Microscopy > Components > Olympus, FCS and FLIM

Olympus, FCS and FLIM Upgrade

The FV1000, FV1200, FV3000 and FVMPE-RS confocal microscopes systems can be easily upgraded to Fluorescence Lifetime Imaging (FLIM) and Fluorescence Correlation Spectroscopy (FCS) capability using photon counting detection. Data are acquired using one of two modalities:

  • the FastFLIM (digital frequency domain); or
  • the TCSPC (time-domain).

FastFLIM (proprietary technology) is the new digital frequency domain (DFD) approach to FLIM measurements. Its advantages, when compared to the TCSPC are twofold:

a) the short time required for data acquisition; and
b) the higher sensitivity of the technique (due to the 100% duty-cycle).

The Olympus FCS and FLIM Upgrade Kit includes the following items:

FastFLIM Unit or TCSPC Unit It accepts the output (via BNC) from up to four PMTs of the confocal unit. The synchronization signal from the Olympus confocal head is connected to the unit.
Detectors Options of:
  • Internal detectors
  • 2-detector unit coupled to the fiber optic port of the microscope
  • 2-detectors coupled to the confocal head of the microscope
Detectors fast PMTs or SPADs.
Laser Launcher Available for 3-, 4- and 6-lasers. The lasers beams are superimposed and the output of the laser launcher is connected to the microscope by using a fiber optic.
Computer Running VistaVision by ISS A separate computer, with a 27" flat monitor

Acquisition and Analysis Software
Fluorescence Fluctuations Spectroscopy (FFS) Module Measurements
  • Fluorescence Correlation Spectroscopy (auto- and cross-correlation)
  • Photon Counting Histogram (PCH)
  • FFS measurements at target XYZ locations in an image
  • FLCS, Fluorescence Lifetime Correlation Spectroscopy
  • Scanning FCS
  • Number & Brightness (N&B)
Imaging Module Measurements
  • Single-point (intensity, polarization, lifetime)
  • Single plan and z-stack (polarization images, ratiometric, FLIM)
Superresolution Module
  • Particle tracking
  • Nanoimaging
FLIM images (digital frequency-domain) (single plane and z-stack)
  • Acquired in digital frequency-domain (DFD). The routine acquires simultaneously a FLIM image and a steady-state image.
FLIM images time-domain (single plane and z-stack)
  • Acquired in time-correlated single photon counting (TCSPC)
Single Molecule Module
  • Burst Analysis
  • FRET and Correlation Methods
  • PIE-FRET Methods
Light Sources
  • Laser diodes: 370 - 1000 nm
  • Ti:Sapphire lasers
Laser Launcher
  • Models for 3-, 4-, 6-lasers.
    Light is delivered to the microscope through a single-mode fiber optic.
Input Channels
  • Two
Detectors
  • Internal
    • (original) PMTs
  • External
    • GaAs PMT (Model H7422P)
    • Hybrid PMT (Model R10467U)
CLK
  • Pixel, Line, Frame
FLIM Image Data Acquisition Minimum Dwell Time
  • 4 µs/pixel
Unit Control
  • USB
Computer
  • 3 GHz, 8GB RAM, 27" monitor 2556x1440 resolution
  • Windows 10, 64-bit

The upgrade can be carried out following one of two modalities:

a) By adding two external PMTs (2-detector unit); the fluorescence signal is collected through the fiber port of the microscope and diverted to the 2-detector unit (Figure 1)
b) Utilize the internal PMTs of the FV1000 (Figure 2)

Figure 1. Schematics of the upgrade package for the Olympus confocal microscope. The part to the right includes the instrument components (PC, control electronics, scanner and laser launcher). The left part of the schematics includes the components provided by ISS with the upgrade package.

Figure 2. Schematics of the upgrade package for the Olympus FV1000. The part to the right includes the instrument components (PC, control electronics, scanner and laser launcher). The signal is collected from the internal detectors of the microscope and diverted to FastFLIM unit (left part) of the schematics which includes the components provided by ISS with the upgrade package.

Two-Photon Intravital Fluorescence Lifetime Imaging of the Kidney Reveals Cell-Type Specific Metabolic Signatures.
Hato, T., Winfree, S., Day, R., Sandoval, R.M., Molitoris, B.A., Yoder, M.C., Wiggins, R.C., Zheng, Y., Dunn, K.W., Dagher, P.C.
J Am Soc Nephrol. 2017 Mar 1. pii: ASN.2016101153. doi: 10.1681/ASN.2016101153. [Epub ahead of print]
Imaging Sensitivity of Quiescent Cancer Cells to Metabolic Perturbations in Bone Marrow Spheroids.
Cavnar, S.P., Xiao, A., Gibbons, A.E., Rickelmann, A.D., Neely, T., Luker, K.E., Takayama, S., Luker, G.D.
Tomography. 2016 Jun;2(2):146-157.
Fluorescence Lifetime Imaging of Apoptosis.
Xiao, A., Gibbons, A.E., Luker, K.E., Luker, G.D.
Tomography. 2015 Dec;1(2):115-124.