APPLICATION NOTE
© 2014 Princeton Instruments, Inc. All rights reserved.

Real-Time Imaging of Singlet Oxygen via Innovative
Microspectroscopy Instrument

New Two-Dimensional InGaAs Detector Thermoelectrically Cooled to –85°C
Facilitates Scientific Research“An innovative experimental
setup developed and used at
Charles University in Prague
now permits researchers
to perform direct, real-time
imaging of singlet oxygen
while addressing the issueof potential spectral overlap
with emitted light from thephotosensitizing agent.”

Introduction
Molecular oxygen is one of the most important molecules in maintaining life as well as inmechanisms by which life is extinguished and materials destroyed. For several decades,
researchers have been intrigued by the physical and chemical properties of molecularoxygen’s lowest excited state, singlet oxygen (1O2). In particular, singlet oxygen has a uniquereactivity that can result in polymer degradation or the death of biological cells. Its role as anintermediate in cell death is exploited by photodynamic therapy (PDT) for cancer, a techniquein which light is utilized as a medical tool1,2.
In PDT, a photosensitizer (PS) is incorporated into abnormal tissues and then irradiated withvisible light so that it transfers energy to ground-state oxygen via the type II photochemical
pathway, producing singlet oxygen (which can be directly detected by its weak 1270 nm
emission)3. Owing to the special interest in elucidating the biochemical action of singlet oxygenon the subcellular level, several high-spatial-resolution methods have been proposed to detect
1O2 luminescence using either a single photomultiplier tube (PMT), a linear InGaAs detector
array, or a two-dimensional InGaAs detector3.
In this application note, a novel microspectroscopy setup that uses a NIRvana:640
InGaAs camera from Princeton Instruments is evaluated by Dr. Marek Scholz of Charles
University (Prague, Czech Republic). Dr. Scholz reports that the new NIRvana® camera’s
two-dimensional InGaAs array has allowed himself and fellow members of the Optical
Spectroscopy Group headed by Prof. Jan Hála to address the issue of potential spectral
overlap of 1O2 phosphorescence with the NIR-extended luminescence of the PS, thus
providing an effective means of distinguishing and separating them for the first time4.

Experimental Setup
The experimental setup utilizes two detection channels (VIS and NIR) to perform realtime imaging of the very weak near-infrared phosphorescence of singlet oxygen andphotosensitizer simultaneously with the visible fluorescence of the photosensitizer. This
innovative setup (see Figure 1) enables acquisition of spectral images based on singlet
oxygen and photosensitizer luminescence from individual cells. One dimension of the image isspatial and the other is spectral, covering a spectral range from 500 to 1700 nm.

1Figure 1.

NIR luminescence microspectroscopy
setup: lower portion of diagram depicts
spectral regions detected by VIS andNIR paths.
DOI: 10.1039/c4pp00121d - Reproduced bypermission of The Royal Society of Chemistry(RSC) on behalf of the European Society for
Photobiology, the European Photochemistry
Association, and RSC. http://pubs.rsc.
org/en/content/articlelanding/2014/pp/
c4pp00121d#!divAbstract

The direct, real-time imaging setup, as illustrated in Figure 1, is built around an Olympus
inverted fluorescence light microscope and uses a 405 nm constant-wavelength (CW) laser
as an excitation source. The laser beam is passed through neutral-density (ND) filters andcoupled by a 500 nm dichroic longpass mirror (DLP) into an NIR-corrected objective (OBJ).
The illuminated spot on the sample (S) is enlarged to about 94 µm by inserting a lens (L1) inthe excitation path. To direct the luminescence emission collected by the objective from thesample to an NIR path for detection, a golden mirror (GM) is inserted. Removing the mirror
directs the emission to a VIS path.
In the NIR path, the signal passes through a combination of NIR longpass and shortpass
filters (F); the filter combination depends on the specific experiment. The signal is thenfocused by an achromatic lens (L2, f = 20 cm) onto the entrance slit of an Acton SpectraPro®
2500i imaging spectrograph from Princeton Instruments. The spectrograph is coupled to a
NIRvana:640 InGaAs camera, also from Princeton Instruments (see Figure 2). To reduce darkcharge, the camera’s NIR-sensitive, two-dimensional focal plane array (FPA) is cooled
to −80°C.

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Figure 2.

The NIR path of the experimental
setup includes a Princeton Instruments
NIRvana:640 two-dimensional InGaAs
detector coupled to a SpectraPro 2500i
imaging spectrograph. Photo courtesyof Dr. Marek Scholz, Charles University.

The 2500i spectrograph is configured either with a grating (150 g/mm, 1.2 µm blazed) for
spectroscopy or a mirror for imaging. To minimize sample photobleaching, the shutter (SH)in the excitation path is controlled by the NIRvana camera and opened only during exposure
times. In the imaging mode, the area of 0.34 x 0.34 μm on the sample is magnified to the
20 x 20 µm pixel size of the NIRvana’s FPA, which corresponds to 58x magnification. At 1275
nm (1O2 phosphorescence band), the spatial resolution due to the diffraction limit is about 1.4µm, according to the Rayleigh criterion, as determined by the objective numerical aperture of
0.55. In the spectroscopy mode, the spectral resolution of the system, determined by the widthof the entrance slit of the spectrograph, is 10 nm.
Alternatively, in the VIS path, a Spec-10:400B back-illuminated CCD camera from Princeton
Instruments is coupled to an Acton SpectraPro 2300i imaging spectrograph. Recently, tosimultaneously detect VIS and NIR spectral regions, the setup was modified by replacing thegolden mirror with a shortpass dichroic mirror (DM). The original set of filters has also been
modified. Refer to Scholz et al. 2014 for further details regarding the experimental setup.

Results and Conclusions
The introduction of the spectral imaging method provides a powerful new tool for
distinguishing and separating potential spectral overlap of 1O2 phosphorescence with the NIRextended luminescence of the photosensitizer. It can be applied to any PS manifesting NIR
luminescence. Some of the data is presented below; more can be found in Scholz et al. 2014.
The images displayed in Figure 3A were captured while continuously irradiating a cell,incubated for 5 hours with 100 μM TMPyP in a D2O-based saline solution, with 1 W/cm2 laser
power. The >1250 nm image (using a combination of 850 and 1250 nm longpass filters) was

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obtained by adding the last four frames (i.e., a total of 20 sec exposure) from a series of
10 consecutive frames with exposure times of 5 sec each. Subsequently, a spectral image
was obtained with a 25 sec exposure. The brightfield and fluorescence images were taken
afterward. The spectrum shows that 1O2 phosphorescence is the dominant spectral feature.
For different cells, it was verified that when a 1350 nm longpass filter was used in place of the1250 nm longpass filter, the >1250 nm image intensity dropped by more than 70%.

Figure 3.

NIR luminescence-based images andspectra accompanied by brightfieldand VIS fluorescence-based imagesof 3T3 mouse fibroblasts incubatedfor 5 hours with 100 μM TMPyP in a
D2O-based saline solution. Frames A
and B represent two different samples.
The green rectangles are defined by
the entrance slit of the spectrographs
and represent the areas from which thespectral images were collected. The
graphs of spectra were obtained byvertical binning of the spectral images.
A Gaussian blur filter with unit radius
was applied to smooth the spectral
image in order to make weak spectralfeatures more distinguishable.
DOI: 10.1039/c4pp00121d - Reproduced bypermission of The Royal Society of Chemistry(RSC) on behalf of the European Society for
Photobiology, the European Photochemistry
Association, and RSC. http://pubs.rsc.
org/en/content/articlelanding/2014/pp/
c4pp00121d#!divAbstract

The images displayed in Figure 3B were captured from different cells incubated using thesame procedure. Four near-infrared images were taken by irradiating these cells with
2.5 W/cm2 laser power. First, the 850–1100 nm image (I1) using a combination of 850 nm
longpass and 1100 nm shortpass filters was acquired with a 1 sec exposure. Next, the >1250
nm image (I2) was taken with a 5 sec exposure. Afterward, the ‘850–1100 nm + NaN3’ image(I3) with a 1 sec exposure and the ‘>1250 nm + NaN3’ image (I4) with a 5 sec exposure wereacquired after adding an aliquot of 10 mM NaN3 to the sample, gently stirring it, and thenleaving it in the dark for 5 minutes. A slight drop in the 850–1100 nm signal was observed,
which is likely indicative of a certain level of photobleaching. In the >1250 nm image (I4),
however, a much larger drop in signal was observed after the addition of the NaN3. The 1O2
signal is assumed to be almost totally quenched in the I4 image, which is formed
predominantly by the NIR background luminescence of the TMPyP. A combination of thesefour images I1–I4 allows the researchers to reconstruct the true image of 1O2 luminescence.
This new experimental setup, which employs an NIR-sensitive, two-dimensional InGaAs focal
plane array as a detector, has proven sufficiently sensitive to yield 1O2 images and spectral
images of individual D2O-treated fibroblast cells incubated with TMPyP. The setup’s overall
efficiency for 1O2 phosphorescence detection was estimated to be 1–3%. The primary limiting
factor was determined to be the numerical aperture of the objective (N.A. = 0.55).

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Enabling Technology
The main advantage of the NIRvana:640 camera (see Figure 4) over previously utilized
1D InGaAs detectors, according to Dr. Scholz, is the NIRvana detection array’s twodimensionality, which leads to a dramatic reduction of acquisition times and avoids some ofthe problems caused by photobleaching of the sample.

Figure 4.

The Princeton Instruments
NIRvana:640 camera features a
two-dimensional InGaAs detection arraythat can be thermoelectrically cooled as
low as –85°C.

The NIRvana:640 has been specially designed by Princeton Instruments for scientificresearch applications requiring superb linearity and excellent near-infrared sensitivity. Its
640 x 512 InGaAs detection array, which delivers response from 0.9 µm to 1.7 µm, can bethermoelectrically cooled as low as –85°C in order to minimize thermally generated noise andimprove signal-to-noise ratio.
Seeking to quantify performance parameters for their new microspectroscopy setup, thePrague group conducted an experiment to determine the dark noise of the NIRvana:640 (at−80°C). The results are presented in Figure 5.

Figure 5.

Dark counts per pixel and standard
deviation for different exposure times
per frame (NIRvana:640 camera).
DOI: 10.1039/c4pp00121d - Reproduced bypermission of The Royal Society of Chemistry(RSC) on behalf of the European Society for
Photobiology, the European Photochemistry
Association, and RSC. http://pubs.rsc.
org/en/content/articlelanding/2014/pp/
c4pp00121d#!divAbstract

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The NIRvana:640 InGaAs camera also provides 16-bit digitization and low read noise foroutstanding dynamic range. Furthermore, the latest Princeton Instruments LightField® 64-bit
data acquisition software, available as an option, affords complete control over all NIRvana
hardware features via an exceptionally intuitive user interface. LightField provides automatic
defect correction, precision exposure control, and a host of innovative functions for easy
capture and export of imaging and spectral data.

Summary
Singlet oxygen, the first excited state of molecular oxygen, is a highly reactive species thatplays an important role in a wide range of biological processes, including cell signaling,
immune response, macromolecule degradation, and elimination of neoplastic tissue during
photodynamic therapy4. Often, a photosensitizing process is employed to produce singlet
oxygen from ground-state oxygen.
An innovative experimental setup developed and used at Charles University in Prague now
permits researchers to perform direct, real-time imaging of singlet oxygen while addressing theissue of potential spectral overlap with emitted light from the photosensitizing agent. This fastdata-acquisition microspectroscopy setup relies on the two-dimensional array and exceptional
near-infrared sensitivity of the Princeton Instruments NIRvana:640 InGaAs detector.

Resources
For more information about the new NIRvana:640 two-dimensional InGaAs detector from
Princeton Instruments, please refer tohttp://www.princetoninstruments.com/products/imcam/nirvana/
To learn about research being performed by Prof. Jan Hála’s Optical Spectroscopy Group at
Charles University in Prague, please visit: http://kchf-45.karlov.mff.cuni.cz/

References
1. Schweitzer C. and Schmidt R. Physical mechanisms of generation and deactivation of
singlet oxygen. Chem. Rev. 103, 1685–1757 (2003).
2. Skovsen, E. Progress report: Non-linear two-photon singlet oxygen emission
microscopy. Department of Chemistry, University of Aarhus, Denmark (2004).

Acknowledgement

Princeton Instruments wishes to thankDr. Marek Scholz for his contributions tothis application note.

3. Hu B., He Y., and Liu Z. NIR area array CCD-based singlet oxygen luminescenceimaging for photodynamic therapy. Journal of Physics: Conference Series 277 (2011).
4.

Scholz M., Dědic R., Valenta J., Breitenbach T., and Hála J. Real-time luminescencemicrospectroscopy monitoring of singlet oxygen in individual cells. Photochem.
Photobiol. Sci. 13, 1203–1212 (2014). DOI: 10.1039/c4pp00121d

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