Overview of Evident (Olympus)
The FV1000, FV1200, FV3000 and FVMPE-RS confocal microscopes systems can be easily expanded to Fluorescence Lifetime Imaging (FLIM) and Fluorescence Correlation Spectroscopy (FCS) capability using the ISS Upgrade Kits. Photon counting detection ensures sensitivity to low signals. Internal detectors can be utilized or external detectors can be added to the system. Data is acquired using one of two modalities: FastFLIM (digital frequency domain); or SWISS card (digital TCSPC, time-domain). While the choice depends upon the user preference, FastFLIM provides directly data in a suitable format for phasor plot analysis. The package includes VistaVision for data acquisition and processing.
The Olympus FCS and FLIM Upgrade Kit includes the following items:
|FastFLIM Unit or TCSPC Unit||Each unit accepts the output signals (via BNC) from the detectors and the synchronization signal from the Olympus confocal head.|
|External Detectors||Fast PMTs, Hybrid PMTs, or SPADs.|
(not required for multiphoton systems)
|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||High-performance computer, 32 GB RAM, with a 32" flat monitor|
Product Specifications for Evident (Olympus)
Acquisition and Analysis Software
- VistaVision by ISS
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)
- Particle tracking
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
- Laser diodes: 370 - 1000 nm
- Ti:Sapphire lasers
- Models for 3-, 4-, 6-lasers.
- Light is delivered to the microscope through a single-mode fiber optic.
- (original) PMTs
- GaAs PMT (Model H7422P)
- Hybrid PMT (Model R10467U)
- Pixel, Line, Frame
FLIM Image Data Acquisition Minimum Dwell Time
- 4 µs/pixel
- High-performance processor, 32 GB RAM, Windows 10, 64-bit
- 32" monitor, 2556 x 1440 resolution
“Single-cell imaging of α and β cell metabolic response to glucose in living human Langerhans islets.” Azzarello, F., Pesce, L., Lorenzi, V.D., Ferri, G., Tesi, M., Guerra, S.D., Marchetti, P. & Cardarelli, F. Communications Biology, 5(1), 2022, Nov. doi: 10.1038/s42003-022-04215-w.
“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. Journal of the American Society of Nephrology, 28(8), pp. 2420–2430, 2017, Mar. doi: 10.1681/asn.2016101153.
“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, 2(2), pp. 146–157, 2016, Jun. doi: 10.18383/j.tom.2016.00157.
“Fluorescence Lifetime Imaging of Apoptosis.” Xiao, A., Gibbons, A.E., Luker, K.E. & Luker, G.D. Tomography, 1(2), pp. 115–124, 2015, Dec. doi: 10.18383/j.tom.2015.00163.
Configurations for Olympus LSM Upgrade
The upgrade can be carried out following one of two modalities:
- By adding two external PMTs (2-detector unit or Mini-TDU); this solution is preferred when the detectors are not available through Olympus (Figure 1).
- Utilize the internal PMTs of the system (Figure 2).