Prime bsi

The emitted fluorescence light was gathered
by the same objective and transmitted through the multichroic mirror
onto a standard Olympus tube lens to create an intermediate image
at the exit of the microscope frame. This image was passed through
a multiband emission filter (FF01–440/521/607/694/809–25,
Semrock, USA) and was then directed into a magnifying telescope (Apo-Rodagon-N
105 mm, Qioptiq GmbH, Germany and Olympus’ wide field tube
lens with 180 mm focal length, #36–401, Edmund Optics, USA),
with two commercial direct vision prisms (117,240, Equascience, France)
placed within the infinity space between the lenses and mounted on
two motorized rotators (8MR190–2–28, Altechna UAB, Lithuania)
controlling the prisms’ angles around the optical axis. The
final image was acquired on a back illuminated sCMOS camera (Prime
BSI, Teledyne Photometrics, USA).
Image acquisition was coordinated
using micromanager software,^{43 (link)} controlling
camera acquisition, laser excitation, XY stage location, and prism
rotator angles. The camera and laser excitation were synchronized
using an in-house-built TTL controller based on an Arduino Uno board
(Arduino AG, Italy).

For differentiation analyses, parasites were incubated in DTM medium at 27°C in the presence or absence of 6mM cis aconitate. Reactivity of PRS antibody was determined using air dried methanol fixed samples reacted with anti-PRS antibody (a kind gift of Peter Butikofer, University of Bern Switzerland)(1: 200 dilution in PBS) followed after washing with PBS with anti-rabbit Alexa488 conjugated secondary antibody and then 4’,6-diamidino-2-phenylindole (DAPI) (100 ng.mL-1) for 2 minutes. Slides were mounted in Fluorimount-G (Invitrogen). The kinetoplast-posterior dimension was determined using ImageJ on a Zeiss Axio Imager Z2 mounted with a Prime BSI (Teledyne Photometrics) camera using a phase contrast objective (x40, x100).

For the assessment of cell cycle status, parasites were airdried on to glass slides and fixed in ice cold methanol for at least 30 minutes. Thereafter, following rehydration in PBS, samples were incubated with 4’,6-diamidino-2-phenylindole (DAPI) (10 μg ml^–1 in PBS) for 2 minutes and then washed for 5 min in PBS. Slides were then mounted with 40 μl Mowiol containing 2.5% 1,4-diazabicyclo(2.2.2)octane (DABCO). Cell cycle status was assessed by scoring the number of kinetoplasts and nuclei within each cell when visualised under a Zeiss Axio Imager Z2 mounted with a Prime BSI (Teledyne Photometrics) camera using a phase contrast objective (x40, x100).