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

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 frag....

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