The Development of a Device for Fluorescent Protein Imaging and Screening
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 macrolenses, 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)
- 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.
- 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.
- 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 macro right light. Photos of BFP, GFPemd and DsRed mice are shown in Figure 3, 4 and 5. For imaging signals other that flurexcence of body fuild is tested and can be detected(Figure 6).
Fig 7-8: Fluorescent signal can be detected by observation through image filter mounted on right light(Fig.7). The same signal can be previewed or recorded via DSLR display(Fig.8). Thus, signals with extreamely low intensity can be detected via long exposure.
Futhermore, discrepancies from human judgment can be shared and standardized, consequently, dataexchange among laboratories become feasible.
The right 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 be deliver stronger excitation energy which may be helpful to detect low level flurescent protein. The whole device is about 1kg in weight.
Conclusion
Friendly optical device for easy imaging is desirable by researches. For imaging signals other than flurescence, the existence of body fluid is tested and can be detected. The intergrated device includes the following features:
- Digital sensor is ideal for flurescence detection for its sensitivity and allows standartisation. With controlled parametrs, comparable observations can be done repeatedly and cross-checked among laboratories. Since optical signals are detected and recorded trough camera. Thus, signals with extremely low intensity can be detected via long exposure. Futhermore, discrepancies from human judgment can be avoided. Since photo parameters can be shared and standartized, consequently, data exchange among laboratories become feasible.
- Longpass filter can be used to allow multiple flurescent proteins observation. Specific settings can be used for simultaneous observation of multiple flurescent protein.
- LED is narrow-band light source which can be advantageous when the excitation and emission wavelength are close to each other. Macro right light can be used as light source without camera.
- the right 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 datect low level flurescent protein.
- 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 barriers.



