips in DME medium (Gibco) containing 25 mM glucose, and supplemented with 10% FCS, penicillin, streptomycin and amphotericin. Mouse cortical primary cultures of neurons were performed with E17 mouse embryos, as previously described [24], [25]. After removing meninges,

Results

Several commercially available models of UV LEDs have been tested in our laboratory over the past few years for their potential usefulness as light source for photolysis. Even though most of them were able to uncage CMNB-caged fluorescein (CMNB-fluorescein), the 100 mW 365 nm UV LED recently released by Nichia was the first to combine the adequate wavelength and power for flash photolysis applications. Therefore, this report will focus on this particular UV LED model.

Discussion

LEDs were first introduced commercially in the 1960s and emitted infrared and then red light. From that point on, the semiconductor industry has put enormous efforts in the development of LEDs with always shorter wavelengths and with improved efficiency and power. The most recent innovations in the LED technology are devices that can emit UV light.

The material used for manufacturing the semiconductor core of modern LEDs, which determines the wavelength of emitted light, is usually a combination

Acknowledgements

The authors wish to thank Michel Saint-Ghislain for his help with fiber cleaving, to Benjamin Rappaz for providing neuron cultures, and to Graham W. Knott for his reading of the manuscript. This work was supported by SNF grant #3100-067116 to J.-Y.C.

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