NIRis: A low-cost, versatile imaging system for NIR fluorescence detection of phototrophic cell colonies used in science and education

A variety of costly research-grade imaging devices are available for the detection of spectroscopic features. Here we present an affordable, open-source and versatile device, suitable for a range of applications. We provide the files to print the imaging chamber with commonly available 3D printers and instructions to assemble it with easily available hardware. The imager is suitable for rapid sample screening in research, as well as for educational purposes. We provide details and results for an already proven set-up which suits the needs of a research group and students interested in UV-induced near-infrared fluorescence detection of microbial colonies grown on Petri dishes. The fluorescence signal confirms the presence of bacteriochlorophyll a in aerobic anoxygenic phototrophic bacteria (AAPB). The imager allows for the rapid detection and subsequent isolation of AAPB colonies on Petri dishes with diverse environmental samples. To this date, 15 devices have been build and more than 7000 Petri dishes have been analyzed for AAPB, leading to over 1000 new AAPB isolates. Parts can be modified depending on needs and budget. The latest version with automated switches and double band pass filters costs around 350€ in materials and resolves bacterial colonies with diameters of 0.5 mm and larger. The low cost and modular build allow for the integration in high school classes to educate students on light properties, fluorescence and microbiology. Computer-aided design of 3D-printed parts and programming of the employed Raspberry Pi computer could be incorporated in computer sciences classes. Students have been also inspired to do agar art with microbes. The device is currently used in seven different high schools in Finland. Additionally, a science education network of Finnish universities has incorporated it in its program for high school students. Video guides have been produced to facilitate easy operation and accessibility of the device.

Answering of research questions often benefits from niche tools, which are consequently 2 not mass-produced as there is no sufficiently-sized market. This results in suitable 3 research-grade devices being expensive and targeted for a broader range of applications. 4 Here we introduce an affordable and open-source imaging system called NIRis 5 (Near-infrared imaging system) which enables rapid screening of Petri dishes for 6 phototrophic bacterial colonies. Building own equipment for the specific needs of 7 research groups has become significantly easier with decreasing prices for electronic 8 hardware and the recent development and increasing number of available and affordable 9 3D printers [1,2]. Consequently, online resources for open-source lab equipment have 10 increased steadily. Examples include various devices for microfluidics, imaging set-ups, 11 plate readers, microscopes, as well as regular labware such as filter holders or 12 pipettes [3][4][5][6][7]. Since its publication in 2012, Raspberry Pi single-board computers have 13 been often integrated into these devices, as they are comparatively affordable and serve 14 the needs for a broad range of applications [9]. Also in NIRis, the image acquisition 15 and camera operation is performed by a Raspberry Pi computer coupled with a 16 near-infrared (NIR)-sensitive camera module distributed by the same company. The 17 various possibilities and advantages of Raspberry Pi computers have been previously 18 described in detail and will not be further discussed in this article [9,10]. Commercially 19 available devices which could analyze samples to a similar level are advanced gel 20 imaging chambers which are advertised to analyze Petri dishes as well. However, these 21 machines are much larger, the customizability concerning emission and excitation light 22 is limited and the price usually starts upwards of 10.000€. NIRis was build out of a 23 need for a simple and efficient fluorescence detection chamber to reliably detect 24 phototrophic bacterial colonies based on the presence of NIR-fluorescent 25 bacteriochlorophyll a (BChl a) [11][12][13]. The system records a reference white light 26 image to capture all colonies, as well as a fluorescence image which selectively shows 27 only phototrophic bacteria (Fig 1) Rapid and low-cost fluorescence detection devices are 28 currently not commercially available, even though the components required to build a 29 suitable device for macroscopic analysis are relatively affordable. More advanced 30 systems, like hyperspectral cameras or spectrometers can characterize fluorescence by 31 providing detailed spectra, showing peak shapes and locations [14]. However, this 32 information is not required if the primary goal is to identify and separate objects with a 33 distinct difference in fluorescence for further analysis and more detailed characterization. 34 Similar to fluorescence microscopy, it is possible to equip the imager with light sources 35 of specific wavelengths or bandpass filters for targeted excitation and bandpass or 36 longpass emission filters to analyze the spectral properties of cells for their classification. 37 The obvious difference between a fluorescence microscope and NIRis is the sample size 38 and resolution. This implies that microorganism need to be cultivated and grown into 39 macroscopic colonies in order to be captured by the employed camera in NIRis. The 40 advantage of the macroscopic approach is that selected colonies can be easily harvested 41 from a Petri dish for further analysis. NIRis is therefore an extremely powerful tool in

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Assembly of the imager for UV-induced NIR fluorescence 52 detection 53 The imaging system can be employed for a wide range of applications due to its 54 modularity. The NIRis version detects the expression of light harvesting complex 1 55 (LH1) with integrated BChl a in the central reaction center of AAPB [12]. NIRis was 56 inspired by a research-grade fluorescence imaging system used by Zeng and colleagues 57 for the same purpose of identifying AAP bacteria [17]. The major difference and 58 advantage of NIRis is the use of low-cost consumer-grade hardware. The fixed sample, 59 light and camera positions ensure repeatable imaging to allow for a more precise 60 analysis of the images and a reduction of reflection problems. Additionally, this design 61 provides portability without altering the imaging result.

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The imager consists of a 3D-printed chamber holding each one consumer-grade white 63 light-emitting diode (LED) and UV LED flashlight. The chamber is ideally printed of 64 black material and can be optionally painted matte black to reduce internal reflections 65 and prevent light leakages. A simple diffuser is permanently placed in front of the white 66 light to improve the illumination and reduce strong reflections from the Petri dish. The 67 diffuser was cut out of readily available surgical masks made of polypropylene fibers. A 68 bottom drawer can be removed from the chamber to insert a Petri dish sample for 69 imaging (Fig 2). The employed 8-megapixel camera module PiNoir V2 (Raspberry Pi,  The 19 parts needed to assemble the device were 3D-printed with glycol-modified 76 polyethylene terephthalate (PETG) since it is known to be stronger than commonly 77 used poly-lactic acid (PLA) filament and still easy to print (Fig 3). Parts were printed 78 with a Prusa i3 Mk2 (Prague, Czech Republic) with a layer height set to 0.35 mm and 79 20 % infill. All parts were assembled with M2 and M4 screws on a wooden board as a  region, from 850 nm to 920 nm (Fig 1, S2 Figure). This filter is placed inside the filter 98 holder and can be operated with a bolt sticking out of the chamber side. Two filters can 99 be stacked to further eliminate false positives from reflection bleed-through as their 100 filtering characteristics multiply (e.g. 0.01 % * 0.01 % Transmission in blocking region). 101 The hardware needed to build this version of the imaging system with two stacked 102 filters and automated switches cost around 350€. The following table specifies the 103 details and prices for the significant individual hardware components (Table 1). 104   BerryBase HLRELM-2 1 08/2021 3.60€ Prices were paid per unit on the indicated purchase date. Different white light LED flashlights were bought locally for around 5€ per piece. Only requirements for the flashlight were a diameter of 2.5 cm and a rear button switch to be able to connect the Raspberry Pi computer in case automated switches should be installed. Additionally, PETG filament for 3D printing and screws are needed for assembly.

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The imager can also record a reference white light image to count all colonies and 105 assess their size, shape and color. Additionally, it is easier to locate fluorescing colonies 106 with a complimentary white light picture for subsequent isolation. For the reference 107 image, the white light is turned on, the filter is pulled away from the camera and a 108 picture is taken (Fig 1). Between the acquisition of both images, the Petri dish stays in 109 the same position and both flashlights remain at the same incident angle for every Petri 110 dish which reduces the amount of variation caused by changing reflections and uneven 111 lighting S1 Video. Additionally, a MATLAB script, which can be found in the  Sample preparation from plant endo-and phyllosphere 121 Detailed extraction and cultivation methods as well as isolation results have been 122 published previously [12]. In short, phyllosphere bacteria were extracted by 3-minute NIRis was primarily used to identify AAP bacteria from environmental samples taken 139 from plant phyllosphere and endosphere. As of now, over 7000 Petri dishes with samples 140 from four countries and more than 20 locations have been imaged, resulting in over 1000 141 AAPB isolates. Operators ranged from high school interns, over B.Sc. and M.Sc. 142 students to professionals indicating its ease of use. UV-induced NIR fluorescence can be 143 employed for the identification of AAPB because it verifies the presence of BChl a 144 which fluoresces at 890 nm, in-vivo [11]. For reference, UV-induced fluorescence spectra 145 of the AAPB S. glacialis strain S2U11 can be found in the supplementary material (S2 146 Figure). Additionally, UV-induced fluorescence spectra of a variety of AABP have been 147 published recently [12]. The fluorescence imaging sucessfully detected very small AAPB 148 colonies reliably, at least from 0.5 mm diameter and larger (Fig 4). The reference white 149 light image taken with NIRis displayed all present colonies for their assessment of size 150 and colour as well as enabling a total colony count (Fig 4). Additionally, it helped to

The imager in education 167
Cost-effective imaging devices that could be used for educational purposes are rare -in 168 particular devices, that could be constructed by high-school students themselves. Our  imaging bacteria which grew after plant leaves were pressed on agar in Petri dishes, a 183 technique first described by William Corpe in 1985, which has been already successfully 184 implemented in teaching environments before [20,21]. Surface bacteria on leaves often 185 include AAP bacteria which can be harvested by the leaf printing method and identified 186 as AAPB by UV-induced NIR fluorescence imaging using NIRis (Fig 4). The project 187 work included sterile sampling, working with Petri dishes and bacterial colonies as well 188 as an introduction to the research question. Eight comprehensive video guides aimed for 189 the teachers and students, along with a shorter video demonstration for this publication 190 have been produced to explain all parts of the process (S1 Video and [8]).

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Additionally, students have been inspired to do agar art with microbes which could 192 be later imaged with the device. For creating agar art, differently colored bacteria 193 strains are used as paints to create images on Petri dishes. There is even more room for 194 creativity, if selected strains are fluorescent which selectively enhances their appearance 195 under excitation light (Fig 4). Here, these strains are AAPB, which will be visible in 196 NIRis imaging when using the fluorescence mode. The topic has been popular -as 197 shown in the yearly agar art competition held by the American Society for 198 Microbiology [23]. Additionally, agar art has been discussed as an educational tool for 199 undergraduate students, for example to teach about fluorescent protein expression 200 [22,23]. Agar art and subsequent imaging with NIRis has also been part of the European 201 Researchers´Night event at the University of Jyväskylä. Participants could paint a 202 picture on an own Petri dish or leave a signature on an agar guest book. After one week 203 of incubation time, images were taken both in white light and fluorescent mode and sent 204 to the participants. In addition to presenting the various colors of microorganisms, the 205 colour palette also demonstrates the difference of pigmentation in the same species 206 Sphingomonas faeni. Patch numbers 1, 5 and 8 have been sequenced to be the same 207 species but show a clear difference in color and UV-induced NIR fluorescence (Fig 4).

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The picture also demonstrates that the fluorescence of chlorophyll in micro algae can 209 show in the UV-induced NIR fluorescenece image as discussed before (Fig 4 patch 4).  [17]. They utilized a research-grade CCD sensor with a similar 217 light and filter arrangement to detect AAPB on Petri dishes. However, they employed 218 LED light in the blue-green spectral region (450 570 nm) with the idea to utilize the 219 effect of energy transfer from carotenoids to BChl a to induce NIR fluorescence [17]. In 220 NIRis the excitation light is around 400 nm where the so-called Soret bands of BChl spectrum of the AAPB S. glacialis strain S2U11 indicated that such a energy transfer 226 was not observable [13]. The difference between the already published open-source 227 imager by Nuñez and colleagues is the light source placement and grade of complexity. 228 While Nuñez and colleagues, as well as Gonzales and colleagues with their agarose gel 229 9/15 imager, opted for a trans-illuminance LED circuit board underneath the Petri dish, the 230 here presented imager utilizes flashlights for white light epi-illumination and UV-light 231 excitation [6,16]. The imaging system employs a simple push-in emission filter to switch 232 between white-light reference and fluorescence imaging modes. 233 An arguably more complex open-source spectral imaging device was published by 234 Lien and colleagues including the possibility for automated hyperspectral imaging of 235 macroscopic samples [18]. However, the use of a hyperspectral camera places it in a 236 different price category. Likewise more sophisticated is an open-source fluorescence 237 imaging device which utilizes filter cubes, an incubation chamber and a consumer-grade 238 digital single-lens reflex (DSLR) camera with macro lens [19] Besides the higher costs, 239 this set-up is restricted to the detection of signals in the visible light range, unless the 240 internal hot mirror of the camera is removed.

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The here provided instructions and CAD files enable anyone with access to a 3D printer 243 to recreate the introduced imaging system. However, we hope that the presented colonies, they are generally equipped with hot mirrors in front of their sensors to filter 252 out NIR and infrared light. This mirror can be manually removed but it is usually more 253 difficult than using the Raspberry Pi solution presented here. However, we encourage 254 research groups which have a need for a recorded emission wavelength in the visible 255 light spectrum to try and utilize regular consumer cameras for an even easier and more 256 portable approach. The here presented imaging system could be assembled even more 257 economically if acrylic NIR-transmitting filters are used instead of research quality 258 filters, as these are the most expensive part. Those filters are easily available as they 259 have been used extensively for shading of remote-control receivers, e.g. in televisions.

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Also, many 3D-printed parts are not vital for the correct operation of the system and 261 can be omitted for more affordable and faster construction. For example, PCB and 262 camera module covers have been only added to guarantee resilience to withstand school 263 use and easy transportation, as well as a neater appearance. Parts that were deemed 264 non-vital for normal operation were marked as such in the parts list (S1 Figure). Of  properties, photosynthesis, fluorescence and sterile microbiology work flows. The device 293 can also easily spark interest in 3D printing, coding and of course in a career in natural 294 sciences. Hands-on learning has been proven to be a successful strategy and can be a 295 welcoming change to theoretical classes. Additionally, we assume that the participation 296 in a research project may enhance the engagement of the students. The main 297 advantages of NIRis are the low cost, simplicity and NIR recording capabilities as well 298 as its portability. While more complex devices may enable additional applications, this 299 imager performs very well for the identification of fluorescent colonies on Petri dishes -300 its primary purpose. Open-source research instruments like this imaging system 301 essentially reduce the costs of scientific work and therefor lower the barrier for research 302 groups with limited funds to contribute and participate in scientific discourse. We 303 believe that developing easily accessible lab instruments and the subsequent publishing 304 of instructions can contribute directly to more democratized and inclusive science.