Multiphoton and Raman microscopes

Multiphoton confocals work by using an infrared laser at double or triple the wavelength necessary to excite a fluorophore.

When two or three photons interact with the fluorophore at the same time, it is excited and emits fluorescence.  The advantage of infrared light is that it can penetrate deep in tissues, unlike visible light which scatters.  Therefore, a multiphoton can image deep in thick samples – provided the emitted signal is strong enough to be captured! It helps if the sample is cleared.

Coherent Raman Microscopes use two coupled infrared lasers to induce bond-specific vibration between atoms in molecules. The resulting deep red to infrared signal is a specific fingerprint that allows to identify specific molecules in an otherwise unstained sample.

Miltenyi TRIM

The Miltenyi TRIM is a multiphoton confocal microscope equipped with two overlapping tuneable infrared lasers (680 to 1300nm) and a fixed line at 1064nm.  This microscope can be configured upright as well inverted, giving complete flexibility of sample access.  It has  a 10x, 20x, 25x and 40x lenses, as well as 11 photoreceptors.  This microscope has multiple filter sets to detect many dyes and fluorescent proteins. The TRIM is also capable of imaging the structure of the imaged tissue using two label-free modes: 1) through second harmonics,  -a process that does not require dyes but relies on autofluorescence; and 2) Raman microscopy -which determines the molecular composition of a sample based on photon-induced specific chemical bond excitation.

Special applications and features:

  1. Multiphoton imaging goes deep in sample
  2. Upright and inverted microscope
  3. Second Harmonics to detect autofluorescence in the tissue and signal context
  4. Raman/CARS microscopy for molecular identification.  Raman works specially well with fatty molecules
  5. Resonant scanner mode for fast imaging

Location: IRR Translational Imaging Hub, contact IRR.Imaging@ed.ac.uk for access.

Leica Stellaris for Coherent Raman Scattering (CRS)

The Leica Stellaris for Coherent Raman Scattering is a fully integrated confocal, multiphoton, and CRS imaging platform.

It is equipped with three lasers, a fixed 405nm laser, a white light laser that can be tunes in 1nm step between 485-685nm and a fast tuneable APE picoEmerald FT infrared laser (660-2340nm).

Its capability includes Stimulated Raman scattering (SRS), coherent anti-Stokes Raman Spectroscopy (CARS), second harmonic generation (SHG) and two photon fluorescence (TPF) as well as confocal microscopy. This inverted microscope is equipped with 10x, 20x, and 63x lenses as well as a 25x and 40x water lenses for long distance work on thick samples. The microscope has an incubation chamber for live cell work.

Special applications and features:

  1. Coherent Raman microscopy for molecular identification of sample components.
  2. Multiphoton imaging goes deep in sample
  3. Second Harmonics to detect autofluorescence in the tissue and signal context
ukri bbsrc square logo
  1. High flexibility in classic confocal mode with the combination of tuneable white laser and receptors: any fluorescent molecule of choice should now be detectable.
  2. Navigator scan for quick mapping with spiral option. The navigator allows for mark and find, as well as tiling of large and complex objects.
  3. FLIM (Fluorescence lifetime imaging microscopy) using TauSense is used to separate signals excited by the same lasers (either a combination of dyes, dyes and background, Raman signal and background). FLIM excites fluorophores by pulses instead of continuously. During interval between pulses, it analyses the speed at which fluorophores emit their photons. The difference in emission speed, which needs to be at least 1ns for TauSense to work, allows for signal separation. Since fluorophore environment also influences fluorescence speed, FLIM can be used to study e.g. metabolism.

Location: IRR Translational Imaging Hub, contact IRR.Imaging@ed.ac.uk for sample imaging.

This article was published on