Fluorescence Lifetime Macro Imager for Biomedical Applications

Summary

This paper describes the use of a new, fast optical imager for the macroscopic photoluminescence lifetime imaging of long decay emitting samples. The integration, image acquisition, and analysis procedures are described, along with the preparation and characterization of the sensor materials for the imaging and the application of the imager in studying biological samples.

Abstract

This paper presents a new photoluminescence lifetime imager designed to map the molecular oxygen (O₂) concentration in different phosphorescent samples ranging from solid-state, O₂-sensitive coatings to live animal tissue samples stained with soluble O₂-sensitive probes. In particular, the nanoparticle-based near-infrared probe NanO2-IR, which is excitable with a 625 nm light-emitting diode (LED) and emits at 760 nm, was used. The imaging system is based on the Timepix3 camera (Tpx3Cam) and the opto-mechanical adaptor, which also houses an image intensifier. O₂ phosphorescence lifetime imaging microscopy (PLIM) is commonly required for various studies, but current platforms have limitations in their accuracy, general flexibility, and usability.

The system presented here is a fast and highly sensitive imager, which is built on an integrated optical sensor and readout chip module, Tpx3Cam. It is shown to produce high-intensity phosphorescence signals and stable lifetime values from surface-stained intestinal tissue samples or intraluminally stained fragments of the large intestine and allows the detailed mapping of tissue O₂ levels in about 20 s or less. Initial experiments on the imaging of hypoxia in grafted tumors in unconscious animals are also presented. We also describe how the imager can be re-configured for use with O₂-sensitive materials based on Pt-porphyrin dyes using a 390 nm LED for the excitation and a bandpass 650 nm filter for emission. Overall, the PLIM imager was found to produce accurate quantitative measurements of lifetime values for the probes used and respective two-dimensional maps of the O₂ concentration. It is also useful for the metabolic imaging of ex vivo tissue models and live animals.

Introduction

O₂ is one of the key environmental parameters for living systems, and knowledge of the distribution of O₂ and its dynamics is important for many biological studies^1,2,3. The assessment of tissue oxygenation by means of phosphorescent probes^4,5,6,7,8 and PLIM^{9,10,11,12,13} are gaining popularity in biological and medical research^{3,9,14,15,16,17,18,19}. This is because PLIM, unlike fluorescence or phosphorescence intensity measurements, is not affected by external factors such as probe concentration, photobleaching, excitation intensity, optical alignment, scattering, and autofluorescence.

However, current O₂ PLIM platforms are limited by their sensitivity, image acquisition speed, accuracy, and general usability. Time-correlated single photon counting (TCSPC), combined with a raster scanning procedure, is frequently used in PLIM and fluorescence lifetime imaging microscopy (FLIM) devices^20,21,22. However, since PLIM requires a long pixel dwell time (in the millisecond range), the time of image acquisition is much longer than what is required for FLIM applications^20,22,23. Other techniques, such as gated CCD/CMOS cameras, lack single photon sensitivity and have low frame rates^20,24,25,26. Moreover, the existing PLIM systems are mostly used in the microscopic format, while macroscopic systems are less common²⁷.

The TCSPC-based PLIM macro imager²⁸ was set up to overcome many of these limitations. The design of the imager was greatly facilitated by the use of a new opto-mechanical adapter, Cricket, which has the following: i) two C-mount adapters, which provide easy coupling of the camera module on the back side and objective lens on the front side; ii) an internal housing for an image intensifier and a power socket for the latter on the outer side of the Cricket; iii) an internal space behind the front-side C-mount adapter where a standard 25 mm emission filter can be housed in front of the intensifier; and iv) a built-in light collimating optics with ring regulators, which allow optical alignment/focusing between the lens and the camera to produce crisp images on the camera chip.

In the assembled imager, the camera module is coupled to the back side of the Cricket adapter, which also houses an image intensifier consisting of a photocathode followed by a microchannel plate (MCP), an amplifier, and a fast scintillator, P47 phosphor. A 760 nm ± 50 nm emission filter is fitted inside the Cricket, and an objective lens, NMV-50M11'', is attached to the front side C-mount adapter. Finally, the lens and the camera are aligned optically with ring regulators.

The role of the intensifier is to detect incoming photons and convert them into fast bursts of light on the camera chip, which are registered and used to generate emission decays and lifetime images. The camera module comprises an advanced TCSPC-based optical sensor array (256 pixels x 256 pixels) and a new generation readout chip^{29,30,31,32,33}, which allow the simultaneous recording of the time of arrival (TOA) and the time over threshold (TOT) of photon bursts at each pixel of the imaging chip with a time resolution of 1.6 ns and an 80 Mpixel/s readout rate.

In this configuration, the camera with the intensifier has single-photon sensitivity. It is data-driven and based on the speedy pixel detector readout (SPIDR) system³⁴. The spatial resolution of the imager was previously characterized with planar phosphorescent O₂ sensors and a resolution plate mask. The instrument response function (IRF) was measured by the imaging of a planar fluorescent sensor under the same settings as used for all the other measurements. The lifetime of the dye of around 2.6 ns was short enough for it to be used for the IRF measurement in PLIM mode. The imager can image objects of up to 18 mm x 18 mm in size with spatial and temporal resolutions of 39.4 µm and 30.6 ns (full width at half-maximum), respectively²⁸.

The following protocols describe the assembly of the macro imager and its subsequent use for mapping the O₂ concentration in biological samples stained with the previously characterized near-infrared O₂ probe, NanO2-IR³⁵. The probe is a bright, photostable, cell-permeable O₂-sensing probe based on platinum (II) benzoporphyrin (PtBP) dye. It is excitable at 625 nm, emits at 760 nm, and provides a robust optical response to O₂ in the physiological range (0%-21% or 0-210 µM of O₂). The imager is also demonstrated to characterize different sensor materials based on Pt(II)-porphyrin dyes. Overall, the imager is compact and flexible, similar to a common photographic camera. In the current setup, the imager is appropriate for different widefield PLIM applications. Substituting the LED with a fast laser source will further improve the performance of the imager and could potentially enable nanosecond FLIM applications.

Protocol

All the procedures with animals were performed under authorizations issued by the Health Products Regulatory Authority (HPRA, Ireland) in accordance with the European Communities Council Directive (2010/63/EU) and were approved by the Animal Experimentation Ethics Committee of the University College Cork.

1. Sample preparation

1. Staining with the probe of live tissue samples ex vivo​
   1. For ex vivo applications, use freshly isolated tissue samples from 4 week old female Balb/c mice.
   2. On the day of the experiment, euthanize a mouse by decapitation, and quickly dissect fragments of the colon (large intestine), approximately 10 mm in size. Wash them immediately with PBS buffer, place in DMEM medium supplemented with 10 mM Hepes buffer (pH 7.2), and incubate at 37 °C³⁶.
   3. For surface staining of the serosal side of the intestine, transfer the live tissue samples into a mini-dish, apply 2 mL of complete DMEM containing 1 mg/mL NanO2-IR probe to cover the tissue samples, and incubate for 30 min at 37 °C.
      NOTE: Cells in post-mortem tissue remain live for many hours in culture. NaNO2-IR shows minor cytotoxicity, so all the experiments were completed within 4 h after tissue isolation.
   4. For deep tissue intraluminal ex vivo staining, transfer the pieces of the intestine to a dry Petri dish, and remove any excess DMEM with filter paper.
   5. Inject 1 µL of DMEM containing 1 mg/mL NanO2-IR³⁵ into the lumen with a Hamilton syringe, and incubate the samples for 15 min or for up to 4 h.
      NOTE: NaNO2-IR shows minor long-term cytotoxic effects; therefore, all the experiments should be completed within 4 h after tissue isolation.
2. Preparation of stained tumor tissue in live animals
   1. For in vivo applications, pre-stain CT26 cells for 18 h in serum-free medium containing NaNO2-IR probe at 0.05 mg/mL.
   2. Take a mouse, shave the area of injection in the right flank, and inject with a syringe 200 µL of a mixture of 1 × 10⁵ non-stained cells and 1 × 10⁵ cells pre-stained with NanO2-IR .
   3. Allow tumors to grow in the mice, monitoring the tumor size with a caliper and the animal weight periodically³⁷. The animals with grafted tumors become ready for imaging on the seventh day of tumor growth.
      NOTE: The tumor volume was calculated using equation (1):
      V = (L × W²)/2     (1)
      ​where L is the diameter of the tumor, and W is the diameter perpendicular to the diameter L.
   4. Sacrifice the animals by cervical dislocation just before the imaging.

2. PLIM imaging setup

1. Take the Cricket adaptor, and remove its back side C-mount adapter to gain access to the intensifier housing inside. Insert the MCP-125 image intensifier into this compartment, and put the C-mount adaptor back.
2. Remove the Cricket's front side C-mount adaptor, insert the 760 nm ± 50 nm emission filter, and fix it by putting back the C-mount.
3. Connect the Tpx3Cam camera module to the back side of the Cricket module via its C-mount adaptor
4. Connect the lens to the front side of the Cricket module via its C-mount adaptor.
5. Mount the whole camera assembly on top of the optical black box, facing down to the stage on which the samples will be imaged (Figure 1).
6. Mount the 624 nm super-bright LED on a post connected to a breadboard inside the black box.
7. Connect the LED to a power supply and a pulse generator. Switch on the LED, and focus it to ensure effective and uniform excitation of imaged samples.
8. Connect the camera to another pulse generator, and synchronize the pulses sent to the camera and the LED³⁸.
9. Using the special cable and socket on the Cricket unit, connect the intensifier to a standard power supply, and set the gain to 2.7 V.
10. Using the focusing capabilities of the lens and Cricket adaptor, focus the camera optics on the sample stage to generate clear images of samples with good contrast and brightness.
11. For imager use with Pt-porphyrin dyes, replace the 625 nm LED with a 390 nm LED for excitation, and replace the 760 nm ± 50 nm filter with a 650 nm ± 50 nm filter in the Cricket module.

3. Image acquisition

1. Place the sample in front of the camera lens.
   NOTE: Use an x-y-z adjustable stage as the sample holder in order to adjust the sample position for good focus.
2. Turn off all the lights in the room.
3. Switch on the Sophy software for tuning the operational parameters, such as the focusing and sample alignment.
   NOTE: Sophy software is provided together with the camera to set up the imaging parameters and record the data. Make sure that the software is connected to the camera by checking the camera code. However, we used a different program for data acquisition.
4. In Modules, select infinite frames, and set the pixel operation mode to time over threshold.
5. In Modules, go to Preview, and select Active module. This opens the Medpix/Timpix Frames window.
6. In this window, change the color scale, and rotate the image to the desired orientation.
7. Switch on the intensifier, and start the recording.
   NOTE: Use the recording screen of the Sophy software to visually confirm the alignment and focus of the sample and to optimize the LED excitation parameters for recording.
8. Stop the recording, and close the Sophy software.
9. Go to the terminal, and use the custom-designed software to acquire the raw data in the binary format and post-process it (https://github.com/svihra/TimePix3).
   1. In the terminal, run the following commands to record the data:
      Cd Document/SPIDR/trunk/Release/
       ls
      ./Tpx3daq - i 1 - b 50 - m - s {Name of the file} - t {acquisition time}
      NOTE: Upon typing "ls," check the list of files in the current directory; confirm that Tpx3daq is seen.
   2. Wait until all the frames are recorded.
   3. To process the data, run the following commands in the terminal:
      cd Documents/DataProcessing/Timepix3/Timepix3/
      root
      .L dataprocess.cpp++
      ​.x DRGUI.C
   4. Wait for an RRGui window to open. Select all the variables on the left and All Data, Single File, and Centroid on the right.
   5. Select the file to process and run the data reducer.
      ​NOTE: All the processed files will appear in the same folder as the raw file.

4. Data analysis

1. Analyze the post-processed data with a dedicated program written in C-language that will write the data into an .ics image file (https://github.com/lmhirvonen/timepix3cam).
2. Open the .ics image files using the freely available Time Resolved Imaging software (see Table of Materials). Use two-exponential functions to fit the phosphorescence decays.
3. Open the fitted .ics image files with the available image analysis software (see Table of Materials).
4. Using Lookup Tables, generate phosphorescence lifetime images, and encode them in pseudocolor scale (e.g., blue color for short lifetimes and red for long lifetimes). Use the Measure function to calculate the average lifetime values for the entire image or specific regions of interest (ROIs).
5. Convert the lifetime values into the oxygen concentration by using the equation obtained from the fitting of the O₂ calibration of the probe³⁶.
   NOTE: Equation (2) was used for this work:
   O₂ [µM] = −86.16 + 770.35 × e^{−0.049 × LT}     (2)

Results

For ex vivo imaging applications, fragments of intestinal tissues were stained by the topical application of the NanO2-IR probe on the serosal side of the tissue. For deeper staining, 1 µL of the probe was injected into the lumen. In the latter case, the 0.2-0.25 mm thick intestinal wall shielded the probe from the camera. The two staining processes are demonstrated in Figure 2A.

The resulting intensity and PLIM images are presented in

Discussion

The above protocols give a detailed description of the assembly of the new imager and its operation in the microsecond FLIM/PLIM mode. The TCSPC-based new generation Tpx3Cam camera, coupled by means of the opto-mechanical adaptor Cricket with the image intensifier, emission filter, and macro-lens, produces a stable, compact, and flexible optical module that is easy to operate. The imager was shown to perform well with a range of different samples and analytical tasks, which included the characterization of phosphorescent...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
627 nm LED	Parts Express		Can be replaced with different LED based on the excitation wavelength of the sensor. Used 390 nm LED for Pt-porphyrin dyes.
760 ± 50 nm emission filter	Edmund Optics	84-788	Can be replaced with different filter based on the emission wavelength of the sensor. Used 650 ± 50 nm bandpass filter for Pt-porphyrin dyes.
Balb/c mice	Envigo, UK	Balb/c
Black box	Thorlabs	XE25C9/M
Cricket Adapter	Photonis	Cricket-2
CT26 cells	ATCC	CT26.WT	https://www.atcc.org/products/crl-2638
DMEM	Sigma-Aldrich	D0697	Other media can also be used
ImageJ Software	ImageJ		Free Image analysis software. Can be downloaded from: https://imagej.nih.gov/ij/index.html
MCP-125 image intensifier with P47 phosphor screen	Photonis	PP0360EF
Mini dishes	Sarstedt	83.3900.300	35 mm diameter
Mylar plastic film, 75 micron	RS Ireland	785-0795	Othe plastic substrates can also be used
NanO2-IR	home-made	n/a	The probe can be synthesised according to the published method 'Tsytsarev V, Arakawa H, Borisov S, Pumbo E, Erzurumlu RS, Papkovsky DB. In vivo imaging of brain metabolism activity using a phosphorescent oxygen-sensitive probe. J Neurosci Methods. 2013 Jun 15;216(2):146-51. doi: 10.1016/j.jneumeth.2013.04.005. Epub 2013 Apr 25. PMID: 23624034; PMCID: PMC3719178.' or provided by our lab.
NMV-50M11” 50 mm lens	Navitar		Other lenses compatibel with C-mount adators can be used
Optical breadboard	Thorlabs	MB1836
Petri Dishes	Sarstedt	82.1472.001	92 mm diameter
Power Supply	Tenma	72-10495
Pulse Generator	Tenma	TGP110
Sophy	Amsterdam Scientific Instruments	n/z	Provided by ASI together with the Tpx3Cam
Tpx3Cam	Amsterdam Scientific Instruments	TPXCAM
Tri2 Software	University of Oxford	n/a	Free Time Resolved Imaging software, can be downloaded from: https://users.ox.ac.uk/~atdgroup/index.shtml
XYZ Translation Stage	Thorlabs	LT3