Overview of Zeiss
The Zeiss LSM 710, LSM 770, LSM 880, and LSM 980 confocal microscopes series can be easily upgraded to Fluorescence Lifetime Imaging (FLIM) and Fluorescence Correlation Spectroscopy (FCS) capability using photon counting detection. Data is acquired using one of two modalities: FastFLIM (digital frequency domain); or SWISS card (digital TCSP, 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 Zeiss FCS and FLIM upgrade package includes the following items:
|FastFLIM Unit or TCSPC Unit||Each unit accepts the output (via BNC) from the detectors and the synchronization signal from the Zeiss system.|
|Detectors||Two detectors coupled to the descanned port of the confocal head of the microscope; or to the non-descanned port (for multiphoton instruments).|
|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 high-performance computer, with a 32” flat monitor, 2556 x 1440 resolution.|
Product Specifications for Zeiss
Acquisition and Analysis Software
- VistaVision by ISS
Fluorescence Fluctuations Spectroscopy (FFS) 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
- Number & Brightness (N&B)
Imaging Module Measurements
- Single-point (intensity, polarization, lifetime)
- Single plan and z-stack (polarization images, Ratiometric, FLIM)
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, 405, 440, 473, 488, 635 nm
- Single-photon pulsed lasers
- Multi-photon lasers
- Models for 3, 4, 6 laser diodes. Light is delivered to the microscope through a single-mode fiber optic.
- GaAs PMT (Model H7422P)
- Hybrid PMTs (Model R10467U)
- Pixel, Line, Frame
FLIM Image Data Acquisition Minimum Dwell Time
- 6 µs/pixel
- High-performance Processor, 32GB RAM, Windows 10, 64-bit
- 32" monitor, 2556 x 1440 resolution
Example Configuration for Zeiss
“Millisecond spatiotemporal dynamics of FRET biosensors by the pair correlation function and the phasor approach to FLIM.” Hinde, E., Digman, M.A., Hahn, K.M. & Gratton, E. Proceedings of the National Academy of Sciences, 110(1), pp. 135–140, 2012, Dec. doi: 10.1073/pnas.1211882110.
“Metabolic trajectory of cellular differentiation in small intestine by Phasor Fluorescence Lifetime Microscopy of NADH.” Stringari, C., Edwards, R.A., Pate, K.T., Waterman, M.L., Donovan, P.J. & Gratton, E. Scientific Reports, 2(1), 2012, Aug. doi: 10.1038/srep00568.
“Label-free separation of human embryonic stem cells and their differentiating progenies by phasor fluorescence lifetime microscopy.” Stringari, C. Journal of Biomedical Optics, 17(4), p. 046012, 2012, Apr. doi: 10.1117/1.jbo.17.4.046012.
“Biosensor Förster resonance energy transfer detection by the phasor approach to fluorescence lifetime imaging microscopy.” Hinde, E., Digman, M.A., Welch, C., Hahn, K.M. & Gratton, E. Microscopy Research and Technique, 75(3), pp. 271–281, 2011, Aug. doi: 10.1002/jemt.21054.