The NMI node IVMSU (Intravital Microscopy at Stockholm University) is equipped for a broad range of intravital microscopy applications that require multi- and/or single-photon microscopy and FLIM.
Facility Organization
The unit consists of a quarantine, an animal housing room, a surgery and an imaging room.
IVM - Intravital microscopy
Animal housing room
Equipped with 240 IVC
Surgery
Equipped with Leica M205 FA fully motorized fluorescence stereo microscope, surgery equipment inclusive isoflurane anesthesia and monitoring
Imaging room
Instruments
MULTIPHOTON LASER – INSIGHT DUAL X3
Pulsed, tunable infrared laser (80 MHz, 680-1300 nm)
Dual excitation lines: tunable 680–1300 nm line and fixed 1045 nm line
Leica SP8 DIVE with 4Tune non-descanned spectral detectors
Inverted and Fixed Stage
Climate box for temperature control
CO2 supply
Isoflurane anesthesia
Leica HYVOLUTION package for lateral resolution of 140 nm
Variable beam expander for deep-tissue imaging
Resonant scanner (12 kHz line scan frequency)
Samples
Model organisms
Organ/tissue culture
Acute slices
Cells
Imaging depth
Up to 1 mm
Cleared tissue 6 mm
Fluorophores
Most fluorescent dyes
Fluorescent proteins
Endogenous fluorophores
INVERTED SYSTEM – SP8 X DIVE
Excitation: multiphoton laser
405 nm laser
Argon laser 458, 477, 488, 514 nm
White Light Laser 470–670 nm
Leica FALCON fluorescence lifetime measurement
DIC and brightfield
FIXED STAGE SYSTEM – SP8 CFS DIVE
Excitation: multiphoton laser
405 nm laser
Solid State lasers (448, 488, 552, 638 nm)
DIC and brightfield
Multiphoton microscopy
Two-photon principle:
In single-photon excitation (SPE), a fluorophore absorbs one high-energy photon (short wavelength, e.g. 400nm) and emits light with longer wavelength (e.g. 500nm), in two-photon excitation (TPE), the energy of two almost simultaneously absorbed low-energy photons (near infrared, e.g. 800nm) is combined to excite the fluorophore.
Second/Third Harmonic Generation (SHG/THG):
SHG is a non-linear optical process, where the energy of two incident photons is not absorbed, instead, scattered and up-converted. This results in light with double frequency and λ/2 of the incident light. SHG occurs in non-centrosymmetric media such as fibrillar collagen, myosin, microtuble bundles and cellulose.
Third Harmonic Generation (THG):
THG occurs when light penetrates media with different refraction indices as lipid bodies or water-protein aggregates. The energy of three incident photons is combined, resulting in light with triple frequency and λ/3 of the incident light.
Excitation volume
A) With single-photon excitation (as in confocal imaging), the whole thickness of the sample is exposed.
B) Excitation occurs only the focal volume when two-photon excitation is applied (arrow and inset).
Foto: Steve Ruzin and Holly Aaron, UC Berkeley
Multiphoton microscopy is suitable for intravital imaging:
•Reduced scattering of excitation and emission light leads to better signal/noise ratio
•High penetration depth of infrared light in living tissue
•Excitation limited to the focal volume reduces photobleaching and phototoxicity
