Biophysics and Micro/Nanostructures Laboratory
Laser Scanning Confocal Microscope (LSCM) Facility with Multi-Photon

Overview

The Laser Scanning Confocal Microscopy is a widely used form of high resolution epi-fluorescence microscopy which allows the acquisition of thin optical sections of finite and controlled thickness.

Hardware

Our facility comprises of a Leica TCS SP2 system upgraded with a Multi-photon Coherent laser unit.  The basic TCS SP2 is an Inverted Leica DM IRB, IRE 2 with 4 objectives:

HC PL Fluotar 10X/0.3
Working distance: 11mm

HCX PL APO 40X/1.25 Oil PH3 CS
Working distance: 0.1 mm

HCX PL APO 63x/1.20 W Corr/0.17 CS
Working distance: 0.22 mm W Corr/ 0.07mm
W/out Corr

HCX APO 100X /1.40-0.70 Oil CS
Working distance: 0.09mm

For visual inspection, there is the choice of the normal Halogen lamp, or an Hg lamp.  There are 3 filters namely, UV, FITC and Rhodamine.


Detailed specifications:

3 channels + 1 Transmitted light detector
Continuously adjustable bandwidth and center wavelength
High resolution z-stage
Ar 457nm, 488nm, 514nm; ArKr 488nm; Kr 568nm; HeNe 633nm
AOTF 4 to 8 channels, visible range; AOTF, UV range
Adjustable pupil illumination
One pinhole, variable diameter size
Line frequency: up to 2000 lines/second
Frame rates: 3 fps (512 x 512 pixels), 25 fps (512 x 32 pixels)
Scan resolution: up to 4096 x 4096 pixels
Scan zoom 1 – 32x
Scan rotation –5 to +95 degrees
Multi-dimensional series acquisition
Emission spectrum recording
Time-lapse recording
Surface reconstruction
Multiple measurement functions
Physiology software
Dye finder software
3D software with multiple reconstruction rendering & functions
Macro developer software

With the above, optical slicing is easily carried and 3D reconstructed (w/out) stereo projections are possible.  Scanning can be in different modes like xyz, xzy, and even xy.

Multi-photon Excitation Microscopy

TCS SP2 can be upgraded with either a UV laser or a Multi-photon unit.
Multi-photon excitation (MPE) microscopy is a powerful tool that can
probe delicate cells and tissues without damaging the sample. The
laser source of choice for MPE microscopy is an ultrafast Ti-Sapphire laser.
This is possible with the combination of the Verdi V5 and the Mira
Optima 900F was purchased. This offers a mode-locked laser with
an output power of about 790mW. The wavelength can be tuned from
700-980nm and has a repetition rate of 76MHz.

Advantages of MPE:

· Higher axial resolution
· Increased depth penetration
· Reduced photo-bleaching of marker dyes
· Increased cell viability
· No bulk fluorescence

Applications of Confocal and Multiple Photon Microscopy

1. Immunolabeling
2. Organelle identification (using suitable dyes)
3. Protein Trafficking
4. Locating genes on chromosomes
5. Analysis of membrane mobility (FRAP, FRET)
6. Multiple labeling
7. Live cell work (with appropriate incubation system)
8. Subcellular functions ( pH gradients, and membrane potentials)
9. Ion Concentrations (e.g. Ca²⁺, Na⁺, Mg²⁺…)

Gallery:

Fibroblast stained with CMX Ros Red Mitotracker (Molecular Probes) Slice 36/75
Reflectance mode – Fibroblast in Collagen type 1 scaffold with Maximum Projection
3D reconstruction and stereo image of the fibroblast
Fibroblasts: Blue: Nuclei, DAPI, Green: Actin, Red: Tubulin, Texas Red
Triple labeled primary heart muscle cells. Courtesy Dr. Elisabeth Ehler, Institute for Cell Biology, ETH, Hönggerberg, Zürich, Switzerland