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Live Cell Multiphoton Imaging: Principles & Experiments

OBJECTIVE: Femtosecond laser pulses, when focused onto a fluorophore, can generate two-photon fluorescence from the focus only due to its very high peak power. Using this property, femtosecond laser pulses are used in multiphoton microscopy. It has better depth resolution as well as less toxicity compared to the confocal microscopy as here a red shifted light is used for excitation.

THEORY: In multiphoton microscope, where mainly two-photon fluorescence is generated and detected, unlike the confocal microscope, no confocal aperture is needed as the two-photon fluorescence is generated only from the focal spot. Moreover, here we can simultaneously excite a whole range of fluorophores using a single laser unlike the confocal microscope as fluorophores have very wide two-photon absorption band compared to its single photon absorption band. The small difference between the multiphoton and confocal microscope is that in the former case we do not need the pinhole, but we need filter (s) to separate the fluorescence coming from the various fluorophores.

Description: ImagepIma_DSP.BMP

Image of a BPAE tissue under two-photon excitation


  1. Excitation Pulsed Laser
  2. Microscope
  3. Microscope Objective
  4. Fluorescent Sample
  5. Dichroic Mirror
  6. Filters
  7. Detector



Note: For user operation and usage no specific software needed.


1.      Turn on the excitation Laser.

2.      Turn on the ‘ML’ button to convert it to the femtosecond pulse Laser.

3.      Place a drop of immersion oil on the oil immersion microscope objective of a specific magnification

4.      Place the microscopic slide of stained live cell on the sample plane.

5.      Tighten it with the clamps.

6.      Turn on the UV lamp.

7.      Move the objective such that the oil placed on it touches the bottom side of the slide.

8.      Let the UV light pass through the objective and get focused on the sample (i.e., microscopic slide) and see the fluorescence coming from the stained live cell through the microscope eye piece.

9.      Focus the sample seeing the fluorescence of the cells.

10.  Block the UV lamp.

11.  Turn on the FLUOVIEW software for image collection.

12.  Fix the power of the excitation Laser beam.

13.  Let the excitation Laser beam pass through the microscope objective.

14.  Set the value of confocal aperture to its maximum value as multiphoton microscope does not need any confocal aperture.

15.  Place proper fluorescence filter (i.e., band pass filter) for selecting the fluorescence coming from the specific region of the cells (e.g., nucleus, microtubules etc.).

16.  Turn on the Photo Multiplier Tube (PMT) detector to collect the fluorescence.

17.  Increase the PMT voltage to have a good signal.

18.  Select the area of the cell to be selected.

19.  Click on the ‘scan once’ button getting the image of the live cell sample.

20.  Save the image by clicking the buttons in the following order: File I/O => Save Image as and then select the file type (.TIFF or .BMP) and where to save the image and then click the ‘save’ button to save the image.