A microfluidic-based microscopy platform for continuous interrogation

Subscriber access provided by Service des bibliothèques | Université de Sherbrooke

From the Bench

A microfluidic-based microscopy platform for continuous interrogation of Trypanosoma brucei during environmental perturbation Charles M Voyton, Jongsu Choi, Yijian Qiu, P Christine Ackroyd, Meredith T. Morris, James C. Morris, and Kenneth A Christensen Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b01269 • Publication Date (Web): 14 Jan 2019 Downloaded from http://pubs.acs.org on January 14, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

A microfluidic-based microscopy platform for continuous interrogation of Trypanosoma brucei during environmental perturbation

Charles M. Voyton1,3, Jongsu Choi3, Yijian Qiu2, Meredith T. Morris2, P. Christine Ackroyd3, James C. Morris2, and Kenneth A. Christensen*1,3

1Department 2Eukaryotic

of Chemistry, Clemson University, Clemson SC 29634

Pathogens Innovation Center, Department of Genetics and Biochemistry, Clemson University, Clemson SC 29634

3Department

of Chemistry and Biochemistry, Brigham Young University, Provo UT 84602

*Corresponding author Email: [email protected]

1 ACS Paragon Plus Environment

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract. The African trypanosome, Trypanosoma brucei, is the causative agent of human African trypanosomiasis (HAT). African trypanosomes are extracellular parasites that possess a single flagellum that imparts a high degree of motility to the microorganisms. In addition, African trypanosomes show significant metabolic and structural adaptation to environmental conditions. Analysis of the ways that environmental cues affect these organisms generally requires rapid perfusion experiments in combination with single cell imaging, which are difficult to apply under conditions of rapid motion. Microfluidic devices have been used previously as a strategy to for trapping small motile cells in a variety of organisms, including trypanosomes; however, in the past, such devices required individual fabrication in a cleanroom, limiting their application. Here we demonstrate that a commercial microfluidic device, typically used for bacterial trapping, can trap bloodstream and procyclic form trypanosomes, allowing for rapid buffer exchange via perfusion. As a result, timelapse single-cell microscopy images of these highly motile parasites were acquired during environmental variations. Using these devices, we have been able to perform and analyze perfusion-based single-cell tracking experiments of parasite responses to changes in glucose availability, which is a major step in resolving the mechanisms of adaptation of kinetoplasts to their individual biological niches; we demonstrate utility of this tool for making measurements of procyclic form trypanosome intracellular glucose levels as a function of changes in extracellular


Page 2 of 28

Page 3 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

glucose concentrations. These experiments demonstrate that cytosolic glucose equilibrates with external conditions as fast as, or faster than, the rate of solution exchange in the instrument.

Introduction Kinetoplastid parasites, including the African trypanosome (Trypanosoma brucei), impact human health worldwide and are causative agents of neglected tropical diseases.1 This group of parasites use unique metabolic and biological mechanisms to adapt to their changing environmental niches.2 Because early advances in cell culture and molecular genetic approaches were largely made in T. brucei, the African trypanosome serves as a model for investigating key processes in these kinetoplastid parasites.3 One important feature of kinetoplastid parasites, including T. brucei, is their ability to adapt their morphology and metabolism to different environmental conditions. For example, T. brucei respond to the changing conditions in the tsetse fly gut and mammalian bloodstream by changing from the procyclic form (PCF), which metabolizes a variety of carbon sources, to the bloodstream form (BSF), which exclusively metabolizes glucose. Hence, the methods by which the parasites sense and respond to their environment, and how their metabolism changes in response to their environment, are of high research interest. A straightforward way to analyze these changes would be to examine individual parasite cells as they are exposed to different solution conditions. However, many of the life stages of kinetoplastid parasites are both non-adherent and highly motile, which makes them difficult to image using traditional microscopic methods. While perfusion experiments that allow rapid buffer exchange are well established, these methods are typically been performed using adherent cells4 and are therefore not easily applied to trypanosomes. The challenge is to


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

identify an analytical method capable of both sufficiently localizing live BSF and PCF form trypanosomes for imaging while still allowing rapid buffer exchange. Gel matrices that impede movement of live parasites in 3-D space have been previously used to image dynamic events.5,6 However, while gel immobilization methods have high image resolution, they do not allow fast buffer exchange, and are therefore not useful for monitoring dynamic cellular responses to changes in the external environment. The applications of microfluidic devices designed for use in live cell imaging applications has increased, largely due to improved lithography methods.7,8 Microfluidic devices can allow for both live cell immobilization and buffer exchange, and have been used to trap a variety of cell types, including bacteria, yeast, mammalian cells, and in limited cases, trypanosomes.9,10,11,12 For example, a microfluidic device has been deployed in combination with optical tweezers to monitor drug dependent motility in bloodstream T. brucei.12,13 Unfortunately, previously applications of microfluidics to trypanosomes required access to photolithography and a cleanroom for device assembly13,12, which has precluded their practicability for most kinetoplastid biologists. Here we describe application of a commercially available microfluidic system for single cell imaging and buffer exchange of T. brucei. The system utilizes ready-made microfluidic plates available in a variety of configurations, does not require assembly in a clean-room, and includes pneumatic-based fluidics for precise monitoring and control of the pressure and flow rates. These characteristics allow localization of different life stages of T. brucei for single cell imaging, and continuous, complete, and rapid buffer perfusion for regulated buffer exchange. Here we describe use of this microfluidic system to monitor the dynamic responses of living trypanosomes to environmental perturbation. Specifically, we observe changes in intracellular glucose 4 ACS Paragon Plus Environment

Page 4 of 28

Page 5 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

concentration in individual T. brucei cells as a function of altered extracellular glucose concentration, in real time.

Methods Materials and Reagents. All buffer and media components used for analysis were purchased from Sigma-Aldrich (Saint Louis, Missouri). Fluorescein powder (40% Fluorescein) was purchased from Fisher Scientific (Pittsburgh, Pennsylvania). Hygromycin, blasticidin, and G418 were purchased from Gold Biotechnology (Saint Louis, Missouri). The CellASIC ONIX2 system was purchased from Merck Millipore (Burlington, Massachusetts, part number CAX2-S0000) along with the temperature controlled CellASIC ONIX2 Manifold XT (part number CAX2-MBC20). CellASIC ONIX microfluidic plates for bacterial cells was also purchased form Merck Millipore (part number B04A-03-5PK). To reduce background fluorescence due to media components, all imaging experiments were carried out in phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 2 mM KH2PO4) supplemented with up to 10 mM glucose.

Trypanosome culture and transfection. Procyclic form (PCF) trypanosomes were continuously cultured in SDM-79 media supplemented with 15% FBS at 29°C and 5% CO2.14 Bloodstream form (BSF) trypanosomes were cultured in HMI-9 media supplemented with 10% FBS.15 To maintain cell viability and ensure a uniform population, PCF and BSF trypanosomes were maintained in log phase (5 x 105-5 x 106 cells/mL for PCF and 5 x 104-5 x 105 for BSF parasites). The fluorescent glucose sensor FLII12Pglu-700μδ6 was cloned into pXS2 and pXS6 allowing for stable 5 ACS Paragon Plus Environment

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

transformation and expression in PCF and BSF parasites, respectively .16,17 For experiments examining the glucose response in the glycosome, FLII12Pglu-700μδ6 was targeted to the glycosomal lumen via an appended PTS1 signal sequence (AKL) via site directed mutagenesis to the C-terminus of the protein.

Microscopy. All fluorescence and bright field images were taken using an IX73 epifluorescence microscope (Olympus Corporation; Tokyo, Japan). DIC images were captured images using a Sutter TLED for bright filed illumination (Sutter; Novato, California) and an Orca Flash V4.0 CMOS camera (Hamamatsu; Shizuoka, Japan). The fluorescent biosensors were excited using a fast switching DG-4 fluorescence light source (Sutter; Novato, California) using a 430/30 filter for excitation of the ECFP and FRET emission. ECFP (480/30) and FRET (530/30) emission spectra were separated using an Andor Tucam (Andor Technology; Belfast, Northern Ireland) equipped with a 500-nm long pass filter. ECFP and sensitized FRET emission were captured simultaneously on a pair of Orca Flash V4.0 CMOS cameras (Hamamatsu; Shizuoka, Japan). Light source switching and camera exposure were all synchronized using a TTL control box and Slidebook 6.0 (Intelligent Imaging Innovation; Denver, Colorado) for synchronization control. All images were captured and analyzed using Slidebook 6.0 software and the FRET module was used for calculating FRET ratios on a pixel per pixel basis for time-lapse movies and data export.

Microfluidic plate preparation and setup. Microfluidic plates were opened aseptically in a biosafety cabinet. The 0.05% azide in PBS solution in all channels (1-8) for one experimental row 6 ACS Paragon Plus Environment

Page 6 of 28

Page 7 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

(A-D) was removed using a sterile vacuum aspirator. To purge channels of any remaining azide, the channel-flushing program was used (5 kPa for 5 minutes) prior to cell loading. Buffers used for the experiment were loaded into wells 1-5 of the plate, channel 6 was loaded with 10mM glucose in PBS for all experiments. Channel 7 was designated as a waste well and was left empty in all experiments. Channel 8 contained the cell suspension used for microscopy experiments. The remaining channels were filled with 0.05% azide in PBS to they did not become contaminated. Figure 1A shows the plate layout.

Parasite Plate Loading. PCF or BSF parasites were washed three times in PBS and resuspended in PBS supplemented with glucose to a final cell density of 1 x 106 cells/mL. The cell suspension was then pipetted into channel 8 of the desired row on the microfluidic plate and the plate was sealed to the fluidics manifold, following the manufactures instructions. A modified cell-loading module was then used to introduce the parasites to the instrument. Briefly, channel 8 containing parasites was pressurized to 15 kPa for 2 minutes, then channels 6 and 8 were pressurized to 15 kPa for 10 minutes to lodge the cells into the capture area of the device, followed by depressurization of channels 7 and 8. After the device was loaded with parasites, buffer (PBS with 19 mM added glucose) was continuously perfused from channel 1 by pressurizing the channel to 10 kPa. Cells were continuously monitored by microscopy for 1 hour to ensure that cell viability was not impacted by being constrained in the device. For long term viability experiments, PCF cells were perfused with full SDM-79 media and continuously monitored via bright field microscopy for 48 hours.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Characterization of perfusion profile using fluorescein solutions. To measure solution mixing using the microfluidic plate, we perfused different concentrations of fluorescein by mixing the buffer channels at varying ratios while holding total system pressure constant. Fluorescein stocks were prepared in PBS at 1 mg/mL and sterilized by filtration through a 0.22 µm filter. A 100-fold dilution was then pipetted into channel 1 of a plate prepared as described above. Channel 2 was filled with PBS and the remaining channels were filled with 0.05% azide in PBS. The plate was then attached to the pneumatic manifold and channels 1 and 2 were purged for five minutes at 10 kPa pressure. After system priming was complete, PBS was perfused by pressurizing channel 2 to 20 kPa for five minutes. To introduce different fluorescein concentrations, channel 1 and 2 were perfused at different pressures (totaling 20 kPa). In order to generate 25% fluorescein, the fluorescein channel was pressurized to 5 kPa while the PBS channel was pressurized to 15 kPa. Other channel pressure ratios were used to vary the percentage of fluorescein. Channels were perfused for five minutes followed by a five-minute PBS wash; five minutes was sufficient for ≥ 90% buffer exchange. Time-lapse fluorescence microscopy was used to monitor the fluorescence (495/20 ex, 530/30 em) for the duration of the perfusion experiment; images were obtained every 5 seconds using 100msec exposure times for fluorescence images. A representative mask was then drawn using Slidebook 6.0 software, mean intensity and standard deviation were then exported for every frame of the fluorescence time-lapse. The perfusion mixing protocol is shown in Table 1.


Page 8 of 28

Page 9 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Table 1. Fluorescein perfusion protocol Plate Preparation

Channel Number 1 2 3,4,5,6,7,8

Perfusion Protocol

Channel Contents 10 g/mL Fluorescein PBS 0.05% azide in PBS

Protocol Step

Length (min)

Channels

Pressure

1

10

1,2

2

5

2

3

5

1,2

4

5

2

5

5

1,2

6

5

2

7

5

1,2

8

5

2

20 kPa

9

5

1

20 kPa

10

5

2

20 kPa

5 kPa, 5 kPa 20 kPa 5 kPa, 15 kPa 20 kPa 10 kPa, 10 kPa 20 kPa 15 kPa, 5 kPa

On-plate glucose mixing to monitor intracellular glucose response. To see how device mixing could be applied to live cell experiments, PCF parasites expressing the glucose sensor FLII12Pglu700μδ6 were perfused with solutions containing variable glucose concentrations generated by mixing multiple reagent channels. PCF parasites expressing the cytosolic fluorescent glucose sensor were washed three times with PBS and resuspended to a final density of 1 x 106 cells/mL in PBS supplemented with 10 mM glucose. Cells were then added into channel 8 of a plate prepared as described above. Channels 1 and 2 were filled with PBS with 10 mM and 0 mM 9 ACS Paragon Plus Environment

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

glucose respectively. All other channels (3-5) were filled with 0.05% azide in PBS. PCF cells were loaded onto the plate as described above and then the pneumatic and temperature control manifold was allowed to equilibrate at 29°C for 30 minutes before perfusion experiments were started. Once the cells were loaded, they were exposed to various external glucose concentrations using the mixing method described previously for fluorescein (i.e. a mix of glucose-free and 10 mM glucose PBS was provided to produce different glucose concentrations). Cells were first incubated for five minutes in PBS without glucose by pressuring channel 1 to 20 kPa, after which constrained cells were exposed to different external glucose concentrations (2.5 - 10 mM glucose in PBS) by perfusing with a mix of channels 1 and 2 at different pressures (Table 2), such that the total pressure was 20 kPa. FRET and ECFP were continuously monitored during perfusion by fluorescence microscopy (430/30 ex, 480/30 and 530/30 em); images were obtained with 100µsec exposure, every 15 seconds. The FRET/ECFP ratio was extracted for each cell and background was subtracted from a cell-free region. Individual FRET responses were then averaged to represent the glucose response of the imaged population for n ≥ 25. FRET/ECFP ratio movies were made using Slidebook 6.0 software’s ratio module and exported as a series movie.


Page 10 of 28

Page 11 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Table 2. Glucose Mixing Protocol Plate Preparation

Channel Number 1,6

Perfusion Protocol

10 mM glucose

2

PBS 0mM glucose

3,4,5

0.05% azide in PBS

8 Cell Loading

Channel Contents

1 x 106 cells/mL

Protocol Step

Length (min)

Channels

Pressure

1

15

8

15 kPa

2

15

6,8

15 kPa

Protocol Step

Length (min)

Channels

1

10

1,2

2

5

2

3

5

1,2

4

5

2

5

5

1,2

6

5

2

7

5

1,2

8

5

2

20 kPa

9

5

1

20 kPa

10

5

2

20 kPa

Pressure 5 kPa, 5 kPa 20 kPa 5 kPa, 15 kPa 20 kPa 10 kPa, 10 kPa 20 kPa 15 kPa, 5 kPa

Unaveraged single cell glucose response time-lapse. To demonstrate the capabilities of the microfluidic device to continuously monitor single trypanosomes, PCF cells expressing glucose sensor were rinsed and loaded into the microfluidic device (following the loading protocol outlined in table 2) in PBS supplemented with 10 mM glucose (the “high glucose” solution) . The FRET/ECFP ratio of cells in this high glucose solution was monitored for 7.5 minutes at 20 kPa of system pressure; solution lacking glucose was perfused for 12.5 minutes followed by perfusion back into 10 mM glucose PBS for 12.5 minutes at 20 kPa. This cycle was repeated to determine 11 ACS Paragon Plus Environment

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

the temporal response to the perfusion conditions. Images were obtained at 15 second intervals, with 100 µsec exposure times.

Subcellular imaging. To explore the utility of the system for resolving subcellular events, PCF parasites expressing FLII12Pglu-700μδ6 in glycosomes were imaged in the microfluidic device; FLII12Pglu-700μδ6 was targeted to the glycosomal lumen via appended a PTS1 signal (AKL) to the C-terminus of the protein via site directed mutagenesis. PCF parasites were loaded into the device as described above and perfused with PBS supplemented with 10mM glucose for the duration of the experiment, with conditions as described above. DIC and fluorescent microscopy images were obtained as described above. To limit phototoxicity, fluorescence images were captured only once for every 5 DIC image captures.

Results and Discussion Mechanism of cell containment and plate layout. The microfluidic system used here is commercially available microfluidic platform that has been used to investigate biological processes in mammalian, yeast and bacterial cells.18 The platform allows for continuous temperature and environmental control for long-term microscopy experiments. In this study, we used microfluidic plates designed to constrain small organisms in a single focal plane and field of view, thus allowing for continuous monitoring via transmitted light and/or fluorescence microscopy during buffer exchange experiments. The general plate layout for the cell-trapping microfluidic device is shown in Figure 1A. Fluids are perfused through the device by pressurizing 12 ACS Paragon Plus Environment

Page 12 of 28

Page 13 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

different channels containing buffer/media of different compositions (Figure 1B). The celltrapping portion of the microfluidic plate consists of a glass bottom with a PDMS top; the chamber height decreases from sample input to output ports, which creates regions of the plate with different heights, which effectively retain cells based on their size (Figure 1C). As a result, cells are mechanically restrained between the glass bottom and the PDMS ceiling. This approach maintains the cells in a constant X-Y orientation in the focal plane for optimal imaging (Figure 1D). The restrained cells can then be imaged during perfusion with buffers of different composition.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. Representation of a CellASIC microfluidic plate (A). Expanded view of the cell-trapping portion of the microfluidic plate (B). The different colored strips represent the different heights of the trapping compartment indicated to the right. Cells enter the device from the bottom (2.3 µm) side and are pushed through the device until they are constrained between the glass bottom (blue bar) and the different ceiling heights (C). Different sized cells are trapped in different portions of the device according to their size (D).


Page 14 of 28

Page 15 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Constraining live trypanosomes in the microfluidic device. Protocols originally developed for use of the microfluidic device with bacteria were not suitable for trypanosomes, largely because channel pressures recommended for bacteria were too high to maintain parasite viability. To load trypanosomes into the microfluidic plate, we used channel pressures 2-4 fold lower than recommended for bacteria. To alter the density of loaded cells, either the density of the cell suspension loaded on the device could be increased, or the cell loading protocol could be run for a longer time. These conditions were suitable for constraining viable cells at sufficient densities for use in a wide range of perfusion experiments (Movie S1, and detailed below).

BSF cells were retained primarily in the 0.9 µm portion of the device while PCF parasites were retained in the 1.1-1.3 µm height regions. Under these buffer and glucose conditions (PBS supplemented with 10 mM glucose), both BSF and PCF parasites remained motile over the entire course of the experiment (at least 2 and 4 hours, respectively). Representative images of retained cells are shown in Figure 2A and 2B. The time-lapse movies that were the source of these images are included in Movie S2A and S2B.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2. Representative DIC image of BSF (A) and PCF (B) trypanosomes tapped in the bacterial microfluidic plate. The line across the center of the image shows the barrier between two different height capture regions, while the dots in the image are pillars used to maintain the height of the device ceiling. Different trap heights are denoted in the image along with the direction of flow. PCF were primarily trapped in the 1.1-1.3um region and BSF were trapped at the 0.9-0.7 um interface. Images were extracted from time-lapse movies found in Supplemental Information. Scale is 10 µm.


Page 16 of 28

Page 17 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Temperature and environmental controls in the microfluidic system should allow for longterm viability. To provide support for this claim, we investigated the viability of constrained cells in the microfluidic device over a period of 48 hours in full SDM-79 media. As shown in Movie S3AC, movies of PCF cells at 0, 24, and 48 hours after being placed in the instrument indicate that parasites remained motile and viable over the 48 hours tested. Indeed, net cell density increased over time (nearly two-fold over 24 hours), consistent with cell division in the instrument. The observed viability and growth suggest that these microfluidic devices are suitable for multipleday imaging experiments. However, due to small fluctuations in room temperature during long term exposures (i.e. > 4 hours), and a lack of focus compensation instrumentation or objective/stage heater, thermal expansion of imaging instrumentation lead to drifts in image focus over time. This thermally-induced shift in focus prevents continuous acquisition of fluorescence images for longer than ~ 4 hours without additional focus correction/compensation instrumentation. When constrained in the device, subcellular organelles in individual cells could be visualized. As a result, subcellular events can be monitored in individual organelles. To demonstrate subcellular spatial resolution, PCF parasites expressing a glycosomally localized fluorescent probe were imaged using the microfluidic platform. During these time-lapse imaging experiments, parasites were maintained in the imaging chamber of the device and did not leave the focal plane. Representative images of these experiments are shown in Figure 3; the complete time-lapse videos are shown in Movie S4. In the resulting images, glycosomes can be clearly distinguished from the surrounding cell (Figure 3).


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3. DIC and fluorescent images of PCF parasites expressing FLII12Pglu-700μδ6-PTS in glycosomes restrained in the microfluidic device. To decrease background from media components cells were rinsed 3 times with PBS with 10mM glucose added to maintain viability. Subcellular features (glycosomes) were visible in images of parasites constrained in position in the microfluidic device. Images are representative of microscopy images found in supplemental data. Background was subtracted from a cell-free region; size bars = 10 µm.

Fluorescein perfusion and mixing in the device. The microfluidic devices used in this study are designed to hold up to five different buffers, thus allowing multiple perfusion conditions. To generate a chemotactic gradient using this plate setup, channels were individually pressurized and then the contents of those channels allowed mixing. To characterize the buffer mixing and perfusion, time-lapse images were collected as different concentrations of fluorescein were mixed with PBS at different pressures (25, 50, 75, and 100% of a 10 µg/mL fluorescein solutions). Figure 4A shows alteration of fluorescence intensity over time as fluorescein buffers were serially exchanged with buffer containing no fluorescein. A real-time video of these images is shown in 18 ACS Paragon Plus Environment

Page 18 of 28

Page 19 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Movie S5). Data in Figure 4A shown that fluorescence intensity after perfusion increased in a concentration dependent manner, as expected for effective buffer exchange. In addition, after each exchange of fluorescein buffer, the fluorescent intensity returned to baseline, indicating that the microfluidic system achieves complete buffer exchange. There was a slight delay from initiation of buffer perfusion to perceptible fluorescence change (somewhat less than 2 minutes), with complete buffer exchange occurring in ~ 3 minutes. This perfusion time-lag is anticipated to be acceptable for most experiments where biological effects are expected to be on the minutes to hours timescale.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. (A). Fluorescein perfusion experiement where different dye concentrations were achived by mixing solutions from different microfluidic plate channels. Mean fluorescence intensity from cells contained in a region of interest was plotted versus time. Vertical lines represent the time point when perfusion into PBS containing different concentration of fluorescein was initiated; concentrations are shown in annotation in upper region of plot. Dye fluorescence was tracked via fluorescence microscopy (490/20 ex. 530/30 em); 100 msec images were taken every 5 seconds. Mean fluorescence intensity from cells contained in a region of


Page 20 of 28

Page 21 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

interest was plotted versus time. Error bars represent the standard deviation of the measurement. (B). Cytosolic FLII12Pglu-700μδ6 biosensor response (FRET/ECFP ratio) in a group of cells upon perfusion into PBS containing different external glucose concentrations.

Cells

containing cytosolic biosensor were sequentially exposed to increasing external glucose concentrations, alternately with PBS containing no glucose. Vertical lines represent the time point when perfusion into PBS of different glucose

concentration was initiated; glucose

concentration for each perfusion are shown in annotation in upper region of plot. Images were obtained every 15 second. Error bars represent the standard deviation of n=25-50 cells.

Monitoring average parasite response to environmental manipulation. Fluorescent glucose biosensors represent an exciting avenue to explore metabolite concentration flux in living parasites. For example, we have previously expressed and characterized fluorescent glucose sensors in the cytosol or glycosomes of PCF and BSF parasites; in these experiments, FRET ratio of the intracellular sensor changes with intracellular glucose cocnentration.19

Traditional

ratiometric microscopy images, including FRET images, are typically acquired by rapidly capturing an image in each fluorescent channel in succession using a filter wheel or similar hardware. Unfortunately, highly motile cells like trypanosomes can move during the two image acquisition steps, leading to low image overlap, which can make acquisition of meaningful FRET/ECFP ratio images challenging. Instead, we have used an Andor Tucam fluorescence emission wavelength splitter (Figure S1), which separates the ECFP and FRET emission with a long pass dichroic mirror and directs it to two cameras, thus allowing for simultaneous ECFP and FRET emission capture. This strategy ensures that the ECFP and FRET images overlap and enhances the quality of the resulting FRET ratios.20 21 ACS Paragon Plus Environment

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 28

Glucose transport and metabolism is critical for survival of T. brucei. As a result, understanding parasite glucose homeostasis mechanisms is of key research importance. Using trypanosomes containing a cytosolic glucose biosensor, we tracked changes in average cytosolic glucose as parasites were exposed to different extracellular glucose concentrations. Figure 4B shows the average FRET/ECFP ratio of cytosolic FLII12Pglu-700μδ6 over time as cells were sequentially exposed to either zero or 10 mM glucose. The average FRET/ECFP ratio of cytosolic FLII12Pglu-700μδ6 increased as external glucose concentrations increased, demonstrating that glucose concentrations in the cytosol rise with extracellular glucose concentration.

This

observation is consistent with glucose uptake previously observed in mammals, yeast, plants, and trypanosomes.18,21,22 Interestingly, average cytosolic glucose approached external glucose concentrations very quickly, nearly approaching the rate of perfusion determined in Figure 4A. These data indicate that glucose uptake into the cytosol is rapid under these conditions. A full time-lapse movie of all images collected for Figure 4B is shown in Movie S6.

Comparison of single cell responses. Data shown in Figure 4B shows how average cytosolic glucose response changes in a group of cells in response to changes in extracellular glucose. However, analysis of single cells can often provide insight into biological phenomena that are overlooked when responses are averaged during bulk population analysis.23 To assess the individual variability possible in a population, we compared single-cell transient responses to removal of extracellular glucose (Figure 5 B). Representative FRET/ECFP ratio images (Figure 5A) were extracted from a FRET/ECFP ratio time-lapse video of individual PCF trypanosome cells expressing FLII12Pglu-700μδ6 as the glucose concentration in the external PBS was cycled from 22 ACS Paragon Plus Environment

Page 23 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

zero to 10 mM (Movie S7). Resulting Images were pseudo colored such that red denotes highest cytosolic FRET ratio (cytosolic levels under conditions of high extracellular glucose) and blue denotes lowest cytosolic FRET ratio (low glucose) (i.e. cytosolic response to glucose starvation). As shown in Movie S7, cytosolic glucose concentrations in live PCF trypanosomes changed rapidly over time as the extracellular concentration was altered, as indicated by the change in pseudocolor. Single cell response (Figure 5B) was very similar to that observed in the population (Figure 4B), indicating that cytosolic glucose response to changes in extracellular glucose concentration is consistent between individual PCF parasites.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5. (A) Representative FRET/ECFP ratio images extracted from a time-lapse fluorescence movie of PCF cells expressing the glucose sensor. Images were obtained with 100 µsec exposure; 45 second intervals between images. Cells were perfused from high (10 mM) to low (0 mM) glucose in PBS, as denoted by the label in the upper left corner of each image. Pseudocolor images were constructed from the intracellular FRET/ECFP ratio, red cells represent high cytosolic glucose and blue represent low glucose. (B) Single cell traces of the FRET/ECFP response as external glucose concentration in PBS is cycled from zero to 10 mM. Traces represent the glucose sensor response (FRET/ECFP ratio) for individual cells versus time.

Conclusion Here we outline a microfluidic method to constrain BSF and PCF African trypanosomes in place to allow for live cell imaging over the course of buffer exchange. Using a commercially available microfluidic system equipped with bacterial trapping plates, we obtained cell images and time-lapse videos of live trypanosomes during perfusion experiments while maintaining cell viability, including parasite division.

Further, this arrangement allowed us to analyze single

parasites expressing a fluorescent glucose sensor to study parasite response to changes in external glucose concentrations. Time-lapse measurements of single cells were acquired and individually analyzed to identify the distribution of responses within a population. Data shown here demonstrates that this method is appropriate for visualization of both whole cells and subcellular organelles. This strategy represents a significant improvement for single cell analysis of trypanosomes and kinetoplasts during perfusion and expands the analytical repertoire available for these and similar cells.


Page 24 of 28

Page 25 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Supporting information. -Movie S1-S5 and S7-S8 are movie files that represent the DIC and/or fluorescence microscopy movies that were used to create all of the figures in this manuscript. -Figure S1 is a representative diagram of the dual channel imaging system used to capture all FRET images and movies.

Acknowledgements. This study was funded by the National Institutes of Health (R21AI105656) to JCM and KAC and in part by the National Institutes of Health Center for Biomedical Excellence (COBRE) grant (P20GM109094). The sensor pRSET FLII12Pglu-700µδ6 was a gift from Wolf Frommer (Addgene plasmid # 13568).


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

References. (1)

Barrett, M. P.; Burchmore, R. J. S.; Stich, A.; Lazzari, J. O.; Frasch, A. C.; Cazzulo, J. J.; Krishna, S. The trypanosomiases. Lancet 2003, 362 (9394), 1469–1480

(2)

Kuile, B. H. Adaptation of metabolic enzyme activities of Trypanosoma brucei promastigotes to growth rate and carbon regimen . Adaptation of Metabolic Enzyme Activities of Trypanosoma brucei Promastigotes to Growth Rate and Carbon Regimen. J. Bacteriol. 1997, 179 (15), 4699–4705.

(3)

Bringaud, F.; Rivière, L.; Coustou, V. Energy metabolism of trypanosomatids: adaptation to available carbon sources. Mol. Biochem. Parasitol. 2006, 149 (1), 1–9

(4)

MacLean, L.; Myburgh, E.; Rodgers, J.; Price, H. P. Imaging African trypanosomes. Parasite Immunol. 2013, 35 (9–10), 283–294

(5)

Glogger, M.; Subota, I.; Pezzarossa, A.; Denecke, A. L.; Carrington, M.; Fenz, S. F.; Engstler, M. Facilitating trypanosome imaging. Exp. Parasitol. 2017, 180, 13–18

(6)

Dong, L.; Cornaglia, M.; Krishnamani, G.; Zhang, J.; Mouchiroud, L.; Lehnert, T.; Auwerx, J.; Gijs, M. A. M. Reversible and long-term immobilization in a hydrogel-microbead matrix for high-resolution imaging of Caenorhabditis elegans and other small organisms. PLoS One 2018, 13 (3), 1–20

(7)

Sackmann, E. K.; Fulton, A. L.; Beebe, D. J. The present and future role of microfluidics in biomedical research. Nature 2014, 507 (7491), 181–189

(8)

Rogers, J. A.; Nuzzo, R. G. Recent progress in soft lithography. Mater. Today 2005, 8 (2), 50–56

(9)

Zare, R. N.; Kim, S. Microfluidic Platforms for Single-Cell Analysis. Annu. Rev. Biomed. Eng. 2010, 12 (1), 187–201

(10)

Guan, A.; Shenoy, A.; Smith, R.; Li, Z. Streamline based design guideline for deterministic microfluidic hydrodynamic single cell traps. Biomicrofluidics 2015, 9 (2)


Page 26 of 28

Page 27 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

(11)

Yin, H.; Marshall, D. Microfluidics for single cell analysis. Curr. Opin. Biotechnol. 2012, 23 (1), 110–119

(12)

Hochstetter, A.; Stellamanns, E.; Deshpande, S.; Uppaluri, S.; Engstler, M.; Pfohl, T. Microfluidics-based single cell analysis reveals drug-dependent motility changes in trypanosomes. Lab Chip 2015, 15 (8), 1961–1968

(13)

Stellamanns, E.; Uppaluri, S.; Hochstetter, A.; Heddergott, N.; Engstler, M.; Pfohl, T. Optical trapping reveals propulsion forces, power generation and motility efficiency of the unicellular parasites Trypanosoma brucei brucei. Sci. Rep. 2014, 4, 1–7

(14)

Brun, R.; Schönenberger. Cultivation and in vitro cloning or procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Short communication. Acta Trop. 1979, 36 (1979), 289–292

(15)

Hirumi, H.; Hirumi, K. Continuous Cultivation of Trypanosoma brucei Blood Stream Forms in a Medium Containing a Low Concentration of Serum Protein without Feeder Cell Layers Author ( s ): Hiroyuki Hirumi and Kazuko Hirumi Published by : Allen Press on behalf of The American Soc. J. Parasitol. 1989, 75 (6), 985–989.

(16)

Wang, Z.; Morris, J. C.; Drew, M. E.; Englund, P. T.; Acad, E. P. N.; Sci, U. S. A. Inhibition of Trypanosoma brucei Gene Expression by RNA Interference Using an Integratable Vector with Opposing T7 Promoters * RNA interference is a powerful method for inhibition. J. Biol. Chem. 2000, 275 (51), 40174–40179

(17)

Burkard, G.; Fragoso, C. M.; Roditi, I. Highly efficient stable transformation of bloodstream forms of Trypanosoma brucei. Mol. Biochem. Parasitol. 2007, 153, 220–223

(18)

Bermejo, C.; Haerizadeh, F.; Takanaga, H.; Chermak, D.; Frommer, W. B. Dynamic analysis of cytosolic glucose and ATP levels in yeast using optical sensors. Biochem. J. 2010, 432 (2), 399–406

(19)

Voyton, C. M.; Morris, M. T.; Ackroyd, P. C.; Morris, J. C.; Christensen, K. A. A FRET Flow Cytometry-Based High Throughput Screening Assay to Identify Disrupters of Glucose 27 ACS Paragon Plus Environment

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Levels in Trypanosoma brucei. ACS Infect. Dis. 2018 (20)

Spiering, D.; Bravo-Cordero, J. J.; Moshfegh, Y.; Veronika; Miskolci; Hodgson, L. Quantitative Ratiometric Imaging of FRET-Biosensors in Living Cells. Methods Cell Biol. 2013, 114, 593–609

(21)

Deuschle, K.; Chaudhuri, B.; Okumoto, S.; Lager, I.; Lalonde, S.; Frommer, W. B. Rapid metabolism of glucose detected with FRET glucose nanosensors in epidermal cells and intact roots of Arabidopsis RNA-silencing mutants. Plant Cell 2006, 18 (9), 2314–2325

(22)

Liemburg-Apers, D. C.; Imamura, H.; Forkink, M.; Nooteboom, M.; Swarts, H. G.; Brock, R.; Smeitink, J. a M.; Willems, P. H. G. M.; Koopman, W. J. H. Quantitative glucose and ATP sensing in mammalian cells. Pharm. Res. 2011, 28 (11), 2745–2757

(23)

Bass, K. E.; Wang, C. C. The in vitro differentiation of pleomorphic Trypanosoma brucei from bloodstream into procyclic form requires neither intermediary nor short-stumpy stage. Mol. Biochem. Parasitol. 1991, 44 (2), 261–270

For Table of Contents Use Only


Page 28 of 28

A microfluidic-based microscopy platform for continuous interrogation

Recommend Documents