The facility has four devices that provide live-cell incubation conditions for cells in multiwell plates (96, 384 or 1536 well) for flexible dynamic fluorescence imaging in wide field or spinning disk format. These instruments are used for High-Content Analysis and High-Throughput Microscopy of live or fixed cells or small model organisms.

High-Capacity Instruments for Live and Fixed Samples

Three of the systems are modified forms of the BD Pathway855 High-Content Analyzer. These offer stimulation/perturbation with on-stage pipetting. Conventional through-objective actuation or alternatively parallel plate-wide optogenetic manipulation is possible when stimulation programmes should be maintained during the imaging of multiple positions. All units can be used for end-point or short dynamic imaging sessions, well by well, or longitudinal parallel imaging of the whole plate or selected sub-fields for hours, days or longer.

One of the three units is integrated to an automated cell incubator, allowing automated plate exchange, as well as replenishment of tips and reagent plates from ambient or cooled stacks.

Excitation light is provided by high-power LEDs precisely shuttered in sync with image acquisition to provide defined light doses, which excludes photoxicity caused by lagging mechanical shutters that have now been completely removed. Light sources have been replaced with open souce hardware (OSH) designs delivering 10x more power than commercial options, with 5µs precision shuttering. LEDs used provide 405nm/20, 460nm/50, 430-600nm (used for the 500-555nm range) and 630nm/20nm light. A 735nm LED has been built and filters are being optimised.

Data management, image processing and single cell data analysis are handled in automated pipelines, which are under continuous development to meet new needs that arise.

The integrated system can be used with the following instruments :

• Integrated live-cell incubator (capacity 42 plates, Thermo Scientific Cytomat 2C-LIN)
• Integrated ambient Twister stacks (capacity 120 plates/tip racks x 2)
• Cooled stack (capacity 40 plates)
• Cooled plate holder
• Barcode Reader
• Cell Plate Washer option (Thermo Fisher Wellwash Versa)
• Reagent Dispenser option (Thermo Fisher Multidrop)
• 3-gas mixer

Objectives available

• 1.25x 0.04 – Olympus PLAPON
• 2x 0.08 – Olympus PLAPON
• 4x NA 0.13 – Olympus UPLSAPO
• 4x NA 0.16 – Olympus UPLSAPO
• 10x NA 0.3 LWD – Olympus UPLFLN
• 10x NA0.4 – Olympus UPLSAPO
• 20x NA 0.45 LWD – Olympus LUCPLFLN
• 20x NA 0.75 – Olympus UPLSAPO
• 40x NA 0.9 – Olympus UPLSAPO
• 40x NA 0.95 – Olympus PLAPO

Filters Installed

Excitation Filters

2 wheels, 8 positions each

• 405/20
• 438/24 BrightLine HC
• D440/10
• D470/40
• 488/10
• ET500/20X
• 525/40
• HQ548/20
• S555/28
• HQ560/55
• 635/20 (S635/20 or ZET635/20)
• additional filters available

Beam-Combiner Dichroics

5 positions

• Open
• Mirror
• 10/40 splitter
• 50/10 splitter
• additional dicroics available

Objective Dichroics

5 positions

• FF01-458-DI01 Semrock
• 515LP (72100bs Chroma)
• 595DCLP Chroma
• quad-band #84100bs or ZT405/488/561/640rpcv2-UF1 Chroma
• additional dicroics available

Emission Filters

8 positions

• AT450/20X Intended for BFP
• 483/32 BrightLine HC Intended for CFP, mTurquoise2, mTFP1.0
• 542/27 BrightLine HC (one instrument only, other use 540/50) Suitable for YFP, Ypet, mNeonGreen, mGreenLantern, Dronpa
• HQ540/50 Intended for YFP, Ypet, mNeonGreen, mGreenLantern, Dronpa; can be used for GFP
• 570LP
• ET620/60X intended for mCherry, mScarlet, jRCaMP1b
• ET700/75m intended for miRFP670 and similar
• additional filters available

Operetta Confocal HCS Instrument (Perkin Elmer)

A compact spinning disc confocal microscope, designed for rapid analyses of fixed and live cell applications in microtiter plates using a mechanically shuttered xenon-arc source. The operetta has a live cell chamber that can maintain environmental conditions (temperature, CO2), and provides a spectrum of kinetic capabilities for time course experiments and observation of cellular dynamics. The included Harmony High Content Imaging and Analysis Software allows quantitative evaluation of morphometric image data and automated image analyses.

The device has been connected to the house 1bar CO2 line on user request. Although this is lower than the special 3bar CO2 line recommended by the manufacturer, the chamber nevertheless indicates that it reaches 5% CO2.

Location: Biocity, 5th floor

Contact person: Michael Courtney