Our protocol provides detailed 2D mapping of phosphorescence lifetime and oxygen concentration in macroscopic objects, particularly live animal tissue and whole animals, by means of dedicated probes and sensing materials. This information is important for many areas of research. Our protocol uses an integrated and compact imaging module that operates in TCSPC mode and provides easy and accurate visualization of lifetime and oxygen distribution in complex biological samples, such as life tissue.
Take the Cricket adapter and inspect it from different sides. Remove the front side C-mount adapter to show the Photonis PP0360EF intensifier housed in the Cricket, and then put the C-mount adapter back. Remove the Cricket's front side C-mount adapter and insert the 650 plus/minus 50 nanometer emission filter.
Fix it by putting back the C-mount. Then, connect the TPX three-cam camera module to the backside of the Cricket module via their ring adapters. Connect the lens to the front side of the Cricket module via the C-mount adapter.
Connect the LED to a power supply and a pulse generator. Now, mount the 390-nanometer super bright LED on a post attached to a breadboard inside the black box. Turn on the LED and focus it to ensure effective and uniform excitation of the sample being imaged.
Mount the camera assembly on top of the optical black box facing down towards the stage where the samples will be imaged. Connect the camera and LED to two-pulse generators and a four-channel oscilloscope. Using settings on the oscilloscope and pulse generators, synchronize the pulses sent to the camera and the LED.
Utilize the special cable and socket on the Cricket unit to connect the intensifier to a standard power supply and set the gain to 2.7 volts. Position the sample before the camera lens. Then, turn on the camera and all the electrical modules, except the intensifier.
Activate the SOFI software to tune the operational parameters, like focusing and sample alignment. In the modules, set frame exposure to 0.01 seconds. Select Infinite frames and set the pixel operation mode to time over threshold.
Then, go to Preview and select Active module to open the Medipix/Timepix frames window. In the next step, after starting the recording, adjust the color scale and rotate the image to the desired orientation. After that, switch off the lights in the room and switch on the intensifier.
Focus the camera optics on the sample stage with the focusing capabilities of the lens and Cricket adapter to generate clear images of samples with good contrast and brightness. Once the focus is complete, close the SOFI software. Then, go to the terminal, execute the commands of the custom-design software to acquire the raw data in the binary format and close the terminal.
Open a new terminal to process the acquired data. Load the data file and run the data reducer. Analyze the post-process data with a dedicated program written in C language that will write the data into a ICS image file.
Subsequently, open the ICS image files using the freely-available Time Resolved Imaging software, and use two exponential functions to fit the phosphorescence decays. Lastly, open the Fitted. ics image files with the available image analysis software.
Generate phosphorescence lifetime images using lookup tables and code them in pseudo color scale. Utilize the Measure function to calculate the average lifetime values for the entire image or regions of interest. Phosphorescence intensity and phosphorescence lifetime imaging microscopy images of the platinum octaethylporphyrin dye sensor spot in the oxygenated and deoxygenated states are shown.
Make sure that the right emission filter is mounted inside the Cricket and the right LED, cable connections, and pulse settings are used. Currently, this protocol is limited to animal models, but potentially can be applied to diagnose and treat pathological states associated with tissue hypoxia, such as cancer, stroke, and inflammation.
