A Quantitative Comparison of Different Scientific CCD Types

Overview

Recently, a team of scientists working in North America and Europe developed an innovative growth and imaging platform, known as GLO-Roots, that allows root architecture and gene expression to be studied in soil-grown plants.

GLO-Roots (Growth and Luminescence Observatory for Roots; U.S. patent application: 13/970,960) is a collaborative effort between the labs of Dr. José Dinneny at the Carnegie Institution for Science (Stanford, California), Dr. Rubén Réllan-Álvarez (Langebio, Mexico), and Dr. Guillaume Lobet at the Université de Liège in Belgium. Designed and built with help from BioImaging Solutions, Inc. (San Diego, California), the researchers’ new growth and imaging platform supports several model organisms, namely, Arabidopsis thaliana, Brachypodium distachyon, Setaria viridis, and Lycopersicon esculentum.¹

GLO-Roots employs luminescence-based reporters and a pair of Princeton Instruments back-illuminated CCD cameras to enable studies of root architecture and gene expression patterns in soil-grown, light-shielded roots. Custom-designed image analysis algorithms allow the spatial integration of soil properties, gene expression, and root system architecture traits. The GLO-Roots developers assert that the new platform can offer great utility for presenting environmental stimuli to roots in ways that evoke natural adaptive responses and in providing tools for investigating the multidimensional nature of such processes.²

This application note will outline some of the research conducted by the GLO-Roots team, which is led by Dr. José Dinneny at the Carnegie Institution for Science’s Department of Plant Biology on the Stanford University campus

GLO-Roots Setup

The GLO-Roots platform comprises four parts: (1) growth vessels, called rhizotrons, that permit plant growth in soil as well as imaging of roots; (2) luminescence-based reporters that allow various aspects of root biology to be tracked in living plants; (3) GLO1, a luminescence-imaging system designed to automatically image the rhizotrons; and (4) GLO-RIA, an image
analysis suite designed to quantify the root systems imaged utilizing GLO1.² An example of an experimental setup using the GLO-Roots platform is presented in Figure 1.

Whereas most commercially available luminescence imaging systems for biomedical research are optimized for imaging horizontally held specimens or samples in microtiter plates, placing the rhizotrons in this position would induce a gravitropic response in plants. Therefore, GLO-Roots utilizes a custom-designed imaging system (known as the Growth and Luminescence Observatory 1, or simply the GLO1) that has been optimized specifically for imaging dual-reporter luciferase expression in the GLO-Roots rhizotrons.²

Two PIXIS:2048 back-illuminated CCD cameras from Princeton Instruments are mounted on top of each other so as to capture partially overlapping images of a rhizotron, while a motorized stage automatically rotates the rhizotron to capture images of both sides (refer to Figure 1E). A composite image is then generated from the images captured of each side. Figure 1F shows that approximately half of the root system is revealed on each side, with a few roots being visible on both sides. The soil sheet is thick enough to block light from portions of the root system, yet thin enough to ensure its continuous structure can be compiled from opposite-face views. The entire GLO1 imaging system is enclosed in a light-tight black box that is equipped with a door to permit the loading and unloading of rhizotrons.²

Not only do the PIXIS:2048B cameras afford the GLO-Roots researchers the desired spatial resolution and field of view, but also the excellent low-light-level sensitivity required to detect the emission wavelengths of the different reporters used. Table 1 lists the luciferases employed for the Arabadopsis-related GLO-Roots study.

Luciferase	Origin	Maximum Wavelength (nm)	Substrate
PpyRE8	Firefly	618	D-luciferin
CBGRed	Click Beetle	615	D-luciferin
Venus-LUC2	FP + Firefly	580	D-luciferin
LUC (+)	Fireflu	578	D-luciferin
CBG99	Click Beetle	537	D-luciferin
Lux operon	A. fischeri	490	Biosynthesis pathway encoded with operon
NanoLUC	Deep sea shrimp	470	Furimazine

Data and Results

Root systems develop different root types, each of which individually senses myriad local environmental cues and integrates its environment’s information with systemic signals. This highly complex multidimensional amalgam of inputs enables continuous adjustment of root growth rates, direction, and metabolic activity that define a dynamic physical network.²

Figure 2 presents time-lapse imaging of Arabidopsis root systems captured with the GLO1 system along with quantification performed using the GLO-RIA image analysis suite.

Here, root traits such as directionality can be observed through later stages of plant development (notice the 35 DAS root system image and the 35 DAS directionality analysis shown in Figures 2A and 2B, respectively). A time series spanning 11 to 21 days after sowing Arabadopsis accession Col-0 roots expressing reporter ProUBQ10:LUC2o is presented in Figure 2A and Figure 3; a color-coded time projection is shown in Figure 2C.

Directionality analysis indicates a progressive change in root system angles, from 0° (vertical) to 55°, as lateral roots take over as the predominant root type. Figure 2D shows the evolution over time of several root traits that can be automatically captured by GLO-RIA (depth, width, area) and others that were manually quantified (primary root growth rate or number of lateral roots per primary root.²

To date, the GLO-Roots team has published data pertaining to the continuous imaging of root growth; the root system architecture of different Arabidopsis accessions; the utilization of spectrally distinct luciferases to capture additional information associated with gene expression patterns, characterization of root system interactions, and microbial colonization; adaptive changes in root system architecture under water deficit, phosphorus deficiency, and light; the suitability of the GLO-Roots platform for studying additional plant species; and more. The research team is also highly interested in discovering how other environmental stimuli affect root growth and whether such responses differ between accessions of Arabidopsis.²

For more data and an in-depth discussion of results, please refer to R. Rellán-Álvarez et al. 2015. “GLO-Roots: an imaging platform enabling multidimensional characterization of soil-grown root systems.” eLife 4 (1): e07597.

Enabling Technology

Each of the PIXIS:2048 cameras integrated in the GLO1 imaging system utilizes a CCD featuring a back-illuminated sensor whose large photosensitive array is composed of 2048 x 2048 pixels (see Figure 3). By employing Princeton Instruments’ exclusive XP cooling technology, these four-megapixel cameras achieve thermoelectric cooling down to -70°C via an all-metal, hermetically sealed design. This innovative cooling technology ensures maintenance-free operation and is backed by the industry’s only lifetime vacuum guarantee.

In addition to the XP-facilitated minimization of thermally generated (dark) noise, very high quantum efficiency and ultra-low-noise electronics make PIXIS:2048 cameras ideal for demanding, low-light-level imaging applications. Dual-speed operation (i.e., 100 kHz or 2 MHz) allows utilization in both steady-state and fast-kinetics studies.

To optimize quantitative scientific imaging performance for applications from the UV to the NIR, the PIXIS:2048 platform supports a front-illuminated CCD format, a back-illuminated format, a back-illuminated format with high UV sensitivity, and a back-illuminated format with high NIR sensitivity (note that Princeton Instruments leverages its own proprietary eXcelon® processing and back-illuminated, deep-depletion technology to deliver the highest sensitivity in the NIR while suppressing the etaloning that occurs in standard back-illuminated CCDs). Several other CCD array sizes, appropriate for various imaging and spectroscopy applications, are supported by the PIXIS series.

Complete control over all PIXIS hardware features is simple with the latest version of Princeton Instruments’ 64-bit LightField® data acquisition software, available as an option. A host of novel functions for easy capture and export of imaging data are provided via the exceptionally intuitive LightField user interface

References

New Two-Dimensional InGaAs Detector Thermoelectrically Cooled to –85°C
Facilitates Scientific Research

Introduction

Molecular oxygen is one of the most important molecules in maintaining life as well as in mechanisms by which life is extinguished and materials destroyed. For several decades, researchers have been intrigued by the physical and chemical properties of molecular oxygen’s lowest excited state, singlet oxygen (¹O₂). In particular, singlet oxygen has a unique reactivity that can result in polymer degradation or the death of biological cells. Its role as an intermediate in cell death is exploited by photodynamic therapy (PDT) for cancer, a technique in which light is utilized as a medical tool^1,2.

In PDT, a photosensitizer (PS) is incorporated into abnormal tissues and then irradiated with visible 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 oxygen on 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 real-time imaging of the very weak near-infrared phosphorescence of singlet oxygen and photosensitizer 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 is spatial and the other is spectral, covering a spectral range from 500 to 1700 nm.

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 and coupled 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) in the excitation path. To direct the luminescence emission collected by the objective from the sample 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 then focused 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 dark charge, the camera’s NIR-sensitive, two-dimensional focal plane array (FPA) is cooled to −80°C.

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 1.4 µm, 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 width of 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, to simultaneously detect VIS and NIR spectral regions, the setup was modified by replacing the golden 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 NIR-extended 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 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 the
1250 nm longpass filter, the >1250 nm image intensity dropped by more than 70%.

The images displayed in Figure 3B were captured from different cells incubated using the same 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 were acquired after adding an aliquot of 10 mM NaN₃ to the sample, gently stirring it, and then leaving 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 these four images I₁–I₁ 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).

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 two-dimensionality, which leads to a dramatic reduction of acquisition times and avoids some of the problems caused by photobleaching of the sample.

The NIRvana:640 has been specially designed by Princeton Instruments for scientific research 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 be thermoelectrically cooled as low as –85°C in order to minimize thermally generated noise and improve signal-to-noise ratio.

Seeking to quantify performance parameters for their new microspectroscopy setup, the Prague group conducted an experiment to determine the dark noise of the NIRvana:640 (at −80°C). The results are presented in Figure 5.

The NIRvana:640 InGaAs camera also provides 16-bit digitization and low read noise for outstanding 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 that plays 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 the issue 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.

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).
3. Hu B., He Y., and Liu Z. NIR area array CCD-based singlet oxygen luminescence imaging for photodynamic therapy. Journal of Physics: Conference Series 277 (2011).
4. Scholz M., Dědic R., Valenta J., Breitenbach T., and Hála J. Real-time luminescence microspectroscopy monitoring of singlet oxygen in individual cells. Photo