Experimental Animal

The Development of a Device for Fluorescent Protein Imaging and Screening

Abstract

Reporter genes are widely used in current animal models in research and analysis. Among the reporter genes, fluorescent proteins are most commonly used due to their availability, stability and diverse color of choice. However, observation and data keeping often become a difficult task due to the pricy equipment, suboptimal photographic quality, and the lack of flexibility for imaging of different fluorescent wavelengths. Not to mention the difficulties in simultaneously observation object with multiple fluorescent wavelengths.

To overcome such obstacles, an imaging device was designed using macro ring light with switchable twin light sources. The macro ring light is compatible with most high-end digital cameras and some internal focusing macro DSLR lenses. To accommodate diverse needs for different fluorescent wavelengths, both light sources can be customized to specific excitation wavelengths. The device is powered by battery/AC duel design to provide flexibility and mobility. For the needs of frequent commute between barriers, the device is also designed to withstand repetitive disinfection.

In addition to the application in fluorescent protein imaging, the device can also be used to detect body fluid in forensic investigation.

Introduction

Fluorescent proteins have many advantageous features such as non-invasiveness, easiness in construct design and detection. Since its first report to use in the research, fluorescent proteins have revolutionized the research in experimentation, data observation and analysis. Tremendous efforts had invested in the improvement of the fluorescent proteins such as their intensity, number of color variant, reduced toxicity which made fluorescent proteins the preferred system to choose for studying gene activities.

Many imaging devices are available for the detection of fluorescent signal. Unfortunately, numerous limitations in the process of fluorescent observation are unavoidable that may consequently influence the data quality. For imaging specific fluorescent signals from a given object, excitation light source and emission light detection device are essential. Furthermore, acceptable imaging outcome often require experience and task, not to mention collecting meaningful data for analysis and standardization for cross laboratories comparisons. Vast majority of the imaging devices for fluorescent detection are either in simplified designed for handheld or bulky structure to ensure required power. Moreover, it can be cumbersome when simultaneous detection of multiple

fluorescent proteins is needed. In addition, most currently available imaging devices rely on human visual judgment for the captured fluorescent signal. Our goal is to overcome obstacles for optical observation. Efforts were made to build an integrated device for easy quality imaging and yet at a fordable price.

Materials and Methods

BFP,GFPemd(Emerald) and DsRed are commonly used in research and thus were used in this study. In addition, body fluids such as urine and semen are also included in this study for wider applications in related fields. Key spectrum properties of these fluorescent proteins are listed in (Table 1)

Optical devices: In order to build a flexible light source, we designed a macro ring light which can be mounted directly to the end of DSLR macro lenses, or to the bayonet ring of most high-end compact digital cameras(Figure 1). The macro ring light can be used without camera for direct observation.

The macro ring light also includes the following features: (Figure 2)

1. Specific wavelength LED: The ring light has two switchable channels thus up to two wavelengths of excitation light sources can be installed into the same device for convenient observation of multiple fluorescent proteins.
2. Light source filter: When peak excitation wavelength is very close to peak emission wavelength, it is necessary to cut off excitation light spectrum to avoid interference of the unwanted fluorescent signals. Often the intensity of excitation light source is much stronger than emission signals.
3. Image filter : In order to improve contrast of signal/noise ratio, the purpose of the image filter is to filter out excitation light from emission signals.

Animal models: Several transgenic mouse lines were generated bearing transgene with constitutive promoter expressing specific fluorescent proteins for this study. (Table 1)

Results

Fig 3-6 :

Camera with modified macro ring light. Photos of BFP, GFPemd and DsRed mice are shown in Figure 3, 4 and 5.For imaging signals other than fluorescence, the existence of body fluid is tested and can be detected (Figure 6).

Fig 7-8 :

Fluorescent signal can be detected by observation through image filter mounted on ring light (Fig 7). The same signal can be previewed or recorded via DSLR display (Fig 8). Thus, signals with extremely low intensity can be detected via long exposure. Furthermore, discrepancies from human judgment can be avoided. Since photo parameters can be shared and standardized, consequently, data exchange among laboratories become feasible.

The ring light used in this study can be powered by AC and rechargeable Li-ion battery which provide flexibility in observation. When powered by AC, a turbo feature can deliver stronger excitation energy which may be helpful to detect low level fluorescent protein.
The whole device is about 1kg in weight.

Discussion

1. For fluorescence signal observation, the sensitivity of the detection device is the most important issue. The color correctness of the fluorescence is usually a secondary concern. The color is the result relies correspondingly on the optical setting used for the imaging. Thus, comparisons of data obtained from different imaging setup should be performed with great care.
2. Single excitation waveform can excite different fluorescent proteins to emit fluorescence, however, with various efficiency. When the excitation light interference is removed, utilization of longpass filter enables simultaneous observation of multiple fluorescent proteins.
3. Controlled imaging parameters are important for reasonable comparison cross different laboratories. Firstly, identical or equivalent camera model is essential. Furthermore, white balance and ISO settings are also important in order to get the same color character between photos. In addition, aperture-priority mode is recommended to get comparable results in fluorescence intensity.
4. For users who need to commute frequently between barriers with imaging equipment, the capability of the hardware to withstand repetitive sterilization is a major concern. Ring light uses anodized aluminum and plastic parts which are durable for most sterilization process. Caution should be taken on the filter coating which is the only part vulnerable for frequent solvent disinfection.

Conclusion

Friendly optical devices for easy imaging is desirable by researchers. For imaging signals other than fluorescence, the existence of body fluid is tested and can be detected. The integrated device includes the following features:

1. Digital sensor is ideal for fluorescence detection for its superb sensitivity and allows standardization. With controlled parameters, comparable observations can be done repeatedly and cross-checked among laboratories. Since optical signals are detected and recorded through camera. Thus, signals with extremely low intensity can be detected via long exposure. Furthermore, discrepancies from human judgment can be avoided. Since photo parameters can be shared and standardized, consequently, data exchange among laboratories become feasible.
2. Longpass filter can be used to allow multiple fluorescent proteins observation. Specific setting can be used for simultaneous observation of multiple fluorescent proteins.
3. LED is narrow-band light source which can be advantageous when the excitation and emission wavelengths are close to each other. Macro ring light can be used as light source without camera.
4. The ring light used in this study can be powered by AC and rechargeable Li-ion battery which provide flexibility in observation. When powered by AC, a turbo feature can deliver stronger excitation energy which may be helpful to detect low level fluorescent protein.
5. Efforts were made to generate an affordable device that provide exchangeable data for cross laboratories comparison. Integrated design allows application flexibilities and can withstand reasonable sterilization for commuting across barriers.