The study of gene function has been greatly facilitated by the ability to conditionally regulate gene expression at different locations and times throughout the life cycle. A variety of genetic tools that allow such temporal and/or spatial control of gene expression have been developed for study of gene function in several model organisms. These include site-specific recombination using cre or flp recombinases [1
], tetracycline inducible systems [4
], and the Gal4/UAS system [5
]. Another system for conditional gene regulation takes advantage of the cellular heat shock response, which leads to the transcription of genes that allow cells to tolerate brief periods of stress, or to activate cell death pathways when these stresses are too extreme [7
]. One particularly well studied heat shock inducible gene is the hsp70l
gene, (formerly known as hsp70
) which encodes a chaperonin that functions in protein folding mechanisms [9
]. The hsp70l
promoter activates transcription when cellular temperatures are raised by 10-15°C and has been used to analyze gene function at different times in the life cycle [e.g. [10
]]. Global heat shock provides temporal control of gene expression [14
], but does not allow the spatial control necessary for the analysis of region, tissue, and cell specific gene function. Methods for local tissue heating would allow the study of gene function in different tissues throughout the life cycle.
Laser-based techniques for controlled heating in small regions of biological samples are desirable, mainly because of the possibility for precise targeting and control of laser light. The most straight-forward approach uses existing microscopy setups to tightly focus laser light onto tissue to induce local heating [10
]. The main drawback of this technique is the low absorption coefficient of biological tissues in the near-infrared wavelength range (close to that of water at ~1 cm-1
), where typical solid state or diode lasers operate. For example, within 10 μm penetration depth only 0.1% of the laser power is deposited. To achieve temperature differences of 10-15°C, a typical laser with power in the Watt range is required, and this comes at a significant cost. In addition, precise control and calibration of the temperature is difficult, since exact laser light absorption of biological materials is not well known and random light scattering in biological samples affects the laser power at the target location. A solution for achieving heating with lower laser powers has been proposed by Zondervan et al., who used a metallic substrate that strongly absorbs laser light to heat the environment [18
]. However, this approach is not very practical for biological studies as it does not allow three-dimensional positioning of the local heat source. Recently, an infrared laser was used to locally activate hsp70
transgene expression in C. elegans
]. This method is effective, but has similar drawbacks, including difficulties in calibrating heating temperatures and high cost.
The zebrafish has emerged as a powerful genetic system for the study of vertebrate development, with the accessible embryo possessing many features that allow real-time observations and experimental manipulations. The zebrafish hsp70l
promoter has been used to temporally regulate transgene expression by raising the temperature to 37°C at any time during development [14
]. Both spatial and temporal regulation of a hsp70l:GFP
transgene was demonstrated using a pulsed blue dye laser focused through a microscope lens [15
]. However, this system has had limited utility due to issues of efficacy and cell viability. Spatio-temporal control of hsp70l
transgene expression was also achieved using a simple soldering iron heating device in zebrafish [20
], but spatial control is limited by the size of the soldering iron tip and this system is most useful for gene activation in superficial cells. In addition, the high thermal mass of this device necessitates general tissue cooling using a reservoir.
Our aim was to develop a simple micron-scale optical heater that would allow reproducible and convenient local heating anywhere in the zebrafish embryo without causing cell death. By combining an optical fiber based approach with a low-power laser source, we created an inexpensive local heat source that has a well-defined temperature and can be precisely positioned in biological tissue. Moreover, the area of local heating can be controlled by "pulling" optical fibers with a standard electrode puller, and these fine tips can then be positioned almost anywhere in the embryo or larvae. The temperature at the fiber tip can be precisely measured by a thermocouple-based thermometer. This microheater is simple, highly controllable, and induces gene expression without causing tissue damage or cell death. The ability to precisely control heating on the micrometer scale will have broad applications for the study of gene function and may have wide-ranging utility in the fields of medicine and materials science.