IVM

IVM - Intravital microscopy

 

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.

 

 

 

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