AFM-FLIM facility

We recently installed and commissioned an integrated atomic force (AFM) and fluorescence lifetime imaging (FLIM) microscope, funded by an Strategic Equipment grant from EPSRC.

FLIM system: PicoQuant Microtime 200

Laser sources: 405 nm, 485 nnm, 532 nm, and 640 nm

Objectives (Olympus):

Visible range: Plan N 20x NA 0.40 (air), Plan N 40x NA 0.65 (water), PlanFL N 100x / NA 0.95 (air) WD = 0.2 mm, UPlanSApo 60x NA 1.20 (water) WD = 0.25 mm

Infrared: LCPlan N 20x NA 0.45 IR WD = 8.3 mm; LCPlan N 100x NA 0.85 IR WD = 1.48 – 1.18 mm (glass)

Epifluorescence filters: DAPI, eGFP, mCherry TexaRed

Major dichroic mirrors

ZT 532/640 rpc-UF3For 531 nm and/or 637 nm lasers
ZT 405-473/532 rpc-UF3For 402 nm and/or 531 nm lasers
ZT 488/532 rpc-UF3For 482 nm and/or  531 nm lasers
ZT 470-488/640 rpc-UF3For 482 nm and/or 637 nm lasers
ZT 405/485 rpc-UF3For 402 nm and/or 482 nm lasers
ZT 405 rdc-UF3For 402 nm laser

Beam splitter optics

100% Mirror (x4)For reflecting 100 % of the incoming light
50/50 BS Plate (x4)For splitting all light into two directions (Polarisation dependent)
50/50 BS CubeFor splitting all light into two directions (Polarisation independent)
H488lpxrReflected: λ < 488 nm Transmitted: λ > 488 nm
T635lpxrReflected: 475 nm < λ < 635 nm Transmitted: 650 nm < λ
BS R830Reflected: 350 nm < λ < 830 nm Transmitted: 845 nm < λ < 1600 nm (For Infrared detector)
Zt532rdc-UF1Reflected: 405 nm < λ < 532 nm Transmitted: 545 nm < λ < 750 nm

Filters

Blank (x17)For 100 % transmission
750 SPTransmitted: λ < 750 nm
425 LPTransmitted: λ > 425 nm
488 LPTransmitted: λ > 488 nm
532 LPTransmitted: λ > 532 nm
937 LPTransmitted: λ > 937 nm
445/30 BPTransmitted: 430 nm < λ < 460 nm
480/20 BPTransmitted: 570 nm < λ < 490 nm
511/20 BPTransmitted: 501 nm < λ < 521 nm
582/64 BPTransmitted: 550 nm < λ < 614 nm
690/70 BPTransmitted: 655 nm < λ < 725 nm

Detectors

Single Photon Counting Module Excelitas SPAD (x2): Red sensitive 400- 1000 nm.

Photomultiplier module PMA Hybrid 40 (x2): Response 300 – 720 nm.

NIR-PMT detector: Spectral range 950 – 1700 nm.

TCSPC electronics: Hydraharp (high time resolution, for fast lifetimes) and MultiHarp (for longer lifetimes).